WO2018030732A1 - Nanovesicles derived from genus bacillus bacteria and use thereof - Google Patents
Nanovesicles derived from genus bacillus bacteria and use thereof Download PDFInfo
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- WO2018030732A1 WO2018030732A1 PCT/KR2017/008497 KR2017008497W WO2018030732A1 WO 2018030732 A1 WO2018030732 A1 WO 2018030732A1 KR 2017008497 W KR2017008497 W KR 2017008497W WO 2018030732 A1 WO2018030732 A1 WO 2018030732A1
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- vesicles
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- cancer
- bacillus
- bacteria
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to nanovesicles derived from Bacillus bacteria and their use, and more particularly to methods of diagnosing cancer, inflammatory diseases, or metabolic diseases using nanovesicles derived from Bacillus bacteria, and preventing or treating the vesicles. It relates to a composition for.
- chronic infectious diseases which were previously recognized as infectious diseases, have become less important, while chronic diseases caused by the incompatibility between humans and microbiomes are the major diseases that determine quality of life and human life. Changed.
- the chronic disease is characterized by chronic inflammation accompanied by immune dysfunction, cancer, chronic inflammatory diseases, metabolic diseases, etc. have become a major problem in public health.
- Inflammation is a local or systemic defense against damage or infection of cells and tissues, primarily by the direct response of humoral mediators that make up the immune system, or by stimulating local or systemic effector systems. It is caused by a cascade of biological reactions that take place.
- Major inflammatory diseases include gastroenteritis, digestive diseases such as inflammatory bowelitis, oral diseases such as periodontitis, asthma, chronic obstructive pulmonary disease (COPD), respiratory diseases such as rhinitis, atopic dermatitis, hair loss, skin diseases such as psoriasis, degenerative arthritis, Arthritis, such as rheumatoid arthritis; And obesity, diabetes mellitus, cirrhosis and the like metabolic diseases.
- COPD chronic obstructive pulmonary disease
- rhinitis atopic dermatitis
- hair loss skin diseases
- skin diseases such as psoriasis, degenerative arthritis
- Arthritis such as rheumatoid arthritis
- obesity
- microbiota refers to a microbial community including microbes, archaea and eukarya that exist in a given settlement, and the intestinal microbiota is important for human physiology. It plays a role and is known to greatly affect human health and disease through interaction with human cells.
- symbiotic bacteria and archaea that symbiotic to our body secrete nanometer-sized vesicles (nanovesicle) to exchange information, such as genes, proteins to other cells.
- the mucous membrane forms a physical protective film that particles larger than 200 nanometers (nm) in size can't pass through. If the bacteria are symbiotic bacteria, the mucosa cannot pass through the mucous membrane, but the bacteria-derived vesicles are 100 nanometers in size or less. It passes through epithelial cells through the mucous membrane and is absorbed by our body.
- Pathogenic bacteria-derived vesicles absorbed by our bodies are inflammatory diseases such as inflammatory skin diseases such as atopic dermatitis, inflammatory respiratory diseases such as chronic rhinitis, asthma and chronic obstructive pulmonary disease (COPD), inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. It is found to be an important factor in the etiology of. In addition, diabetes, obesity and the like has been attracting attention as it is recently found that there is a close relationship with the occurrence of metabolic diseases and solid cancers, such as lung cancer, stomach cancer, colon cancer. However, there have been no cases of diagnosing cancer, chronic inflammatory diseases, or metabolic diseases by quantifying Bacillus bacteria-derived vesicles in clinical samples.
- Bacillus is a Gram-positive bacillus belonging to the Firmicutes portal, which grows in anaerobic as well as aerobic environments, and can survive for a long time in the form of spores in stress situations.
- Bacillus bacteria Bacillus subtilis is a Gram-positive bacillus that can grow in non-pathogenic and aerobic environments, and is widely distributed in the natural world such as dry grass, sewage, soil, and dust.
- subtypes of Bacillus subtilis natto Bacillus subtilis natto (Bacillus subtilis natto) can grow in anaerobic environment, attach to crested soybeans when they make cheonggukjang, and multiply by blocking the air with heat formed near 40 °C.
- Cheonggukjang is a traditional Korean food made from fermented soybeans, similar to Japanese natto. It takes several months to ferment soybean paste but fermented soybeans can be eaten in 2-3 days. In other words, the soybeans can be made on the shortest date of fermented soybeans. Cheonggukjang made by natural fermentation is soaked in hot water for 10-20 hours, then poured with water and boiled. Put a few straws in a bowl, put it in a bowl, cool it to 60 °C, put it in a warm place and cover it with blankets or blankets, and keep it at 45 °C, and then the natto bacteria such as natto breed and become fermented substances.
- compositions used to treat or prevent chronic inflammatory diseases are largely divided into steroidal and nonsteroidal compositions, many of which are accompanied by various side effects.
- TNF- ⁇ is increasing in the etiology of inflammatory diseases such as rheumatoid arthritis, hair loss, inflammatory bowel disease, and the like, and TNF- ⁇ inhibitors have been spotlighted as therapeutic agents for rheumatoid arthritis.
- it has not been used to diagnose cancer, chronic inflammatory disease, or metabolic disease by quantifying genes in vesicles secreted from bacteria of Bacillus or Bacillus subtilis, or to prevent or treat inflammatory diseases through the vesicles. to be.
- the present inventors earnestly studied to solve the above-mentioned conventional problems, and as a result of analyzing the bacteria-derived vesicle metagenome present in human samples, solid cancers such as liver cancer, bladder cancer, breast cancer, and ovarian cancer, asthma, and atopic dermatitis
- solid cancers such as liver cancer, bladder cancer, breast cancer, and ovarian cancer
- asthma and atopic dermatitis
- the contents of the bacterium derived from Bacillus bacterium were significantly reduced, and the vesicles were isolated by culturing Bacillus bacteria, especially Bacillus subtilis, in vitro.
- the present invention was completed based on this.
- an object of the present invention is to provide an information providing method for diagnosing cancer, chronic inflammatory diseases, and metabolic diseases.
- Another object of the present invention is to provide a composition for preventing or treating cancer, chronic inflammatory diseases, and metabolic diseases, including the bacterium-derived vesicles as an active ingredient.
- the present invention provides a method for providing information for the diagnosis of cancer, chronic inflammatory diseases, or metabolic diseases comprising the following steps.
- the cancer may be liver cancer, bladder cancer, breast cancer, or ovarian cancer.
- the chronic inflammatory disease may be asthma, chronic hepatitis, or atopic dermatitis.
- the metabolic disease may be diabetes or cirrhosis of the liver.
- the patient derived sample may be blood, urine, or stool.
- the present invention also provides a pharmaceutical composition for the prevention or treatment of cancer, inflammatory diseases, or metabolic diseases, comprising the bacterium-derived vesicles as an active ingredient.
- the present invention provides a health functional food composition for improving cancer, inflammatory disease, or metabolic disease, comprising the bacterium-derived vesicles as an active ingredient.
- the present invention provides an inhalant composition for preventing or treating cancer, inflammatory disease, or metabolic disease, comprising a bacterium-derived vesicle derived as an active ingredient.
- the present invention provides a cosmetic composition for improving inflammatory disease, comprising a bacterium-derived vesicle derived as an active ingredient.
- Bacillus bacteria-derived vesicles may be vesicles derived from Bacillus subtilis.
- Bacillus subtilis-derived vesicles may be natto-derived vesicles.
- the cancer is selected from the group consisting of liver cancer, bladder cancer, breast cancer, and ovarian cancer;
- the inflammatory disease is selected from the group consisting of asthma, chronic hepatitis, hair loss, rheumatoid arthritis, inflammatory growth inflammation, and atopic dermatitis;
- the metabolic disease may be diabetes or cirrhosis of the liver.
- the inflammatory disease may be one or more diseases selected from the group consisting of rheumatoid arthritis, hair loss, and inflammatory growth inflammation in which the inflammatory cytokine IL-6 or TNF- ⁇ is involved in the pathogenesis.
- the vesicles may have an average diameter of 10 to 200 nm.
- the vesicles may be secreted naturally or artificially from bacteria of the genus Bacillus.
- the vesicles may be isolated from the culture medium of bacteria in Bacillus.
- the present invention also provides a method for preventing or treating cancer, inflammatory diseases, or metabolic diseases, comprising administering to a subject a pharmaceutical composition comprising a bacterium-derived bacterium-derived vesicle as an active ingredient.
- the present invention also provides a preventive or therapeutic use for inflammatory diseases of vesicles derived from Bacillus genus.
- the present inventors confirmed that the intestinal bacteria are not absorbed into the body, but the bacteria-derived vesicles are absorbed into the body and distributed systemically and excreted externally through the kidneys, liver, and lungs.
- Bacterial-derived bacteria of Bacillus present in the blood of patients with metabolic diseases such as solid cancers such as liver cancer, bladder cancer, breast cancer, ovarian cancer, asthma, atopic dermatitis, diabetes, liver cirrhosis, etc. It was confirmed that the vesicles were significantly reduced compared to the normal persons.
- the bacterium-derived vesicles according to the present invention are useful for the diagnosis or prediction method of cancer, chronic inflammatory disease, or metabolic disease, and for the prevention or treatment of food, inhalants, cosmetics, or drugs.
- a standard strain of Bacillus subtilis a subtilis of Bacillus subtilis and Natto, a subtype of Bacillus subtilis
- the bacterium-derived vesicles according to the present invention are useful for the diagnosis or prediction method of cancer, chronic inflammatory disease, or metabolic disease, and for the prevention or treatment of food, inhalants, cosmetics, or drugs.
- Figure 1a is a photograph of the distribution of bacteria and vesicles over time after the oral administration of bacteria and bacteria-derived vesicles (EV) to the mouse.
- Figure 1b is a 12 hours after oral administration, blood, kidneys, liver, and various organs were extracted to evaluate the distribution of bacteria and vesicles in the body.
- FIG. 2 is a diagram schematically illustrating a method of analyzing bacterial-derived vesicle metagenome in human derivatives.
- Figure 3 is a result of comparing the distribution of bacteria-derived vesicles of the genus Bacillus after the analysis of bacteria-derived vesicles metagenome present in liver cancer patients and normal blood.
- Figure 4 is a result of comparing the distribution of bacteria-derived vesicles of the genus Bacillus after the analysis of bacteria-derived vesicles metagenome present in bladder cancer patients and normal blood.
- 5 is a result of comparing the distribution of bacteria-derived vesicles of the genus Bacillus after analyzing the bacteria-derived vesicles metagenome present in breast cancer patients and normal blood.
- Figure 6 is a result of comparing the distribution of bacteria-derived vesicles of the genus Bacillus after the analysis of bacterial-derived vesicles metagenome present in ovarian cancer patients and normal blood.
- 11 is a result of comparing the distribution of bacteria-derived vesicles of the genus Escherichia after the analysis of bacteria-derived vesicles metagenome present in diabetic patients and normal blood.
- Figure 12a is a result of measuring the form and size of Bacillus subtilis derived from Bacillus subtilis in the Bacillus subtilis culture medium by ultracentrifugation method (a).
- Figure 12b is a result of measuring the form and size of Bacillus subtilis derived from Bacillus subtilis through the dynamic light scattering method (b) for the vesicles separated by the ultracentrifugation method in Bacillus subtilis culture.
- FIG. 13A shows E. coli vesicles (E. coli EV), a causative factor in cancer, inflammatory disease and metabolic disease, as a positive control group, Bacillus subtilis EV and Natto bacteria-derived vesicles ( Bacillus subtilis natto EV). ) Is treated with macrophages (Raw 264.7) at various concentrations, and IL-6 concentration is measured by ELISA to compare the degree of secretion of inflammatory mediators [NC: negative control (PBS), EV: extracellular vesicle].
- NC negative control
- EV extracellular vesicle
- E. coli EV E. coli vesicles
- a causative factor in cancer inflammatory diseases, and metabolic diseases
- Bacillus subtilis EVs and Bacillus subtilis natto EVs Bacillus subtilis natto EVs
- EC E. coli vesicles
- FIG. 14A is for evaluating the anti-inflammatory effects of Bacillus subtilis and Bacillus subtilis Natto strains (Lactobacillus), a macrophage line (Raw 264.7) as a control drug Lactobacillus plantarum vesicles ( L. plantarum EV) , Bacillus subtilis EV and Natto-derived vesicles ( Bacillus subtilis natto EV) were pretreated and treated with E. coli-derived vesicles as a causative agent of inflammation after 12 hours. Measurement results [NC: negative control (PBS), PC: positive control, EV: extracellular vesicle].
- NBS negative control
- PC positive control
- EV extracellular vesicle
- 14B is for evaluating the anti-inflammatory effect of Bacillus subtilis and Bacillus subtilis Natto strains (Lactobacillus), a macrophage line (Raw 264.7) as a control drug Lactobacillus plantarum vesicles ( L. plantarum EV) , Bacillus subtilis EV and Natto-derived vesicles ( Bacillus subtilis natto EV) were pretreated and treated with E. coli-derived vesicles as a causative agent of inflammation after 12 hours, and then TNF- ⁇ concentration was determined by ELISA. Measurement results [NC: negative control (PBS), PC: positive control, EV: extracellular vesicle].
- the present invention relates to vesicles derived from bacteria of the genus Bacillus and uses thereof.
- the present inventors conducted meta-genome analysis of bacterium-derived vesicles from Bacillus-derived samples from patients with metabolic diseases such as liver cancer, bladder cancer, breast cancer, ovarian cancer, solid cancers such as asthma, atopic dermatitis, diabetes and liver cirrhosis.
- metabolic diseases such as liver cancer, bladder cancer, breast cancer, ovarian cancer, solid cancers such as asthma, atopic dermatitis, diabetes and liver cirrhosis.
- the standard bacterium belonging to the bacterium of Bacillus and the natto bacteria-derived vesicles were isolated and administered to inflammatory cells.
- the present invention was completed based on this.
- the present invention provides a method for providing information for the diagnosis of cancer, chronic inflammatory diseases, and metabolic diseases comprising the following steps.
- the present invention provides information for diagnosing diabetes comprising the following steps Provide the method of providing.
- Diagnosis means, in a broad sense, to determine the actual condition of a patient in all aspects. The content of the judgment is the name of the disease, the etiology, the type of disease, the seriousness, the detailed mode of the condition, the presence or absence of complications, and the prognosis. Diagnosis in the present invention is to determine the onset of cancer, chronic inflammatory diseases, or metabolic diseases and the level of the disease.
- the present invention provides a food, cosmetic, inhalant, or drug composition for preventing or treating cancer, chronic inflammatory disease, or metabolic disease, comprising a bacterium-derived vesicle of Bacillus as an active ingredient. .
- prophylaxis means any action that inhibits or delays the development of cancer, chronic inflammatory diseases, or metabolic diseases by administration of a food, cosmetic, inhalant, or drug composition according to the present invention.
- treatment means any action in which symptoms for cancer, chronic inflammatory diseases, or metabolic diseases are improved or beneficially changed by administration of a food, cosmetic, inhalant, or drug composition according to the present invention.
- the Bacillus bacteria-derived vesicles of the present invention may be isolated from a culture of Bacillus bacteria or food fermented with Bacillus bacteria, preferably from a culture solution of Bacillus subtilis or natto bacteria or from foods fermented with Bacillus subtilis or natto bacteria. And, it may be natural or artificial secreted from Bacillus subtilis or natto bacteria, but is not limited thereto.
- the method for separating the vesicles from the culture solution or fermented food of the bacterium of the present invention is not particularly limited as long as the vesicles.
- centrifugation, ultra-fast centrifugation, filtration by filter, gel filtration chromatography, pre-flow electrophoresis, or capillary electrophoresis, and combinations thereof may be used to separate the vesicles and also remove impurities. It may further include a process for washing, concentration of the obtained vesicles and the like.
- the vesicles separated by the method in the present invention may have an average diameter of 10 to 1000 nm, more preferably 40 to 100 nm, but is not limited thereto.
- the pharmaceutical composition according to the present invention includes a bacterium-derived vesicle derived from Bacillus as an active ingredient, and may include a pharmaceutically acceptable carrier.
- Such pharmaceutically acceptable carriers are conventionally used in the preparation, and include, but are not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and the like. If necessary, other conventional additives such as antioxidants and buffers may be further included.
- diluents, dispersants, surfactants, binders, lubricants and the like may be additionally added to formulate injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions and the like.
- Suitable pharmaceutically acceptable carriers and formulations can be preferably formulated according to the individual components using methods disclosed in Remington's literature.
- the pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection, inhalant, external preparation for skin, oral ingestion, and the like.
- the pharmaceutical composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, skin, nasal, airways) according to the desired method, and the dosage is determined by the condition and weight of the patient, disease Depending on the degree, drug form, route of administration, and time, it may be appropriately selected by those skilled in the art.
- the composition according to the invention is administered in a pharmaceutically effective amount.
- the pharmaceutically effective amount means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to the medical treatment, and the effective dose level refers to the type, severity, drug activity, and drug of the patient. Sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts.
- the composition according to the present invention may be administered as a separate therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.
- the effective amount of the composition according to the present invention may vary depending on the age, sex, and weight of the patient, and generally 0.001 to 150 mg, preferably 0.01 to 100 mg per kg of body weight is administered daily or every other day or 1 It can be administered in 1 to 3 times a day.
- the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.
- the present invention provides a health functional food composition for improving cancer, inflammatory disease, or metabolic disease, comprising the bacterium-derived vesicles as an active ingredient.
- improvement means any action that at least reduces the parameters associated with the condition being treated, for example the extent of symptoms.
- the active ingredient may be added to the food as it is, or used together with other foods or food ingredients, and may be appropriately used according to a conventional method.
- the mixing amount of the active ingredient can be suitably determined according to the purpose of use (prevention or improvement).
- the compositions of the invention are added in amounts of up to 15% by weight, preferably up to 10% by weight relative to the raw materials.
- the amount may be below the above range.
- the health functional food composition of the present invention in addition to containing the active ingredient as an essential ingredient in the indicated ratio, is not particularly limited to other ingredients, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks.
- natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.
- natural flavoring agents such as, tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.
- the proportion of the natural carbohydrate can be appropriately determined by the choice of those skilled in the art.
- the nutraceutical composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavors such as synthetic and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like.
- these components can be used independently or in combination.
- the proportion of such additives may also be appropriately selected by those skilled in the art.
- the present invention provides an inhalant composition for preventing or treating inflammatory diseases, which comprises a bacterium-derived vesicle as an active ingredient.
- the active ingredient may be added to the inhalant as it is, or may be used together with other ingredients, and may be appropriately used according to conventional methods.
- the amount of the active ingredient to be mixed may be suitably determined depending on the purpose of use (prophylactic or therapeutic).
- the present invention provides a cosmetic composition for improving inflammatory disease or hair loss comprising a bacterium-derived vesicles as an active ingredient.
- the cosmetic composition of the present invention may include not only vesicles derived from bacteria of the genus Bacillus, but also components commonly used in cosmetic compositions, for example, conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances. And a carrier.
- composition of the present invention in addition to the vesicles derived from the bacterium of the genus Bacillus, may be used by mixing the organic sunscreens that have been conventionally used as long as it does not impair the skin protection effect by reacting with the bacterium-derived vesicles.
- organic sunscreen examples include glyceryl pava, drometrizole trisiloxane, drometrizole, digaloyltrioleate, disodium phenyldibenzimidazole tetrasulfonate, diethylhexyl butamidotriazone, diethylamino Hydroxybenzoylhexylbenzoate, die-methoxycinnamate, a mixture of lowson and dihydroxyacetone, methylenebis-benzotriazolyltetramethylbutylphenol, 4-methylbenzylidene camphor, menthyl anthranilate, benzophenone -3 (oxybenzone), benzophenone-4, benzophenone-8 (dioxyphenbenzone), butylmethoxydibenzoylmethane, bisethylhexyloxyphenol methoxyphenyltriazine, synoxate, ethyldihydroxypropylpava, Oct
- Examples of products to which the cosmetic composition of the present invention may be added include, for example, cosmetics such as astringent cosmetics, soft cosmetics, nourishing cosmetics, various creams, essences, packs, foundations, and the like, cleansing agents, soaps, treatments, and essences.
- Specific formulations of the cosmetic composition of the present invention include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack, Formulations such as soaps, shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, emulsions, lipsticks, makeup bases, foundations, press powders, loose powders, eye shadows and the like.
- the content of the bacterium-derived vesicles of the genus Bacillus of the present invention is 0.00001-30% by weight, preferably 0.5-20%, more preferably 1.0-10% by weight based on the total weight of the composition. to be.
- the content of the bacterium-derived vesicles of the Bacillus is less than 0.00001% by weight, the ultraviolet absorbing effect is greatly reduced, and when it exceeds 30% by weight, skin irritation may occur, and formulation problems may occur.
- the present invention provides a method for preventing or treating cancer, inflammatory disease, or metabolic disease comprising administering to a subject a composition comprising a bacterium-derived vesicle derived as an active ingredient.
- an individual means a subject in need of treatment of a disease, and more specifically, a mammal such as a primate, mouse, rat, dog, cat, horse, and cow, which is human or non-human. Means.
- Cancer refers to a malignant tumor that grows rapidly while infiltrating surrounding tissues and spreads or metastasizes to various parts of the body and threatens life.
- Cells the smallest unit of the body, divide and grow normally under the control function of the cells themselves, and die off at the end of their life or damage to maintain the balance of the overall number, but for a number of reasons If a problem occurs in the cell's own regulatory function, abnormal cells that normally must die will overgrow, invading surrounding tissues and organs, forming a mass, and destroying or modifying existing structures.
- the cancer may preferably be liver cancer, bladder cancer, breast cancer, or ovarian cancer, but is not limited thereto.
- inflammatory disease refers to a disease caused by an inflammatory response in a mammalian body.
- respiratory inflammatory diseases such as asthma, chronic obstructive pulmonary disease and rhinitis
- Skin inflammatory diseases such as atopic dermatitis, psoriasis, acne, contact dermatitis, and hair loss
- Gastrointestinal inflammatory diseases such as gastritis, peptic ulcer, and inflammatory bowel disease
- vaginitis Arthritis, such as osteoarthritis and rheumatoid arthritis; And complications thereof.
- the inflammatory disease is used in the sense including cancer associated with the inflammatory response, in addition to the general inflammatory disease, for example, includes breast cancer, ovarian cancer, bladder cancer, liver cancer and the like.
- the inflammatory disease is used in the sense including metabolic diseases related to the inflammatory response, in addition to the general inflammatory disease, for example, diabetes, obesity, cirrhosis of the liver and the like.
- chronic inflammatory diseases preferably mean, but are not limited to, asthma, chronic hepatitis, hair loss, or atopic dermatitis.
- metabolic disease refers to a disease in which complications occur in various organs due to metabolic disorders in a mammal's body.
- carbohydrate metabolic disorders such as diabetes and complications thereof may be cited as cirrhosis.
- metabolic diseases preferably include, but are not limited to, diabetes and cirrhosis.
- nanovesicles refers to the structure of the nano-size membrane secreted by various bacteria.
- Gram-negative bacteria-derived vesicles or outer membrane vesicles contain toxic proteins, bacterial DNA and RNA as well as lipopolysaccharides, and gram-positive bacteria-derived vesicles.
- OMVs outer membrane vesicles
- proteins and nucleic acids it also contains peptidoglycan and lipoteichoic acid, which are components of bacterial cell walls.
- nanovesicles or vesicles are naturally secreted or artificially produced by bacteria of the genus Bacillus, and have a spherical shape and have an average diameter of 10 to 200 nm.
- the term "metagenome” is also referred to as a "gunoelectric", and means a total of a genome including all viruses, bacteria, fungi, and the like in an isolated region such as soil and animal intestine, and is not mainly cultured. It is used as a concept of genome to explain the identification of many microorganisms at once using sequencer to analyze microorganisms.
- the metagenome does not refer to one genome or genome, but to a kind of mixed dielectric as the genome of all species of one environmental unit. This is a term from the point of view of defining a species in the course of the evolution of biology in terms of functional species as well as various species that interact with each other to create a complete species.
- rapid sequencing is used to analyze all DNA and RNA, regardless of species, to identify all species in one environment, and to identify interactions and metabolism.
- Bacterial-derived vesicles in the present invention is centrifugation, ultra-fast centrifugation, extrusion, sonication, cell lysis, homogenization, freeze-thaw, electroporation, mechanical degradation, chemical treatment, filtration by filter, gel containing the bacteria
- the separation can be carried out using one or more methods selected from the group consisting of filtration chromatography, pre-flow electrophoresis, and capillary electrophoresis.
- it may further include a process for washing to remove impurities, concentration of the obtained vesicles and the like.
- the bacterial and bacterial-derived vesicles are orally administered to the mice to evaluate the absorption, distribution, and excretion of the bacteria and vesicles in the body. It was confirmed that it is absorbed and distributed systemically and excreted through the kidney, liver, and the like (see Example 1).
- bacterial metagenome analysis was performed using vesicles isolated from blood of normal, matched age and gender to liver cancer, bladder cancer, breast cancer, and ovarian cancer patients. As a result, it was confirmed that bacterial vesicles of Bacillus spp. Significantly reduced in samples of patients with liver cancer, bladder cancer, breast cancer, and ovarian cancer compared to normal samples (see Examples 3, 4, 5, and 6).
- bacterial metagenomic analysis was performed using vesicles isolated from a sample of asthma and atopic dermatitis patients and normal subjects matching the age and gender of the patient. As a result, it was confirmed that the vesicle-derived bacteria of Bacillus spp. Significantly decreased in the samples of asthma and atopic dermatitis patients compared to the normal samples (see Examples 7 and 8).
- bacterial metagenomic analysis was performed using vesicles isolated from a sample of a diabetic and cirrhosis patient and a normal person matching the age and sex of the patient. As a result, it was confirmed that the vesicles derived from Bacillus spp. Significantly reduced in the samples of diabetic and cirrhosis patients compared to the normal samples (see Examples 9 and 10).
- vesicles derived from Bacillus subtilis (standard strain)
- Naxos-derived vesicles which are subtypes of Bacillus subtilis
- there was little inflammation-inducing effect compared to E. coli-derived vesicles and when pretreatment of vesicles or Natto-derived vesicles effectively suppressed the inflammatory response caused by Escherichia coli-derived vesicles.
- the present invention was completed based on this observation (see Examples 13 and 14).
- the blood-labeled 50 ⁇ g of bacteria and bacteria-derived vesicles were administered in the same manner as described above, and then 12 hours after administration
- the heart, liver, kidneys, spleen, fat and muscles were taken.
- bacteria-derived vesicles were distributed in blood, heart, lung, liver, kidney, spleen, fat, muscle, and kidney, but the bacteria were not absorbed (see FIG. 1B).
- DNA extracted by the above method was amplified using the above 16S rDNA primers, followed by sequencing (Illumina MiSeq sequencer), and the results were outputted in a Standard Flowgram Format (SFF) file, using GS FLX software (v2.9).
- SFF Standard Flowgram Format
- GS FLX Standard Flowgram Format
- OTU operational taxonomy unit
- clustering is performed according to sequence similarity using UCLUST and USEARCH, genus 94%, family 90%, order 85%, class 80%, phylum 75% sequence similarity
- Clustering is based on the phylum, class, order, family, and genus levels of each OTU, and BLASTN and GreenGenes' 16S RNA sequence database (108,453 sequences) is used to identify bacteria with greater than 97% sequence similarity at the genus level.
- Was profiled QIIME).
- Example 3 Blood-derived vesicles derived from liver cancer patients Metagenome Through analysis Bacillus Reduction of Bacterial-Derived Vesicles in Genus
- Example 7 Blood-derived vesicles from asthma patients Metagenome Through analysis Bacillus Reduction of Bacterial-Derived Vesicles in Genus
- Example 8 Vesicles derived from blood bacteria of atopic dermatitis patients Metagenome Through analysis Bacillus Reduction of Bacterial-Derived Vesicles in Genus
- Example 10 Blood Bacteria Derived from Liver Cirrhosis Patients Metagenome Through analysis Bacillus Reduction of Bacterial-Derived Vesicles in Genus
- Example 11 Blood-derived vesicles from diabetic patients Metagenome Analysis of Escherichia Genus Bacteria-Derived Vesicles
- vesicles were isolated from the culture medium of Bacillus subtilis strains.
- 2 L of autoclaved brain heart infusion broth (BD 237500) was isolated. After inoculation and incubation in a chamber so that the absorbance (OD) value was 1.5 for 72 hours, the culture solution was centrifuged at high speed (10,000 ⁇ g) for 20 minutes to obtain a supernatant except for the bacterial precipitation pellets. The supernatant was sequentially filtered using a 0.45 ⁇ m filter and a 0.22 ⁇ m filter, and then cultured about 14-fold concentrated using a Quixstand benchtop system. The culture medium was again subjected to ultrafast centrifugation for 2 hours at 150,000 ⁇ g, 4 ° C. to obtain pellets, and then dissolved in sterile saline (PBS) to quantify the protein.
- PBS sterile saline
- the form and size of Bacillus subtilis obtained from Bacillus subculture were observed and measured.
- 50 ⁇ g / ml sample was obtained by protein quantification and observed with a JEM 1011 electron microscope (Jeol, Japan). As shown in FIG. 12A, it was confirmed that the vesicle-derived vesicles were spherical. .
- the size of Bacillus subtilis derived from Bacillus subtilis was measured by dynamic light scattering (DLS) through zetasizer nano ZS (Malverk, UK) with a sample of 50 ⁇ g / ml, as shown in FIG. 12B. It was found that the average diameter of the derived vesicles was in the range of 40 to 100 nm.
- Escherichia coli Eschericahia coli
- Eschericahia coli is a major bacterium that lives not only in the large intestine but also in the surrounding environment, and is highly resistant to antibiotics.
- E. coli-derived vesicles are found to be a major cause of chronic obstructive airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), it has become known as a major cause of chronic inflammatory diseases. Therefore, the present invention was intended to evaluate the anti-inflammatory effect of Bacillus subtilis-derived vesicles on the occurrence of inflammation caused by E. coli-derived vesicles.
- B. subtilis EV 0.1, 1, 10 ⁇ g / ml, or B.
- subtilis natto EV 0.1, 1, 10 ⁇ g, in the macrophage line (Raw 264.7).
- Ml Ml
- E. coli vesicles 1 ⁇ g / ml
- the negative control group (NC) was treated with PBS in the same manner as in Example 13, the positive control group (PC) was treated only with E.
- Lactobacillus plantarum vesicles 1 ⁇ g / ml
- lactobacillus plantarum EV 1 ⁇ g / ml
- the present inventors confirmed that the intestinal bacteria are not absorbed into the body, but the bacteria-derived vesicles are absorbed into the body and distributed systemically and excreted externally through the kidneys, liver, and lungs.
- Bacterial-derived bacteria of Bacillus present in the blood of patients with metabolic diseases such as solid cancers such as liver cancer, bladder cancer, breast cancer, ovarian cancer, asthma, atopic dermatitis, diabetes, liver cirrhosis, etc. It was confirmed that the vesicles were significantly reduced compared to the normal persons.
- the bacterium-derived vesicles according to the present invention are useful for the diagnosis or prediction method of cancer, chronic inflammatory disease, or metabolic disease, and for the prevention or treatment of food, inhalants, cosmetics, or drugs.
- a standard strain of Bacillus subtilis a subtilis of Bacillus subtilis and Natto, a subtype of Bacillus subtilis
- the bacterium-derived vesicles according to the present invention are useful for the diagnosis or prediction method of cancer, chronic inflammatory disease, or metabolic disease, and for the prevention or treatment of food, inhalants, cosmetics, or drugs.
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Abstract
The present invention relates to vesicles derived from the genus Bacillus bacteria and a use thereof. The present inventors have confirmed that the vesicles are significantly reduced in samples of patients with cancers, such as liver cancer, bladder cancer, breast cancer, and ovarian cancer; inflammatory diseases, such as asthma and atopic dermatitis; or metabolic diseases, such as diabetes and cirrhosis, compared with normal persons, and the vesicles inhibited the secretion of inflammatory mediators by pathogenic vesicles, such as E. coli-derived vesicles, which are causative factors of inflammatory diseases, diabetes, and the like. Therefore, the vesicles derived from the genus Bacillus bacteria according to the present invention can be advantageously used for the purpose of developing a diagnostic method for cancer, inflammatory diseases, and metabolic diseases, and a preventive or therapeutic composition therefor.
Description
본 발명은 바실러스 속 세균 유래 나노소포 및 이의 용도에 관한 것으로, 보다 구체적으로 바실러스 속 세균에서 유래하는 나노소포를 이용한 암, 염증질환, 또는 대사질환의 진단방법, 및 상기 소포를 포함하는 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to nanovesicles derived from Bacillus bacteria and their use, and more particularly to methods of diagnosing cancer, inflammatory diseases, or metabolic diseases using nanovesicles derived from Bacillus bacteria, and preventing or treating the vesicles. It relates to a composition for.
21세기에 들어서면서 과거 전염병으로 인식되던 급성 감염성질환의 중요성이 덜해지는 반면, 인간과 마이크로바이옴과의 부조화에 의해 발생하는 만성질환이 삶의 질과 인간 수명을 결정하는 주요 질환으로 질병패턴이 바뀌었다. 상기 만성질환은 면역기능 이상을 동반한 만성염증을 특징으로 하고, 암, 만성염증질환, 대사질환 등이 국민보건에 큰 문제가 되고 있다.In the 21st century, chronic infectious diseases, which were previously recognized as infectious diseases, have become less important, while chronic diseases caused by the incompatibility between humans and microbiomes are the major diseases that determine quality of life and human life. Changed. The chronic disease is characterized by chronic inflammation accompanied by immune dysfunction, cancer, chronic inflammatory diseases, metabolic diseases, etc. have become a major problem in public health.
염증(Inflammation)은 세포 및 조직의 손상이나 감염에 대한 국부적 또는 전신적인 방어기작으로, 주로 면역계를 이루는 체액성 매개체(humoral mediator)가 직접 반응하거나, 국부적 또는 전신적 작동 시스템(effector system)을 자극함으로써 일어나는 연쇄적인 생체반응에 의해 유발된다. 주요 염증성 질환으로는 위염, 염증성 장염 등의 소화기질환, 치주염 등의 구강 질환, 천식, 만성폐쇄성폐질환(COPD), 비염 등의 호흡기질환, 아토피 피부염, 탈모, 건선 등의 피부질환, 퇴행성관절염, 류마티스 관절염 등과 같은 관절염; 및 비만, 당뇨병, 간경화증 등이 대사질환이 포함된. 또한, 다양한 연구들을 통해 지속적인 염증이 암을 유발할 수 있다는 결과들이 보고되어 왔다(Cancers 2014, 6, 926-957). Inflammation is a local or systemic defense against damage or infection of cells and tissues, primarily by the direct response of humoral mediators that make up the immune system, or by stimulating local or systemic effector systems. It is caused by a cascade of biological reactions that take place. Major inflammatory diseases include gastroenteritis, digestive diseases such as inflammatory bowelitis, oral diseases such as periodontitis, asthma, chronic obstructive pulmonary disease (COPD), respiratory diseases such as rhinitis, atopic dermatitis, hair loss, skin diseases such as psoriasis, degenerative arthritis, Arthritis, such as rheumatoid arthritis; And obesity, diabetes mellitus, cirrhosis and the like metabolic diseases. In addition, various studies have reported that persistent inflammation can cause cancer (Cancers 2014, 6, 926-957).
인체에 공생하는 미생물은 100조에 이르러 인간 세포보다 10배 많으며, 미생물의 유전자수는 인간 유전자수의 100배가 넘는 것으로 알려지고 있다. 미생물총(microbiota 혹은 microbiome)은 주어진 거주지에 존재하는 진정세균(bacteria), 고세균(archaea), 진핵생물(eukarya)을 포함한 미생물 군집(microbial community)을 말하고, 장내 미생물총은 사람의 생리현상에 중요한 역할을 하며, 인체 세포와 상호작용을 통해 인간의 건강과 질병에 큰 영향을 미치는 것으로 알려져 있다. The microorganisms symbiotic to the human body reaches 100 trillion times more than human cells, and the number of genes of microorganisms is known to be more than 100 times the number of human genes. Microbiota (microbiota or microbiome) refers to a microbial community including microbes, archaea and eukarya that exist in a given settlement, and the intestinal microbiota is important for human physiology. It plays a role and is known to greatly affect human health and disease through interaction with human cells.
우리 몸에 공생하는 진정세균 및 고세균은 다른 세포로의 유전자, 단백질 등의 정보를 교환하기 위하여 나노미터 크기의 소포(nanovesicle)를 분비한다. 점막은 200 나노미터(nm) 크기 이상의 입자는 통과할 수 없는 물리적인 방어막을 형성하여 점막에 공생하는 세균인 경우에는 점막을 통과하지 못하지만, 세균 유래 소포는 크기가 100 나노미터 크기 이하라서 비교적 자유롭게 점막을 통하여 상피세포를 통과하여 우리 몸에 흡수된다. 우리 몸에 흡수되는 병원성 세균유래 소포는 아토피피부염과 같은 염증성 피부질환, 만성비염, 천식, 만성폐쇄성폐질환 (COPD) 등의 염증성 호흡기질환, 궤양성대장염, 크론씨병 등의 염증성 장질환 등과 같은 염증성질환의 병인에 중요한 인자임이 밝혀지고 있다. 또한, 당뇨병, 비만 등이 대사질환, 및 폐암, 위암, 대장암 등의 고형암의 발생과도 긴밀한 관계가 있음이 최근 밝혀지면서 주목을 받고 있다. 그러나 아직까지 임상샘플에서 바실러스 속 세균유래 소포를 정량하여 암, 만성염증질환, 또는 대사질환을 진단하는 사례는 전무한 실정이다.Symbiotic bacteria and archaea that symbiotic to our body secrete nanometer-sized vesicles (nanovesicle) to exchange information, such as genes, proteins to other cells. The mucous membrane forms a physical protective film that particles larger than 200 nanometers (nm) in size can't pass through. If the bacteria are symbiotic bacteria, the mucosa cannot pass through the mucous membrane, but the bacteria-derived vesicles are 100 nanometers in size or less. It passes through epithelial cells through the mucous membrane and is absorbed by our body. Pathogenic bacteria-derived vesicles absorbed by our bodies are inflammatory diseases such as inflammatory skin diseases such as atopic dermatitis, inflammatory respiratory diseases such as chronic rhinitis, asthma and chronic obstructive pulmonary disease (COPD), inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. It is found to be an important factor in the etiology of. In addition, diabetes, obesity and the like has been attracting attention as it is recently found that there is a close relationship with the occurrence of metabolic diseases and solid cancers, such as lung cancer, stomach cancer, colon cancer. However, there have been no cases of diagnosing cancer, chronic inflammatory diseases, or metabolic diseases by quantifying Bacillus bacteria-derived vesicles in clinical samples.
바실러스 속 세균은 Firmicutes 문에 속하는 그람양성 간균으로서, 호기성 환경뿐만 아니라 혐기성 환경에서도 자라고, 스트레스 상황에서는 spore를 형성하여 오랜 기간 동안 생존할 수 있다. 바실러스 속 세균중에서 고초균(Bacillus subtilis)은 비병원성, 호기성 환경에서 자랄 수 있고, 공기 중은 물론, 마른 풀, 하수, 토양, 먼지 등 자연계에 널리 분포하는 그람양성간균이다. 또한, 고초균 아형인 낫토균 (Bacillus
subtilis
natto)은 혐기성 환경에서도 자랄 수 있으며, 볏집에 많이 붙어살기에 청국장을 만들 때 볏집을 꼽아 놓고, 40℃ 근방까지 열이 형성된 상태로 공기가 차단되면서 대량 증식한다. 청국장은 콩을 발효시켜 만든 한국 전통음식으로, 일본의 낫토와 흡사하다. 된장은 발효시켜서 먹기까지 몇 달이 걸리지만 청국장은 담가서 2~3일이면 먹을 수 있다. 즉, 콩 발효 식품류 중 가장 짧은 기일에 만들 수 있는 장이 청국장인 것이다. 자연발효에 의한 청국장은 메주콩을 10～20시간 더운 물에 불렸다가 물을 붓고 푹 끓여 물씬하게 익힌 다음 보온만으로 띄운 것이다. 그릇에 짚을 몇 가닥씩 깔면서 퍼 담아 60℃까지 식힌 다음 따뜻한 곳에 놓고 담요나 이불을 씌워 45 ℃로 보온하면 낫토균 등의 고초균이 번식하여 발효물질로 변한다. Bacillus is a Gram-positive bacillus belonging to the Firmicutes portal, which grows in anaerobic as well as aerobic environments, and can survive for a long time in the form of spores in stress situations. Among Bacillus bacteria, Bacillus subtilis is a Gram-positive bacillus that can grow in non-pathogenic and aerobic environments, and is widely distributed in the natural world such as dry grass, sewage, soil, and dust. Furthermore, subtypes of Bacillus subtilis natto (Bacillus subtilis natto) can grow in anaerobic environment, attach to crested soybeans when they make cheonggukjang, and multiply by blocking the air with heat formed near 40 ℃. Cheonggukjang is a traditional Korean food made from fermented soybeans, similar to Japanese natto. It takes several months to ferment soybean paste but fermented soybeans can be eaten in 2-3 days. In other words, the soybeans can be made on the shortest date of fermented soybeans. Cheonggukjang made by natural fermentation is soaked in hot water for 10-20 hours, then poured with water and boiled. Put a few straws in a bowl, put it in a bowl, cool it to 60 ℃, put it in a warm place and cover it with blankets or blankets, and keep it at 45 ℃, and then the natto bacteria such as natto breed and become fermented substances.
만성염증질환을 치료 또는 예방하기 위하여 사용되고 있는 일반적인 조성물로는 크게 스테로이드성 및 비스테로이드성 조성물로 구분되어지며, 이중 대부분이 여러 가지 부작용을 수반하는 경우가 많다. 또한, 류마티스관절염, 탈모, 염증성장염 등의 염증질환의 병인에 TNF-α의 중요성이 부각되고 있고, 류마티스관절염 치료제로 TNF-α 억제제가 각광을 받고 있다. 그러나 아직까지 바실러스 속 세균 혹은 고초균에서 분비되는 소포 내 유전자 정량을 통해 암, 만성염증질환, 또는 대사질환을 진단하거나, 또는 상기 소포를 통해 염증질환의 예방, 또는 치료를 위해 이용된 경우는 없는 실정이다.Common compositions used to treat or prevent chronic inflammatory diseases are largely divided into steroidal and nonsteroidal compositions, many of which are accompanied by various side effects. In addition, the importance of TNF-α is increasing in the etiology of inflammatory diseases such as rheumatoid arthritis, hair loss, inflammatory bowel disease, and the like, and TNF-α inhibitors have been spotlighted as therapeutic agents for rheumatoid arthritis. However, it has not been used to diagnose cancer, chronic inflammatory disease, or metabolic disease by quantifying genes in vesicles secreted from bacteria of Bacillus or Bacillus subtilis, or to prevent or treat inflammatory diseases through the vesicles. to be.
본 발명자들은 상기와 같은 종래의 문제점을 해결하기 위해 예의 연구한 결과, 사람 샘플 내에 존재하는 세균 유래 소포 메타게놈 분석을 통해 정상인에 비하여 간암, 방광암, 유방암, 난소암 등의 고형암, 천식, 아토피피부염 등의 만성염증질환, 및 당뇨병, 간경화 등의 대사질환 환자 유래 샘플에서 바실러스 속 세균 유래 소포의 함량이 현저히 감소되어 있음 확인하였고, 바실러스 속 세균, 특히 고초균을 체외에서 배양하여 소포를 분리하여 소포의 치료효능을 평가한 결과, 항염증 효과가 탁월함을 확인한 바, 이에 기초하여 본 발명을 완성하였다. The present inventors earnestly studied to solve the above-mentioned conventional problems, and as a result of analyzing the bacteria-derived vesicle metagenome present in human samples, solid cancers such as liver cancer, bladder cancer, breast cancer, and ovarian cancer, asthma, and atopic dermatitis In the samples from patients with chronic inflammatory diseases such as diabetes and metabolic diseases such as cirrhosis and cirrhosis, the contents of the bacterium derived from Bacillus bacterium were significantly reduced, and the vesicles were isolated by culturing Bacillus bacteria, especially Bacillus subtilis, in vitro. As a result of evaluating the therapeutic efficacy, it was confirmed that the anti-inflammatory effect is excellent, the present invention was completed based on this.
이에, 본 발명은 암, 만성염증질환, 및 대사질환의 진단을 위한 정보제공방법을 제공하는 것을 목적으로 한다. Accordingly, an object of the present invention is to provide an information providing method for diagnosing cancer, chronic inflammatory diseases, and metabolic diseases.
또한, 본 발명은 바실러스 속 세균 유래 소포를 유효성분으로 포함하는 암, 만성염증질환, 및 대사질환의 예방 또는 치료용 조성물을 제공하는 것을 다른 목적으로 한다. Another object of the present invention is to provide a composition for preventing or treating cancer, chronic inflammatory diseases, and metabolic diseases, including the bacterium-derived vesicles as an active ingredient.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.
상기와 같은 본 발명의 목적을 달성하기 위하여, 본 발명은 하기의 단계를 포함하는, 암, 만성염증질환, 또는 대사질환의 진단을 위한 정보제공방법을 제공한다.In order to achieve the object of the present invention as described above, the present invention provides a method for providing information for the diagnosis of cancer, chronic inflammatory diseases, or metabolic diseases comprising the following steps.
(a) 정상인 및 피검자 유래 샘플에서 분리한 소포로부터 DNA를 추출하는 단계;(a) extracting DNA from vesicles isolated from normal and subject derived samples;
(b) 상기 추출한 DNA에 대하여 16S rDNA 서열에 존재하는 바실러스 속 세균 유래 소포 검출 프라이머 쌍을 이용하여 PCR을 수행하여 각각의 PCR 산물을 수득하는 단계; 및(b) performing PCR on the extracted DNA using a Bacillus bacteria-derived vesicle detection primer pair present in the 16S rDNA sequence to obtain respective PCR products; And
(c) 상기 PCR 산물의 정량분석을 통하여 정상인에 비하여 바실러스 속 세균 유래 소포의 함량이 낮을 경우 암, 염증질환, 또는 대사질환으로 판정하는 단계.(c) determining the cancer, inflammatory disease, or metabolic disease when the content of the bacterium-derived vesicles in Bacillus is lower than that of the normal person through quantitative analysis of the PCR product.
본 발명의 일 구현예로, 상기 암은 간암, 방광암, 유방암, 또는 난소암일 수 있다.In one embodiment of the present invention, the cancer may be liver cancer, bladder cancer, breast cancer, or ovarian cancer.
본 발명의 또 다른 구현예로, 상기 만성염증질환은 천식, 만성간염, 또는 아토피피부염일 수 있다. In another embodiment of the present invention, the chronic inflammatory disease may be asthma, chronic hepatitis, or atopic dermatitis.
본 발명의 또 다른 구현예로, 상기 대사질환은 당뇨병 또는 간경화증일 수 있다. In another embodiment of the present invention, the metabolic disease may be diabetes or cirrhosis of the liver.
본 발명의 또 다른 구현예로, 상기 환자 유래 샘플은 혈액, 소변, 또는 대변인 것일 수 있다. In another embodiment of the invention, the patient derived sample may be blood, urine, or stool.
또한, 본 발명은 바실러스 속 세균 유래 소포를 유효성분으로 포함하는, 암, 염증질환, 또는 대사질환의 예방 또는 치료용 약학적 조성물을 제공한다. The present invention also provides a pharmaceutical composition for the prevention or treatment of cancer, inflammatory diseases, or metabolic diseases, comprising the bacterium-derived vesicles as an active ingredient.
본 발명의 일 구현예로, 바실러스 속 세균 유래 소포를 유효성분으로 포함하는, 암, 염증질환, 또는 대사질환 개선용 건강기능성 식품 조성물을 제공한다.In one embodiment, the present invention provides a health functional food composition for improving cancer, inflammatory disease, or metabolic disease, comprising the bacterium-derived vesicles as an active ingredient.
본 발명의 다른 구현예로, 바실러스 속 세균 유래 소포를 유효성분으로 포함하는, 암, 염증질환, 또는 대사질환 예방 또는 치료용 흡입제 조성물을 제공한다.In another embodiment, the present invention provides an inhalant composition for preventing or treating cancer, inflammatory disease, or metabolic disease, comprising a bacterium-derived vesicle derived as an active ingredient.
본 발명의 다른 구현예로, 바실러스 속 세균 유래 소포를 유효성분으로 포함하는, 염증질환 개선용 화장료 조성물을 제공한다. In another embodiment, the present invention provides a cosmetic composition for improving inflammatory disease, comprising a bacterium-derived vesicle derived as an active ingredient.
본 발명의 다른 구현예로, 상기 바실러스 속 세균 유래 소포는 고초균(Bacillus subtilis) 유래 소포일 수 있다. In another embodiment of the present invention, the Bacillus bacteria-derived vesicles may be vesicles derived from Bacillus subtilis.
본 발명의 다른 구현예로, 상기 고초균 유래 소포는 낫토균 유래 소포일 수 있다. In another embodiment of the present invention, the Bacillus subtilis-derived vesicles may be natto-derived vesicles.
본 발명의 다른 구현예로, 상기 암은 간암, 방광암, 유방암, 및 난소암으로 이루어지는 군으로부터 선택되고; 상기 염증질환은 천식, 만성간염, 탈모, 류마티스관절염, 염증성장염, 및 아토피피부염으로 이루어지는 군으로부터 선택되며; 상기 대사질환은 당뇨병 또는 간경화증일 수 있다. In another embodiment of the invention, the cancer is selected from the group consisting of liver cancer, bladder cancer, breast cancer, and ovarian cancer; The inflammatory disease is selected from the group consisting of asthma, chronic hepatitis, hair loss, rheumatoid arthritis, inflammatory growth inflammation, and atopic dermatitis; The metabolic disease may be diabetes or cirrhosis of the liver.
본 발명의 다른 구현예로, 상기 염증질환은 염증성 사이토카인인 IL-6 또는 TNF-α가 병인에 관여하는 류마티스관절염, 탈모, 염증성장염으로 이루어진 군으로부터 선택되는 하나 이상의 질환일 수 있다. In another embodiment of the present invention, the inflammatory disease may be one or more diseases selected from the group consisting of rheumatoid arthritis, hair loss, and inflammatory growth inflammation in which the inflammatory cytokine IL-6 or TNF-α is involved in the pathogenesis.
본 발명의 다른 구현예로, 상기 소포는 평균 직경이 10 내지 200 nm인 것일 수 있다. In another embodiment of the present invention, the vesicles may have an average diameter of 10 to 200 nm.
본 발명의 다른 구현예로, 상기 소포는 바실러스 속 세균에서 자연적으로 또는 인공적으로 분비되는 것일 수 있다. In another embodiment of the present invention, the vesicles may be secreted naturally or artificially from bacteria of the genus Bacillus.
본 발명의 다른 구현예로, 상기 소포는 바실러스 속 세균의 배양액에서 분리되는 것일 수 있다. In another embodiment of the present invention, the vesicles may be isolated from the culture medium of bacteria in Bacillus.
또한, 본 발명은 바실러스 속 세균 유래 소포를 유효성분으로 포함하는 약학적 조성물을 개체에 투여하는 단계를 포함하는, 암, 염증질환, 또는 대사질환의 예방 또는 치료방법을 제공한다. The present invention also provides a method for preventing or treating cancer, inflammatory diseases, or metabolic diseases, comprising administering to a subject a pharmaceutical composition comprising a bacterium-derived bacterium-derived vesicle as an active ingredient.
또한, 본 발명은 바실러스 속 세균 유래 소포의, 염증질환 예방 또는 치료용도를 제공한다. The present invention also provides a preventive or therapeutic use for inflammatory diseases of vesicles derived from Bacillus genus.
본 발명자들은 장내 세균인 경우에는 체내에 흡수되지 않지만, 세균유래 소포인 경우에는 체내에 흡수되어, 전신적으로 분포하고, 콩팥, 간, 폐를 통해 체외로 배설됨을 확인하였고, 환자 혈액, 소변, 대변 등에 존재하는 세균유래 소포 메타게놈 분석을 통해 간암, 방광암, 유방암, 난소암 등의 고형암, 천식, 아토피피부염 등의 만성염증질환, 당뇨병, 간경화 등의 대사질환 환자의 혈액에 존재하는 바실러스 속 세균유래 소포가 정상인에 비하여 유의하게 감소되어 있음을 확인하였다. The present inventors confirmed that the intestinal bacteria are not absorbed into the body, but the bacteria-derived vesicles are absorbed into the body and distributed systemically and excreted externally through the kidneys, liver, and lungs. Bacterial-derived bacteria of Bacillus present in the blood of patients with metabolic diseases such as solid cancers such as liver cancer, bladder cancer, breast cancer, ovarian cancer, asthma, atopic dermatitis, diabetes, liver cirrhosis, etc. It was confirmed that the vesicles were significantly reduced compared to the normal persons.
또한, 프로테우스 속 세균의 한 종인 고초균 표준균주 및 고초균의 아형인 낫토균을 체외에서 배양하여 소포를 분리하여, 체외에서 염증세포에 투여하였을 때, 병원성 소포에 의한 염증매개체 분비를 유의하게 억제함을 관찰하였는 바, 본 발명에 따른 바실러스 속 세균 유래 소포는 암, 만성염증질환, 또는 대사질환의 진단 또는 예측방법, 및 식품, 흡입제, 화장품, 혹은 약물 등의 예방용 혹은 치료용 조성물에 유용하게 이용될 수 있을 것이다. In addition, when the vesicles were isolated by culturing in vitro and incubated with a standard strain of Bacillus subtilis, a subtilis of Bacillus subtilis and Natto, a subtype of Bacillus subtilis, it significantly inhibited the secretion of inflammatory mediators caused by pathogenic vesicles. As observed, the bacterium-derived vesicles according to the present invention are useful for the diagnosis or prediction method of cancer, chronic inflammatory disease, or metabolic disease, and for the prevention or treatment of food, inhalants, cosmetics, or drugs. Could be.
도 1a은, 마우스에 세균과 세균유래 소포 (EV)를 구강으로 투여한 후, 시간별로 세균과 소포의 분포양상을 촬영한 사진이다. Figure 1a is a photograph of the distribution of bacteria and vesicles over time after the oral administration of bacteria and bacteria-derived vesicles (EV) to the mouse.
도 1b는 구강으로 투여한 후 12시간째에, 혈액, 콩팥, 간, 및 여러 장기를 적출하여, 세균과 소포의 체내 분포양상을 평가한 그림이다.Figure 1b is a 12 hours after oral administration, blood, kidneys, liver, and various organs were extracted to evaluate the distribution of bacteria and vesicles in the body.
도 2는, 인체 유래물에서 세균유래 소포 메타게놈 분석 방법을 모식화한 그림이다.2 is a diagram schematically illustrating a method of analyzing bacterial-derived vesicle metagenome in human derivatives.
도 3은, 간암 환자 및 정상인 혈액에 존재하는 세균유래 소포 메타게놈 분석을 실시한 후, 바실러스 속 세균유래 소포의 분포를 비교한 결과이다. Figure 3 is a result of comparing the distribution of bacteria-derived vesicles of the genus Bacillus after the analysis of bacteria-derived vesicles metagenome present in liver cancer patients and normal blood.
도 4는, 방광암 환자 및 정상인 혈액에 존재하는 세균유래 소포 메타게놈 분석을 실시한 후, 바실러스 속 세균유래 소포의 분포를 비교한 결과이다. Figure 4 is a result of comparing the distribution of bacteria-derived vesicles of the genus Bacillus after the analysis of bacteria-derived vesicles metagenome present in bladder cancer patients and normal blood.
도 5는, 유방암 환자 및 정상인 혈액에 존재하는 세균유래 소포 메타게놈 분석을 실시한 후, 바실러스 속 세균유래 소포의 분포를 비교한 결과이다. 5 is a result of comparing the distribution of bacteria-derived vesicles of the genus Bacillus after analyzing the bacteria-derived vesicles metagenome present in breast cancer patients and normal blood.
도 6은, 난소암 환자 및 정상인 혈액에 존재하는 세균유래 소포 메타게놈 분석을 실시한 후, 바실러스 속 세균유래 소포의 분포를 비교한 결과이다. Figure 6 is a result of comparing the distribution of bacteria-derived vesicles of the genus Bacillus after the analysis of bacterial-derived vesicles metagenome present in ovarian cancer patients and normal blood.
도 7은, 천식 환자 및 정상인 혈액에 존재하는 세균유래 소포 메타게놈 분석을 실시한 후, 바실러스 속 세균유래 소포의 분포를 비교한 결과이다. 7 is a result of comparing the distribution of bacteria-derived vesicles of Bacillus genus after analyzing the bacteria-derived vesicles metagenome present in asthma patients and normal blood.
도 8은, 아토피피부염 환자 및 정상인 혈액에 존재하는 세균유래 소포 메타게놈 분석을 실시한 후, 바실러스 속 세균유래 소포의 분포를 비교한 결과이다. 8 is a result of comparing the distribution of bacteria-derived vesicles of the genus Bacillus after the analysis of bacterial-derived vesicles metagenome present in atopic dermatitis patients and normal blood.
도 9는, 당뇨병 환자 및 정상인 혈액에 존재하는 세균유래 소포 메타게놈 분석을 실시한 후, 바실러스 속 세균유래 소포의 분포를 비교한 결과이다. 9 is a result of comparing the distribution of bacteria-derived vesicles of the genus Bacillus after the analysis of bacteria-derived vesicles metagenome present in diabetic patients and normal blood.
도 10은, 간경화증 환자 및 정상인 혈액에 존재하는 세균유래 소포 메타게놈 분석을 실시한 후, 바실러스 속 세균유래 소포의 분포를 비교한 결과이다. 10 is a result of comparing the distribution of bacterium-derived vesicles of the genus Bacillus after analyzing the bacterial-derived vesicles metagenome present in liver cirrhosis patients and normal blood.
도 11은, 당뇨병 환자 및 정상인 혈액에 존재하는 세균유래 소포 메타게놈 분석을 실시한 후, Escherichia 속 세균유래 소포의 분포를 비교한 결과이다. 11 is a result of comparing the distribution of bacteria-derived vesicles of the genus Escherichia after the analysis of bacteria-derived vesicles metagenome present in diabetic patients and normal blood.
도 12a는, 고초균 배양액에서 초원심분리법으로 분리한 소포에 대하여 전자현미경(a)을 통해 고초균 유래 소포의 형태 및 크기를 측정한 결과이다.Figure 12a is a result of measuring the form and size of Bacillus subtilis derived from Bacillus subtilis in the Bacillus subtilis culture medium by ultracentrifugation method (a).
도 12b는, 고초균 배양액에서 초원심분리법으로 분리한 소포에 대하여 동적광산랍법(b)을 통해 고초균 유래 소포의 형태 및 크기를 측정한 결과이다.Figure 12b is a result of measuring the form and size of Bacillus subtilis derived from Bacillus subtilis through the dynamic light scattering method (b) for the vesicles separated by the ultracentrifugation method in Bacillus subtilis culture.
도 13a는, 암, 염증질환, 대사질환에 원인인자인 대장균 유래 소포(E. coli EV)를 양성대조군으로 하고, 고초균 표준균주 유래 소포(Bacillus
subtilis EV) 및 낫토균 유래 소포(Bacillus
subtilis natto EV)를 대식세포주(Raw 264.7)에 다양한 농도로 처리한 후, 염증매개체인 IL-6농도를 ELISA법으로 측정하여, 염증매개체 분비 정도를 비교한 결과이다[NC: negative control(PBS), EV: extracellular vesicle].FIG. 13A shows E. coli vesicles (E. coli EV), a causative factor in cancer, inflammatory disease and metabolic disease, as a positive control group, Bacillus subtilis EV and Natto bacteria-derived vesicles ( Bacillus subtilis natto EV). ) Is treated with macrophages (Raw 264.7) at various concentrations, and IL-6 concentration is measured by ELISA to compare the degree of secretion of inflammatory mediators [NC: negative control (PBS), EV: extracellular vesicle].
도 13b는, 암, 염증질환, 대사질환에 원인인자인 대장균 유래 소포(E. coli EV)를 양성대조군으로 하고, 고초균 표준균주 유래 소포(Bacillus
subtilis EV) 및 낫토균 유래 소포(Bacillus
subtilis natto EV)를 대식세포주(Raw 264.7)에 다양한 농도로 처리한 후, 염증매개체인 TNF-α 농도를 ELISA법으로 측정하여, 염증매개체 분비 정도를 비교한 결과이다[NC: negative control(PBS), EV: extracellular vesicle]. 13B is a positive control group of E. coli vesicles (E. coli EV), a causative factor in cancer, inflammatory diseases, and metabolic diseases, and Bacillus subtilis EVs and Bacillus subtilis natto EVs ( Bacillus subtilis natto EV). ) Was treated with macrophages (Raw 264.7) at various concentrations, and TNF-α concentration was measured by ELISA to compare the degree of secretion of inflammatory mediators [NC: negative control (PBS), EV: extracellular vesicle].
도 14a는, 고초균 표준균주 및 고초균 낫토균주(낫토균) 유래 소포의 항염증 효과를 평가하기 위한 것으로서, 대식세포주(Raw 264.7)에 대조 약물로서 락토바실러스 플란타룸 유래 소포(L.
plantarum EV), 고초균 표준균주 유래 소포(Bacillus subtilis EV), 및 낫토균 유래 소포 (Bacillus
subtilis natto EV)를 전처리하고, 12시간 후에 염증유발 원인인자로서 대장균 유래 소포를 처리한 후 ELISA법으로 IL-6 농도를 측정한 결과이다[NC: negative control(PBS), PC: positive control, EV: extracellular vesicle].FIG. 14A is for evaluating the anti-inflammatory effects of Bacillus subtilis and Bacillus subtilis Natto strains (Lactobacillus), a macrophage line (Raw 264.7) as a control drug Lactobacillus plantarum vesicles ( L. plantarum EV) , Bacillus subtilis EV and Natto-derived vesicles ( Bacillus subtilis natto EV) were pretreated and treated with E. coli-derived vesicles as a causative agent of inflammation after 12 hours. Measurement results [NC: negative control (PBS), PC: positive control, EV: extracellular vesicle].
도 14b는, 고초균 표준균주 및 고초균 낫토균주(낫토균) 유래 소포의 항염증 효과를 평가하기 위한 것으로서, 대식세포주(Raw 264.7)에 대조 약물로서 락토바실러스 플란타룸 유래 소포(L.
plantarum EV), 고초균 표준균주 유래 소포(Bacillus subtilis EV), 및 낫토균 유래 소포 (Bacillus
subtilis natto EV)를 전처리하고, 12시간 후에 염증유발 원인인자로서 대장균 유래 소포를 처리한 후 ELISA법으로 TNF-α 농도를 측정한 결과이다[NC: negative control(PBS), PC: positive control, EV: extracellular vesicle].14B is for evaluating the anti-inflammatory effect of Bacillus subtilis and Bacillus subtilis Natto strains (Lactobacillus), a macrophage line (Raw 264.7) as a control drug Lactobacillus plantarum vesicles ( L. plantarum EV) , Bacillus subtilis EV and Natto-derived vesicles ( Bacillus subtilis natto EV) were pretreated and treated with E. coli-derived vesicles as a causative agent of inflammation after 12 hours, and then TNF-α concentration was determined by ELISA. Measurement results [NC: negative control (PBS), PC: positive control, EV: extracellular vesicle].
본 발명은 바실러스 속 세균 유래 소포 및 이의 용도에 관한 것이다. The present invention relates to vesicles derived from bacteria of the genus Bacillus and uses thereof.
본 발명자들은 메타게놈 분석을 통해 정상인에 비하여 간암, 방광암, 유방암, 난소암 등의 고형암, 천식, 아토피피부염 등의 만성염증질환, 당뇨병, 간경화 등의 대사질환 환자 유래 샘플에서 바실러스 속 세균유래 소포의 함량이 현저히 감소되어 있음을 확인하였고, 또한, 바실러스 속 세균에 속하는 고초균 표준균주 및 고초균의 아형인 낫토균 유래 소포를 분리하여 염증세포에 투여하였을 때, 병원성 소포인 대장균 유래 소포에 의한 염증반응을 효율적으로 억제함을 확인한 바, 이에 기초하여 본 발명을 완성하였다.The present inventors conducted meta-genome analysis of bacterium-derived vesicles from Bacillus-derived samples from patients with metabolic diseases such as liver cancer, bladder cancer, breast cancer, ovarian cancer, solid cancers such as asthma, atopic dermatitis, diabetes and liver cirrhosis. When the contents of the bacterium were significantly reduced, the standard bacterium belonging to the bacterium of Bacillus and the natto bacteria-derived vesicles were isolated and administered to inflammatory cells. When it was confirmed that it was suppressed efficiently, the present invention was completed based on this.
이에, 본 발명은 하기의 단계를 포함하는 암, 만성염증질환, 및 대사질환의 진단을 위한 정보제공방법을 제공한다. Accordingly, the present invention provides a method for providing information for the diagnosis of cancer, chronic inflammatory diseases, and metabolic diseases comprising the following steps.
(a) 정상인 및 피검자 유래 샘플에서 분리한 소포로부터 DNA를 추출하는 단계;(a) extracting DNA from vesicles isolated from normal and subject derived samples;
(b) 상기 추출한 DNA에 대하여 16S rDNA 서열에 존재하는 바실러스 속 세균 유래 소포 검출 프라이머 쌍을 이용하여 PCR을 수행하여 각각의 PCR 산물을 수득하는 단계; 및(b) performing PCR on the extracted DNA using a Bacillus bacteria-derived vesicle detection primer pair present in the 16S rDNA sequence to obtain respective PCR products; And
(c) 상기 PCR 산물의 정량분석을 통하여 정상인에 비하여 바실러스 속 세균 유래 소포의 함량이 낮을 경우 암, 염증질환, 또는 대사질환으로 판정하는 단계.(c) determining the cancer, inflammatory disease, or metabolic disease when the content of the bacterium-derived vesicles in Bacillus is lower than that of the normal person through quantitative analysis of the PCR product.
또한, 본 발명자들은 메타게놈 분석을 통해 정상인에 비하여 당뇨병 환자 유래 샘플에서 Escherichia 속 세균유래 소포의 함량이 현저히 증가되어 있음을 확인하였는바, 본 발명은 하기의 단계를 포함하는 당뇨병의 진단을 위한 정보제공방법을 제공한다. In addition, the inventors have confirmed that the metagenome analysis has significantly increased the content of Escherichia bacteria-derived vesicles in a sample derived from a diabetic patient compared to a normal person, the present invention provides information for diagnosing diabetes comprising the following steps Provide the method of providing.
(a) 정상인 및 피검자 유래 샘플에서 분리한 소포로부터 DNA를 추출하는 단계;(a) extracting DNA from vesicles isolated from normal and subject derived samples;
(b) 상기 추출한 DNA에 대하여 16S rDNA 서열에 존재하는 Escherichia 속 세균 유래 소포 검출 프라이머 쌍을 이용하여 PCR을 수행하여 각각의 PCR 산물을 수득하는 단계; 및(b) performing PCR on the extracted DNA using a pair of Escherichia bacteria-derived vesicle detection primers present in the 16S rDNA sequence to obtain respective PCR products; And
(c) 상기 PCR 산물의 정량분석을 통하여 정상인에 비하여 Escherichia 속 세균 유래 소포의 함량이 높을 경우 당뇨병으로 판정하는 단계.(c) Determining diabetes mellitus when the content of the vesicles derived from the genus Escherichia is higher than the normal person through quantitative analysis of the PCR product.
본 발명에서 사용되는 용어, 진단이란 넓은 의미로는 환자의 병의 실태를 모든 면에 걸쳐서 판단하는 것을 의미한다. 판단의 내용은 병명, 병인, 병형, 경중, 병상의 상세한 양태, 합병증의 유무, 및 예후 등이다. 본 발명에서 진단은 암, 만성염증질환, 또는 대사질환의 발병 여부 및 질환의 수준 등을 판단하는 것이다. As used herein, the term "diagnosis" means, in a broad sense, to determine the actual condition of a patient in all aspects. The content of the judgment is the name of the disease, the etiology, the type of disease, the seriousness, the detailed mode of the condition, the presence or absence of complications, and the prognosis. Diagnosis in the present invention is to determine the onset of cancer, chronic inflammatory diseases, or metabolic diseases and the level of the disease.
또한, 본 발명의 다른 양태로서, 본 발명은 바실러스 속 세균 유래 소포를 유효성분으로 포함하는, 암, 만성염증질환, 또는 대사질환의 예방 또는 치료용 식품, 화장품, 흡입제, 또는 약물 조성물을 제공한다. In still another aspect, the present invention provides a food, cosmetic, inhalant, or drug composition for preventing or treating cancer, chronic inflammatory disease, or metabolic disease, comprising a bacterium-derived vesicle of Bacillus as an active ingredient. .
본 발명에서 사용되는 용어, 예방이란 본 발명에 따른 식품, 화장품, 흡입제, 또는 약물 조성물의 투여에 의해 암, 만성염증질환, 또는 대사질환을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.As used herein, the term "prophylaxis" means any action that inhibits or delays the development of cancer, chronic inflammatory diseases, or metabolic diseases by administration of a food, cosmetic, inhalant, or drug composition according to the present invention.
본 발명에서 사용되는 용어, 치료란 본 발명에 따른 식품, 화장품, 흡입제, 또는 약물 조성물의 투여에 의해 암, 만성염증질환, 또는 대사질환에 대한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다. As used herein, the term "treatment" means any action in which symptoms for cancer, chronic inflammatory diseases, or metabolic diseases are improved or beneficially changed by administration of a food, cosmetic, inhalant, or drug composition according to the present invention.
본 발명의 상기 바실러스 속 세균 유래 소포는 바실러스 속 세균 배양액 또는 바실러스 속 세균으로 발효시킨 식품에서 분리될 수 있으며, 바람직하게는 고초균 또는 낫토균의 배양액 또는 고초균 또는 낫토균으로 발효시킨 식품에서 분리될 수 있으며, 고초균 또는 낫토균으로부터 자연적 또는 인공적으로 분비된 것일 수 있으나, 이에 제한되는 것은 아니다. The Bacillus bacteria-derived vesicles of the present invention may be isolated from a culture of Bacillus bacteria or food fermented with Bacillus bacteria, preferably from a culture solution of Bacillus subtilis or natto bacteria or from foods fermented with Bacillus subtilis or natto bacteria. And, it may be natural or artificial secreted from Bacillus subtilis or natto bacteria, but is not limited thereto.
본 발명의 상기 바실러스 속 세균의 배양액 또는 발효식품에서 소포를 분리하는 방법은 소포를 포함한다면 특별히 제한되지 않는다. 예컨대 원심분리, 초고속 원심분리, 필터에 의한 여과, 겔 여과 크로마토그래피, 프리-플로우 전기영동, 또는 모세관 전기영동 등의 방법, 및 이들의 조합을 이용하여 소포를 분리할 수 있으며, 또한 불순물의 제거를 위한 세척, 수득된 소포의 농축 등의 과정을 추가로 포함할 수 있다. The method for separating the vesicles from the culture solution or fermented food of the bacterium of the present invention is not particularly limited as long as the vesicles. For example, centrifugation, ultra-fast centrifugation, filtration by filter, gel filtration chromatography, pre-flow electrophoresis, or capillary electrophoresis, and combinations thereof may be used to separate the vesicles and also remove impurities. It may further include a process for washing, concentration of the obtained vesicles and the like.
본 발명에서 상기 방법에 의하여 분리된 소포는 평균 직경이 10~1000 nm일 수 있으며, 보다 바람직하게는 40~100nm일 수 있으나, 이에 제한되는 것은 아니다. The vesicles separated by the method in the present invention may have an average diameter of 10 to 1000 nm, more preferably 40 to 100 nm, but is not limited thereto.
본 발명에 따른 약학적 조성물은 바실러스 속 세균 유래 소포를 유효성분으로 포함하며, 약학적으로 허용 가능한 담체를 포함할 수 있다. 상기 약학적으로 허용 가능한 담체는 제제 시에 통상적으로 이용되는 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 사이클로덱스트린, 덱스트로즈 용액, 말토덱스트린 용액, 글리세롤, 에탄올, 리포좀 등을 포함하지만 이에 한정되지 않으며, 필요에 따라 항산화제, 완충액 등 다른 통상의 첨가제를 더 포함할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제, 윤활제 등을 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립, 또는 정제로 제제화할 수 있다. 적합한 약학적으로 허용되는 담체 및 제제화에 관해서는 레밍턴의 문헌에 개시되어 있는 방법을 이용하여 각 성분에 따라 바람직하게 제제화할 수 있다. 본 발명의 약학적 조성물은 제형에 특별한 제한은 없으나 주사제, 흡입제, 피부 외용제, 또는 경구 섭취제 등으로 제제화할 수 있다. The pharmaceutical composition according to the present invention includes a bacterium-derived vesicle derived from Bacillus as an active ingredient, and may include a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are conventionally used in the preparation, and include, but are not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and the like. If necessary, other conventional additives such as antioxidants and buffers may be further included. In addition, diluents, dispersants, surfactants, binders, lubricants and the like may be additionally added to formulate injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions and the like. Suitable pharmaceutically acceptable carriers and formulations can be preferably formulated according to the individual components using methods disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection, inhalant, external preparation for skin, oral ingestion, and the like.
본 발명의 약학적 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구투여(예를 들어, 정맥 내, 피하, 피부, 비강, 기도에 적용)할 수 있으며, 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 시간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다.The pharmaceutical composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, skin, nasal, airways) according to the desired method, and the dosage is determined by the condition and weight of the patient, disease Depending on the degree, drug form, route of administration, and time, it may be appropriately selected by those skilled in the art.
본 발명에 따른 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에 있어서, 약학적으로 유효한 양은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명에 따른 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The composition according to the invention is administered in a pharmaceutically effective amount. In the present invention, the pharmaceutically effective amount means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to the medical treatment, and the effective dose level refers to the type, severity, drug activity, and drug of the patient. Sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts. The composition according to the present invention may be administered as a separate therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.
구체적으로, 본 발명에 따른 조성물의 유효량은 환자의 나이, 성별, 체중에 따라 달라질 수 있으며, 일반적으로는 체중 1 kg 당 0.001 내지 150 mg, 바람직하게는 0.01 내지 100 mg을 매일 또는 격일 투여하거나 1일 1 내지 3회로 나누어 투여할 수 있다. 그러나 투여 경로, 비만의 중증도, 성별, 체중, 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.Specifically, the effective amount of the composition according to the present invention may vary depending on the age, sex, and weight of the patient, and generally 0.001 to 150 mg, preferably 0.01 to 100 mg per kg of body weight is administered daily or every other day or 1 It can be administered in 1 to 3 times a day. However, the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.
본 발명의 다른 양태로서, 본 발명은 바실러스 속 세균 유래 소포를 유효성분으로 포함하는 암, 염증질환, 또는 대사질환 개선용 건강기능성 식품 조성물을 제공한다. As another aspect of the present invention, the present invention provides a health functional food composition for improving cancer, inflammatory disease, or metabolic disease, comprising the bacterium-derived vesicles as an active ingredient.
본 발명에서 사용되는 용어, 개선이란, 치료되는 상태와 관련된 파라미터, 예를 들면 증상의 정도를 적어도 감소시키는 모든 행위를 의미한다.As used herein, the term improvement means any action that at least reduces the parameters associated with the condition being treated, for example the extent of symptoms.
본 발명의 건강기능식품 조성물에서 유효성분을 식품에 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합량은 그의 사용 목적(예방 또는 개선용)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조 시에 본 발명의 조성물은 원료에 대하여 15 중량% 이하, 바람직하게는 10 중량% 이하의 양으로 첨가된다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있다.In the nutraceutical composition of the present invention, the active ingredient may be added to the food as it is, or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient can be suitably determined according to the purpose of use (prevention or improvement). Generally, in the preparation of food or beverages the compositions of the invention are added in amounts of up to 15% by weight, preferably up to 10% by weight relative to the raw materials. However, in the case of prolonged intake for health and hygiene purposes or health control purposes, the amount may be below the above range.
본 발명의 건강기능식품 조성물은 지시된 비율로 필수 성분으로서 상기 유효성분을 함유하는 것 외에 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 당업자의 선택에 의해 적절하게 결정될 수 있다.The health functional food composition of the present invention, in addition to containing the active ingredient as an essential ingredient in the indicated ratio, is not particularly limited to other ingredients, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. have. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrate can be appropriately determined by the choice of those skilled in the art.
상기 외에 본 발명의 건강기능식품 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율 또한 당업자에 의해 적절히 선택될 수 있다. In addition to the above, the nutraceutical composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavors such as synthetic and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like. These components can be used independently or in combination. The proportion of such additives may also be appropriately selected by those skilled in the art.
본 발명의 또 다른 양태로서, 본 발명은 바실러스 속 세균 유래 소포를 유효성분으로 포함하는 염증질환 예방 또는 치료용 흡입제 조성물을 제공한다. As another aspect of the present invention, the present invention provides an inhalant composition for preventing or treating inflammatory diseases, which comprises a bacterium-derived vesicle as an active ingredient.
본 발명의 흡입제 조성물에서 유효성분을 흡입제에 그대로 첨가하거나 다른 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합량은 그의 사용 목적(예방 또는 치료용)에 따라 적합하게 결정될 수 있다.In the inhalant composition of the present invention, the active ingredient may be added to the inhalant as it is, or may be used together with other ingredients, and may be appropriately used according to conventional methods. The amount of the active ingredient to be mixed may be suitably determined depending on the purpose of use (prophylactic or therapeutic).
본 발명의 또 다른 양태로서, 본 발명은 바실러스 속 세균 유래 소포를 유효성분으로 포함하는 염증질환 또는 탈모 개선용 화장료 조성물을 제공한다.As another aspect of the present invention, the present invention provides a cosmetic composition for improving inflammatory disease or hair loss comprising a bacterium-derived vesicles as an active ingredient.
본 발명의 상기 화장료 조성물은 바실러스 속 세균 유래 소포뿐만 아니라, 화장료 조성물에 통상적으로 이용되는 성분들을 포함할 수 있으며, 예컨대 항산화제, 안정화제, 용해화제, 비타민, 안료, 및 향료와 같은 통상적인 보조제, 그리고 담체를 포함할 수 있다. The cosmetic composition of the present invention may include not only vesicles derived from bacteria of the genus Bacillus, but also components commonly used in cosmetic compositions, for example, conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances. And a carrier.
또한, 본 발명의 조성물은 바실러스 속 세균 유래 소포 이외에, 바실러스 속 세균 유래 소포와 반응하여 피부보호 효과를 손상시키지 않는 한도에서 종래부터 사용되어오던 유기 자외선 차단제를 혼합하여 사용할 수도 있다. 상기 유기 자외선 차단제로는 글리세릴파바, 드로메트리졸트리실록산, 드로메트리졸, 디갈로일트리올리에이트, 디소듐페닐디벤즈이미다졸테트라설포네이트, 디에틸헥실부타미도트리아존, 디에틸아미노하이드록시벤조일헥실벤조에이트, 디이에이-메톡시신나메이트, 로우손과 디하이드록시아세톤의 혼합물, 메틸렌비스-벤조트리아졸릴테트라메칠부틸페놀, 4-메틸벤질리덴캠퍼, 멘틸안트라닐레이트, 벤조페논-3(옥시벤존),벤조페논-4, 벤조페논-8(디옥시페벤존), 부틸메톡시디벤조일메탄, 비스에틸헥실옥시페놀메톡시페닐트리아진, 시녹세이트, 에틸디하이드록시프로필파바, 옥토크릴렌, 에틸헥실디메틸파바, 에틸헥실메톡시신나메이트, 에틸헥실살리실레이트, 에틸헥실트리아존, 이소아밀-p-메톡시신나메이트, 폴리실리콘-15(디메치코디에틸벤잘말로네이트), 테레프탈릴리덴디캠퍼설포닉애씨드 및 그 염류, 티이에이-살리실레이트 및 아미노벤조산(파바)으로 이루어진 군으로부터 선택된 1종 이상을 사용할 수 있다. In addition, the composition of the present invention, in addition to the vesicles derived from the bacterium of the genus Bacillus, may be used by mixing the organic sunscreens that have been conventionally used as long as it does not impair the skin protection effect by reacting with the bacterium-derived vesicles. Examples of the organic sunscreen include glyceryl pava, drometrizole trisiloxane, drometrizole, digaloyltrioleate, disodium phenyldibenzimidazole tetrasulfonate, diethylhexyl butamidotriazone, diethylamino Hydroxybenzoylhexylbenzoate, die-methoxycinnamate, a mixture of lowson and dihydroxyacetone, methylenebis-benzotriazolyltetramethylbutylphenol, 4-methylbenzylidene camphor, menthyl anthranilate, benzophenone -3 (oxybenzone), benzophenone-4, benzophenone-8 (dioxyphenbenzone), butylmethoxydibenzoylmethane, bisethylhexyloxyphenol methoxyphenyltriazine, synoxate, ethyldihydroxypropylpava, Octocrylene, ethylhexyldimethylpava, ethylhexylmethoxycinnamate, ethylhexylsalicylate, ethylhexyltrizone, isoamyl-p-methoxycinnamate, polysilicon-15 (dimethicodiethylbenzalmal Ronate), terephthalylidene dicamphor sulfonic acid and salts thereof, thy-salicylate and aminobenzoic acid (Pava) can be used.
본 발명의 화장료 조성물을 첨가할 수 있는 제품으로는, 예를 들어, 수렴화장수, 유연화장수, 영양화장수, 각종 크림, 에센스, 팩, 파운데이션 등과 같은 화장품류와 클렌징, 세안제, 비누, 트리트먼트, 미용액 등이 있다. 본 발명의 화장료 조성물의 구체적인 제형으로서는 스킨로션, 스킨 소프너, 스킨토너, 아스트린젠트, 로션, 밀크로션, 모이스쳐 로션, 영양로션, 마사지크림, 영양크림, 모이스쳐 크림, 핸드크림, 에센스, 영양에센스, 팩, 비누, 샴푸, 클렌징폼, 클렌징로션, 클렌징크림, 바디로션, 바디클렌저, 유액, 립스틱, 메이크업 베이스, 파운데이션, 프레스파우더, 루스파우더, 아이섀도 등의 제형을 포함한다. Examples of products to which the cosmetic composition of the present invention may be added include, for example, cosmetics such as astringent cosmetics, soft cosmetics, nourishing cosmetics, various creams, essences, packs, foundations, and the like, cleansing agents, soaps, treatments, and essences. Etc. Specific formulations of the cosmetic composition of the present invention include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack, Formulations such as soaps, shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, emulsions, lipsticks, makeup bases, foundations, press powders, loose powders, eye shadows and the like.
본 발명의 바람직한 구현예에 따르면, 본 발명의 바실러스 속 세균 유래 소포의 함량은 조성물 총 중량에 대하여 0.00001-30 중량%이며, 바람직하게는 0.5-20%이며, 보다 바람직하게는 1.0-10 중량%이다. 상기 바실러스 속 세균 유래 소포의 함량이 0.00001 중량% 미만이면 자외선 흡수 효과가 크게 감소되고, 30 중량%를 초과하는 경우에는 피부 자극을 초래할 수 있으며, 제형상의 문제점이 발생할 수 있다. According to a preferred embodiment of the present invention, the content of the bacterium-derived vesicles of the genus Bacillus of the present invention is 0.00001-30% by weight, preferably 0.5-20%, more preferably 1.0-10% by weight based on the total weight of the composition. to be. When the content of the bacterium-derived vesicles of the Bacillus is less than 0.00001% by weight, the ultraviolet absorbing effect is greatly reduced, and when it exceeds 30% by weight, skin irritation may occur, and formulation problems may occur.
본 발명의 또 다른 양태로서, 본 발명은 바실러스 속 세균 유래 소포를 유효성분으로 포함하는 조성물을 개체에 투여하는 단계를 포함하는 암, 염증질환, 또는 대사질환의 예방 또는 치료방법을 제공한다. As another aspect of the present invention, the present invention provides a method for preventing or treating cancer, inflammatory disease, or metabolic disease comprising administering to a subject a composition comprising a bacterium-derived vesicle derived as an active ingredient.
본 발명에서 개체란 질병의 치료를 필요로 하는 대상을 의미하고, 보다 구체적으로는 인간 또는 비-인간인 영장류, 생쥐(mouse), 쥐(rat), 개, 고양이, 말, 및 소 등의 포유류를 의미한다. In the present invention, an individual means a subject in need of treatment of a disease, and more specifically, a mammal such as a primate, mouse, rat, dog, cat, horse, and cow, which is human or non-human. Means.
본 발명의 진단 대상 질병인 암(cancer)은 주위 조직에 침윤하면서 빠르게 성장하고 신체 각 부위에 확산되거나 전이되어 생명을 위협하는 악성종양(malignant tumor)을 의미한다. 신체를 구성하는 가장 작은 단위인 세포(cell)는 정상적으로는 세포 자체의 조절 기능에 의해 분열 및 성장하고, 수명이 다하거나 손상되면 스스로 사멸하여 전반적인 수의 균형을 유지하나, 여러 가지 원인에 의해 이러한 세포 자체의 조절 기능에 문제가 생기면 정상적으로는 사멸해야 할 비정상 세포들이 과다 증식하게 되며, 주위 조직 및 장기에 침입하여 종괴를 형성하고 기존의 구조를 파괴하거나 변형시키게 된다. 본 발명에 있어서, 암은 바람직하게는 간암, 방광암, 유방암, 또는 난소암일 수 있으나, 이에 제한되는 것은 아니다. Cancer, a diagnostic disease of the present invention, refers to a malignant tumor that grows rapidly while infiltrating surrounding tissues and spreads or metastasizes to various parts of the body and threatens life. Cells, the smallest unit of the body, divide and grow normally under the control function of the cells themselves, and die off at the end of their life or damage to maintain the balance of the overall number, but for a number of reasons If a problem occurs in the cell's own regulatory function, abnormal cells that normally must die will overgrow, invading surrounding tissues and organs, forming a mass, and destroying or modifying existing structures. In the present invention, the cancer may preferably be liver cancer, bladder cancer, breast cancer, or ovarian cancer, but is not limited thereto.
본 발명에서 사용되는 용어, 염증질환(inflammatory disease)은, 포유동물 체내의 염증 반응에 의하여 유발되는 질환을 의미하며, 대표적인 예로는, 천식, 만성폐쇄성폐질환, 비염 등의 호흡기계 염증질환; 아토피피부염, 건선, 여드름, 접촉피부염, 탈모 등의 피부 염증질환; 위염, 소화기궤양, 염증성장염 등의 소화기 염증질환; 질염; 골관절염, 류마티스관절염 등의 관절염; 및 이의 합병증 등을 포함한다. 또한, 상기 염증성 질환은 일반적인 염증성 질환 이외에도, 염증 반응과 관련된 암을 포함하는 의미로 사용되며, 예컨대, 유방암, 난소암, 방광암, 간암 등을 포함한다. 또한, 상기 염증성 질환은 일반적인 염증성 질환 이외에도, 염증 반응과 관련된 대사질환을 포함하는 의미로 사용되며, 예컨대, 당뇨병, 비만, 간경화증 등을 포함한다. 본 발명에 있어서, 만성염증질환은 바람직하게는 천식, 만성간염, 탈모, 또는 아토피피부염을 의미하나, 이에 제한되는 것은 아니다.As used herein, the term "inflammatory disease" refers to a disease caused by an inflammatory response in a mammalian body. Representative examples include respiratory inflammatory diseases such as asthma, chronic obstructive pulmonary disease and rhinitis; Skin inflammatory diseases such as atopic dermatitis, psoriasis, acne, contact dermatitis, and hair loss; Gastrointestinal inflammatory diseases such as gastritis, peptic ulcer, and inflammatory bowel disease; vaginitis; Arthritis, such as osteoarthritis and rheumatoid arthritis; And complications thereof. In addition, the inflammatory disease is used in the sense including cancer associated with the inflammatory response, in addition to the general inflammatory disease, for example, includes breast cancer, ovarian cancer, bladder cancer, liver cancer and the like. In addition, the inflammatory disease is used in the sense including metabolic diseases related to the inflammatory response, in addition to the general inflammatory disease, for example, diabetes, obesity, cirrhosis of the liver and the like. In the present invention, chronic inflammatory diseases preferably mean, but are not limited to, asthma, chronic hepatitis, hair loss, or atopic dermatitis.
본 발명에서 사용되는 용어, 대사질환(metabolic disease)은, 포유동물의 체내에서 대사장애에 의해 여러 장기에 합병증이 발생하는 질환을 의미하며, 예컨대, 당뇨과 같은 탄수화물 대사장애 및 이의 합병증으로 간경화증 등을 포함하며, 본 발명에 있어서, 대사질환은 바람직하게 당뇨병 및 간경화증을 포함하나, 이에 제한되는 것은 아니다. As used herein, the term "metabolic disease" refers to a disease in which complications occur in various organs due to metabolic disorders in a mammal's body. For example, carbohydrate metabolic disorders such as diabetes and complications thereof may be cited as cirrhosis. In the present invention, metabolic diseases preferably include, but are not limited to, diabetes and cirrhosis.
본 발명에서 사용되는 용어, 나노소포(Nanovesicle)혹은 “소포(Vesicle)”란, 다양한 세균에서 분비되는 나노크기의 막으로 된 구조물을 의미한다. 그람음성균(gram-negative bacteria) 유래 소포, 또는 외막 소포체(outer membrane vesicles, OMVs)는 내독소(lipopolysaccharide) 뿐만 아니라 독성 단백질 및 세균 DNA와 RNA도 가지고 있고, 그람양성균(gram-positive bacteria) 유래 소포는 단백질과 핵산 외에도 세균의 세포벽 구성성분인 펩티도글리칸(peptidoglycan)과 리포테이코산(lipoteichoic acid)도 가지고 있다. 본 발명에 있어서, 나노소포 혹은 소포는 바실러스 속 세균에서 자연적으로 분비되거나 또는 인공적으로 생산하는 것으로, 구형의 형태이며, 10 내지 200 nm의 평균 직경을 가지고 있다.The term used in the present invention, nanovesicles (Nanovesicle) or "vesicles (Vesicles), refers to the structure of the nano-size membrane secreted by various bacteria. Gram-negative bacteria-derived vesicles or outer membrane vesicles (OMVs) contain toxic proteins, bacterial DNA and RNA as well as lipopolysaccharides, and gram-positive bacteria-derived vesicles. In addition to proteins and nucleic acids, it also contains peptidoglycan and lipoteichoic acid, which are components of bacterial cell walls. In the present invention, nanovesicles or vesicles are naturally secreted or artificially produced by bacteria of the genus Bacillus, and have a spherical shape and have an average diameter of 10 to 200 nm.
본 발명에서 사용되는 용어, 메타게놈이란 '군유전체'라고도 하며, 흙, 동물의 장 등 고립된 지역 내의 모든 바이러스, 세균, 곰팡이 등을 포함하는 유전체의 총합을 의미하는 것으로, 주로 배양이 되지 않는 미생물을 분석하기 위해서 서열분석기를 사용하여 한꺼번에 많은 미생물을 동정하는 것을 설명하는 유전체의 개념으로 쓰인다. 특히, 메타게놈은 한 종의 게놈, 유전체를 말하는 것이 아니라, 한 환경단위의 모든 종의 유전체로서 일종의 혼합유전체를 말한다. 이는 오믹스적으로 생물학이 발전하는 과정에서 한 종을 정의할 때 기능적으로 기존의 한 종뿐만 아니라, 다양한 종이 서로 상호작용하여 완전한 종을 만든다는 관점에서 나온 용어이다. 기술적으로는 빠른 서열분석법을 이용해서, 종에 관계없이 모든 DNA, RNA를 분석하여, 한 환경 내에서의 모든 종을 동정하고, 상호작용, 대사작용을 규명하는 기법의 대상이다.As used herein, the term "metagenome" is also referred to as a "gunoelectric", and means a total of a genome including all viruses, bacteria, fungi, and the like in an isolated region such as soil and animal intestine, and is not mainly cultured. It is used as a concept of genome to explain the identification of many microorganisms at once using sequencer to analyze microorganisms. In particular, the metagenome does not refer to one genome or genome, but to a kind of mixed dielectric as the genome of all species of one environmental unit. This is a term from the point of view of defining a species in the course of the evolution of biology in terms of functional species as well as various species that interact with each other to create a complete species. Technically, rapid sequencing is used to analyze all DNA and RNA, regardless of species, to identify all species in one environment, and to identify interactions and metabolism.
본 발명에서 세균 유래 소포는 세균을 포함하는 배양액을 원심분리, 초고속 원심분리, 압출, 초음파분해, 세포 용해, 균질화, 냉동-해동, 전기천공, 기계적 분해, 화학물질 처리, 필터에 의한 여과, 겔 여과 크로마토그래피, 프리-플로우 전기영동, 및 모세관 전기영동으로 이루어진 군에서 선택된 하나 이상의 방법을 사용하여 분리할 수 있다. 또한, 불순물의 제거를 위한 세척, 수득된 소포의 농축 등의 과정을 추가로 포함할 수 있다. Bacterial-derived vesicles in the present invention is centrifugation, ultra-fast centrifugation, extrusion, sonication, cell lysis, homogenization, freeze-thaw, electroporation, mechanical degradation, chemical treatment, filtration by filter, gel containing the bacteria The separation can be carried out using one or more methods selected from the group consisting of filtration chromatography, pre-flow electrophoresis, and capillary electrophoresis. In addition, it may further include a process for washing to remove impurities, concentration of the obtained vesicles and the like.
본 발명의 실시예에서는 세균 및 세균유래 소포를 마우스 경구로 투여하여 세균 및 소포의 체내 흡수, 분포, 및 배설 양상을 평가하여, 세균인 경우에는 장점막을 통해 흡수되지 않는데 비해 소포는 투여 5분 이내에 흡수되어 전신적으로 분포하고, 신장, 간 등을 통해 배설됨을 확인하였다(실시예 1 참조).In an embodiment of the present invention, the bacterial and bacterial-derived vesicles are orally administered to the mice to evaluate the absorption, distribution, and excretion of the bacteria and vesicles in the body. It was confirmed that it is absorbed and distributed systemically and excreted through the kidney, liver, and the like (see Example 1).
본 발명의 일 실시예에서는, 간암, 방광암, 유방암, 및 난소암 환자, 및 상기 암환자에 연령과 성별을 매칭한 정상인의 혈액에서 분리한 소포를 이용하여 세균 메타게놈 분석을 실시하였다. 그 결과, 정상인 샘플에 비하여, 간암, 방광암, 유방암, 및 난소암 환자의 샘플에 바실러스 속 세균유래 소포가 유의하게 감소되어 있음을 확인하였다(실시예 3, 4, 5, 및 6 참조).In one embodiment of the present invention, bacterial metagenome analysis was performed using vesicles isolated from blood of normal, matched age and gender to liver cancer, bladder cancer, breast cancer, and ovarian cancer patients. As a result, it was confirmed that bacterial vesicles of Bacillus spp. Significantly reduced in samples of patients with liver cancer, bladder cancer, breast cancer, and ovarian cancer compared to normal samples (see Examples 3, 4, 5, and 6).
본 발명의 다른 실시예에서는, 천식 및 아토피피부염 환자, 및 상기 환자의 연령 및 성별을 매칭한 정상인의 샘플에서 분리한 소포를 이용하여 세균 메타게놈 분석을 실시하였다. 그 결과, 정상인 샘플에 비하여, 천식 및 아토피피부염 환자의 샘플에 바실러스 속 세균유래 소포가 유의하게 감소되어 있음을 확인하였다(실시예 7 및 8 참조).In another embodiment of the present invention, bacterial metagenomic analysis was performed using vesicles isolated from a sample of asthma and atopic dermatitis patients and normal subjects matching the age and gender of the patient. As a result, it was confirmed that the vesicle-derived bacteria of Bacillus spp. Significantly decreased in the samples of asthma and atopic dermatitis patients compared to the normal samples (see Examples 7 and 8).
본 발명의 다른 실시예에서는, 당뇨병 및 간경화증 환자, 및 상기 환자의 연령 및 성별을 매칭한 정상인의 샘플에서 분리한 소포를 이용하여 세균 메타게놈 분석을 실시하였다. 그 결과, 정상인 샘플에 비하여, 당뇨병 및 간경화증 환자의 샘플에 바실러스 속 세균유래 소포가 유의하게 감소되어 있음을 확인하였다(실시예 9 및 10 참조).In another embodiment of the present invention, bacterial metagenomic analysis was performed using vesicles isolated from a sample of a diabetic and cirrhosis patient and a normal person matching the age and sex of the patient. As a result, it was confirmed that the vesicles derived from Bacillus spp. Significantly reduced in the samples of diabetic and cirrhosis patients compared to the normal samples (see Examples 9 and 10).
본 발명의 다른 실시예에서는, 당뇨병환자, 및 연령 및 성별을 매칭한 정상인의 샘플에서 분리한 소포를 이용하여 세균 메타게놈 분석을 실시하였다. 그 결과, 정상인 샘플에 비하여, 당뇨병 환자의 샘플에 Escherichia 속 세균유래 소포가 유의하게 증가되어 있음을 확인하였다(실시예 11 참조).In another embodiment of the present invention, bacterial metagenome analysis was performed using vesicles isolated from a sample of a diabetic and a normal person matching age and sex. As a result, it was confirmed that the bacterial vesicles of the genus Escherichia were significantly increased in the samples of diabetics compared to the normal samples (see Example 11).
또한, 본 발명의 다른 실시예에서는, 바실러스 속 세균 유래 소포가 암, 만성염증질환, 및 대사질환의 원인인자에 의해 염증반응을 억제하는지를 평가하기 위하여 예의 연구한 결과, 고초균(표준균주) 유래 소포 및 고초균의 아형인 낫토균 유래 소포인 경우, 대장균 유래 소포에 비해 염증 유발효과가 거의 없었고, 고초균 또는 낫토균 유래 소포를 전처리하였을 때 병원성 원인인자인 대장균 유래 소포에 의한 염증반응을 효율적으로 억제함을 관찰하였는 바, 이에 기초하여 본 발명을 완성하였다(실시예 13 및 14 참조).Further, in another embodiment of the present invention, as a result of intensive studies to evaluate whether the bacterium-derived vesicles of Bacillus bacteria inhibit the inflammatory response caused by cancer, chronic inflammatory diseases, and metabolic diseases, vesicles derived from Bacillus subtilis (standard strain) In the case of Naxos-derived vesicles, which are subtypes of Bacillus subtilis, there was little inflammation-inducing effect compared to E. coli-derived vesicles, and when pretreatment of vesicles or Natto-derived vesicles effectively suppressed the inflammatory response caused by Escherichia coli-derived vesicles. The present invention was completed based on this observation (see Examples 13 and 14).
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.
[실시예]EXAMPLE
실시예 1. 세균 및 세균 유래 소포의 체내 흡수, 분포, 및 배설 양상 분석Example 1 Analysis of Absorption, Distribution, and Excretion of Bacteria and Bacterial-Derived Vesicles
바실러스 속 세균 및 유래 소포가 위장관을 통해 전신적으로 흡수되는 지를 평가하기 위하여 다음과 같은 방법으로 실험을 수행하였다. 마우스의 위장에 형광으로 표지한 세균과 세균유래 소포를 각각 50 μg의 용량으로 위장관으로 투여하고, 0분, 5분, 3시간, 6시간, 12시간 후에 형광을 측정하였다. 마우스 전체 이미지를 관찰한 결과,세균인 경우에는 전신적으로 흡수되지 않았지만, 세균유래 소포인 경우에는, 투여 후 5분에 전신적으로 흡수되었고, 투여 30분에는 간 및 방광에 형광이 진하게 관찰되어, 소포가 간 및 비뇨기계로 배설됨을 알 수 있었다. 또한, 소포는 투여 12시간까지 체내에 존재함을 알 수 있었다(도 1a 참조). In order to evaluate whether Bacillus bacteria and derived vesicles are absorbed systemically through the gastrointestinal tract, the experiment was performed as follows. Fluorescently labeled bacteria and bacterial-derived vesicles were administered to the gastrointestinal tract at a dose of 50 μg, respectively, and the fluorescence was measured after 0 minutes, 5 minutes, 3 hours, 6 hours, and 12 hours. As a result of observing the entire image of the mouse, bacteria were not absorbed systemically, but in the case of bacterial-derived vesicles, they were absorbed systemically 5 minutes after administration, and fluorescence was strongly observed in the liver and bladder at 30 minutes after administration. Were excreted in the liver and urinary system. In addition, the vesicles were found to exist in the body up to 12 hours after administration (see FIG. 1A).
세균과 세균유래 소포가 전신적으로 흡수된 후, 여러 장기로 침윤된 양상을 평가하기 위하여, 형광으로 표지한 50 μg의 세균과 세균유래 소포를 상기의 방법과 같이 투여한 후, 투여 12시간 후에 혈액, 심장, 간, 신장, 비장, 지방, 근육을 채취하였다. 채취한 조직에서 형광을 관찰한 결과, 세균 유래 소포가 혈액, 심장, 폐, 간, 콩팥, 비장, 지방, 근육, 신장에 분포하였으나, 세균은 흡수되지 않음을 알 수 있었다(도 1b 참조).After the systemic uptake of bacteria and bacteria-derived vesicles systemically, in order to evaluate the infiltration into various organs, the blood-labeled 50 μg of bacteria and bacteria-derived vesicles were administered in the same manner as described above, and then 12 hours after administration The heart, liver, kidneys, spleen, fat and muscles were taken. As a result of fluorescence observation in the collected tissue, bacteria-derived vesicles were distributed in blood, heart, lung, liver, kidney, spleen, fat, muscle, and kidney, but the bacteria were not absorbed (see FIG. 1B).
실시예 2. 임상샘플에서 세균 유래 소포 메타게놈 분석Example 2 Bacterial-derived Vesicular Metagenome Analysis in Clinical Samples
혈액 등의 임상샘플을 먼저 10 ml 튜브에 넣고 원심분리법(3,500 x g, 10min, 4℃)으로 부유물을 가라앉히고 상등액만을 새로운 10 ml 튜브에 옮겼다. 0.22㎛ 필터를 사용하여 세균 및 이물질을 제거한 후, 센트리프랩튜브 (centripreigugal filters 50 kD)에 옮겨서 1500 x g, 4℃에서 15분간 원심분리하여 50 kD 보다 작은 물질은 버리며 10 ml 까지 농축 시켰다. 다시 한 번 0.22㎛ filter를 사용하여 세균 및 이물질을 제거한 후, 소포를 분리하여 생리식염수(PBS)로 녹였다. Clinical samples such as blood were first placed in 10 ml tubes and the suspension was allowed to settle by centrifugation (3,500 x g, 10 min, 4 ° C.) and only the supernatant was transferred to a new 10 ml tube. After removing bacteria and foreign substances using a 0.22㎛ filter, it was transferred to centripreigugal filters (50 kD) and centrifuged at 1500 x g and 4 ° C for 15 minutes to discard materials smaller than 50 kD and concentrated to 10 ml. Once again by removing the bacteria and foreign substances using a 0.22㎛ filter, the vesicles were separated and dissolved in physiological saline (PBS).
상기 방법으로 분리한 소포 100㎕를 100℃에서 끓여서 내부의 DNA를 지질 밖으로 나오게 하고 그 후 얼음에 5분 동안 식혔다. 그리고 남은 부유물을 제거하기 위하여 10,000 x g, 4℃에서 30분간 원심분리하고 상등액만을 모았다. 그리고 Nanodrop을 이용하여 DNA 양을 정량하였다. 이후, 상기 추출된 DNA에 세균 유래 DNA가 존재하는지 확인하기 위하여 하기 표 1에 나타낸 16s rDNA primer로 PCR을 수행하여 상기 추출된 유전자에 세균 유래 유전자가 존재하는 것을 확인하였다.100 μl of the vesicles separated by the above method was boiled at 100 ° C. to let the internal DNA come out of the lipid and then cooled on ice for 5 minutes. And centrifuged at 10,000 x g, 4 ℃ 30 minutes to remove the remaining suspended solids and collected only the supernatant. The amount of DNA was quantified using Nanodrop. Thereafter, PCR was performed with the 16s rDNA primer shown in Table 1 to confirm whether the bacteria-derived DNA exists in the extracted DNA, and it was confirmed that the bacteria-derived gene exists in the extracted gene.
| primerprimer | 서열order | 서열번호SEQ ID NO: | |
| 16S rDNA16S rDNA | 16S_V3_F16S_V3_F | 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3'5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3 ' | 1One |
| 16S_V4_R16S_V4_R | 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-35'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3 | 22 | |
상기 방법으로 추출한 DNA를 상기의 16S rDNA 프라이머를 사용하여 증폭을 한 다음 시퀀싱을 수행하고 (Illumina MiSeq sequencer), 결과를 Standard Flowgram Format (SFF) 파일로 출력하고 GS FLX software (v2.9)를 이용하여 SFF 파일을 sequence 파일 (.fasta)과 nucleotide quality score파일로 변환한 다음 리드의 신용도 평가를 확인하고, window (20 bps) 평균 base call accuracy가 99% 미만 (Phred score <20)인 부분을 제거하였다. Operational Taxonomy Unit (OTU) 분석을 위해서는 UCLUST와 USEARCH를 이용하여 시퀀스 유사도에 따라 클러스터링을 수행하고, genus는 94%, family는 90%, order는 85%, class는 80%, phylum은 75% 시퀀스 유사도를 기준으로 클러스터링을 하고 각 OTU의 phylum, class, order, family, genus 레벨의 분류를 수행하고, BLASTN와 GreenGenes의 16S RNA 시퀀스 데이터베이스 (108,453 시퀀스)를 이용하여 속 수준에서 97% 이상의 시퀀스 유사도 갖는 세균을 프로파일링 하였다 (QIIME).DNA extracted by the above method was amplified using the above 16S rDNA primers, followed by sequencing (Illumina MiSeq sequencer), and the results were outputted in a Standard Flowgram Format (SFF) file, using GS FLX software (v2.9). After converting the SFF file into a sequence file (.fasta) and a nucleotide quality score file, the credit rating of the lead is checked, and the window (20 bps) has an average base call accuracy of less than 99% (Phred score <20). It was. For operational taxonomy unit (OTU) analysis, clustering is performed according to sequence similarity using UCLUST and USEARCH, genus 94%, family 90%, order 85%, class 80%, phylum 75% sequence similarity Clustering is based on the phylum, class, order, family, and genus levels of each OTU, and BLASTN and GreenGenes' 16S RNA sequence database (108,453 sequences) is used to identify bacteria with greater than 97% sequence similarity at the genus level. Was profiled (QIIME).
실시예Example
3. 간암환자 혈액 세균 유래 소포 3. Blood-derived vesicles derived from liver cancer patients
메타게놈Metagenome
분석을 통한 Through analysis
바실러스Bacillus
속 세균 유래 소포의 감소 확인 Reduction of Bacterial-Derived Vesicles in Genus
실시예 2의 방법으로 간암환자 91명의 혈액과 성별과 연령을 매칭한 정상대조군 99명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 바실러스 속 세균유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 위암환자의 혈액에 바실러스 속 세균유래 소포가 유의하게 감소되어 있음을 확인하였다 (정상인 vs 간암환자: 0.37% vs 0.13%; fold change: 0.37; p=0.0002) (도 3 참조).After the genes were extracted from the vesicles present in the blood and subjected to metagenomic analysis, the blood of 91 hepatic cancer patients and the blood of 99 normal controls matched with gender and age were analyzed by the method of Example 2 The distribution of was evaluated. As a result, it was confirmed that bacterium-derived vesicles of Bacillus were significantly reduced in the blood of gastric cancer patients compared to normal blood (normal vs liver cancer: 0.37% vs 0.13%; fold change: 0.37; p = 0.0002) (FIG. 3) Reference).
실시예Example
4. 방광암환자 혈액 세균 유래 소포 4. Blood-derived vesicles from bladder cancer patients
메타게놈Metagenome
분석을 통한 Through analysis
바실러스Bacillus
속 세균 유래 소포의 감소 확인 Reduction of Bacterial Derived Vesicles
실시예 2의 방법으로 방광암환자 96명의 혈액과 성별과 연령을 매칭한 정상대조군 184명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 바실러스 속 세균유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 방광암환자의 혈액에 바실러스 속 세균유래 소포가 유의하게 감소되어 있음을 확인하였다 (정상인 vs 방광암환자: 0.38% vs 0.10%; fold change: 0.27; p=0.001) (도 4 참조).In the method of Example 2, the blood of 96 bladder cancer patients and 184 blood of normal control group matched with sex and age were extracted from the vesicles in the blood and subjected to metagenomic analysis. The distribution of was evaluated. As a result, it was confirmed that bacterium-derived vesicles of Bacillus were significantly reduced in blood of bladder cancer patients compared to normal blood (normal vs bladder cancer patients: 0.38% vs 0.10%; fold change: 0.27; p = 0.001) (FIG. 4). Reference).
실시예Example
5. 유방암환자 혈액 세균 유래 소포 5. Blood-derived vesicles from breast cancer patients
메타게놈Metagenome
분석을 통한 Through analysis
바실러스Bacillus
속 세균 유래 소포의 감소 확인 Reduction of Bacterial-Derived Vesicles in Genus
실시예 2의 방법으로 유방암환자 96명의 혈액과 성별과 연령을 매칭한 정상대조군 192명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 바실러스 속 세균유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 유방암환자의 혈액에 바실러스 속 세균유래 소포가 유의하게 감소되어 있음을 확인하였다 (정상인 vs 유방암환자: 0.59% vs 0.26%; fold change: 0.44; p=0.0006) (도 5 참조).After the genes were extracted from the vesicles present in the blood and subjected to a metagenome analysis, the blood of 96 breast cancer patients and 192 blood of a normal control group matched with sex and age were analyzed. The distribution of was evaluated. As a result, it was confirmed that bacterium-derived vesicles of Bacillus were significantly reduced in the blood of breast cancer patients compared to normal blood (normal vs breast cancer patients: 0.59% vs 0.26%; fold change: 0.44; p = 0.0006) (FIG. 5). Reference).
실시예Example
6. 난소암환자 혈액 세균 유래 소포 6. Blood Bacteria Derived from Ovarian Cancer Patients
메타게놈Metagenome
분석을 통한 Through analysis
바실러스Bacillus
속 세균 유래 소포의 감소 확인 Reduction of Bacterial-Derived Vesicles in Genus
실시예 2의 방법으로 난소암환자 137명의 혈액과 성별과 연령을 매칭한 정상대조군 139명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 바실러스 속 세균유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 난소암환자의 혈액에 바실러스 속 세균유래 소포가 유의하게 감소되어 있음을 확인하였다 (정상인 vs 난소암환자: 0.61% vs 0.27%; fold change: 0.44; p=0.01) (도 6 참조).In the method of Example 2, 137 blood of ovarian cancer patients and 139 blood of the normal control group matched with sex and age were extracted from vesicles in the blood and subjected to metagenome analysis. The distribution of vesicles was evaluated. As a result, it was confirmed that bacterium-derived vesicles of Bacillus were significantly decreased in blood of ovarian cancer patients compared to normal blood (normal vs ovarian cancer patients: 0.61% vs 0.27%; fold change: 0.44; p = 0.01) ( 6).
실시예Example
7. 천식환자 혈액 세균 유래 소포 7. Blood-derived vesicles from asthma patients
메타게놈Metagenome
분석을 통한 Through analysis
바실러스Bacillus
속 세균 유래 소포의 감소 확인 Reduction of Bacterial-Derived Vesicles in Genus
실시예 2의 방법으로 천식환자 277명의 혈액과 성별과 연령을 매칭한 정상대조군 246명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 바실러스 속 세균유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 천식환자의 혈액에 바실러스 속 세균유래 소포가 유의하게 감소되어 있음을 확인하였다 (정상인 vs 천식환자: 0.47% vs 0.09%; fold change: 0.20; p<0.000001) (도 7 참조).In the method of Example 2, 277 blood of asthma patients and 246 blood of the normal control group matched with gender and age were extracted from the vesicles in the blood and subjected to metagenomic analysis. The distribution of was evaluated. As a result, it was confirmed that the bacterium-derived vesicles of Bacillus were significantly reduced in the blood of asthma patients compared to normal blood (normal vs asthma patients: 0.47% vs 0.09%; fold change: 0.20; p <0.000001) (FIG. 7) Reference).
실시예Example
8. 아토피피부염환자 혈액 세균 유래 소포 8. Vesicles derived from blood bacteria of atopic dermatitis patients
메타게놈Metagenome
분석을 통한 Through analysis
바실러스Bacillus
속 세균 유래 소포의 감소 확인 Reduction of Bacterial-Derived Vesicles in Genus
실시예 2의 방법으로 아토피피부염환자 25명의 혈액과 성별과 연령을 매칭한 정상대조군 138명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 바실러스 속 세균유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 아토피피부염환자의 혈액에 바실러스 속 세균유래 소포가 유의하게 감소되어 있음을 확인하였다 (정상인 vs 아토피피부염환자: 0.10% vs 0.04%; fold change: 0.44; p=0.01) (도 8 참조).Blood of 25 atopic dermatitis patients and blood of 138 normal controls in matched sex and age were extracted by metagenenomic analysis of vesicles present in the blood, followed by bacterium of Bacillus spp. The distribution of vesicles was evaluated. As a result, it was confirmed that the bacterium derived from Bacillus bacteria in blood of atopic dermatitis patients was significantly reduced compared to normal blood (normal vs. atopic dermatitis patients: 0.10% vs 0.04%; fold change: 0.44; p = 0.01) ( 8).
실시예Example
9. 당뇨병환자 혈액 세균 유래 소포 9. Blood-derived vesicles from diabetic patients
메타게놈Metagenome
분석을 통한 Through analysis
바실러스Bacillus
속 세균 유래 소포의 감소 확인 Reduction of Bacterial-Derived Vesicles in Genus
실시예 2의 방법으로 당뇨병환자 73명의 혈액과 성별과 연령을 매칭한 정상대조군 146명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 바실러스 속 세균유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 당뇨병환자의 혈액에 바실러스 속 세균유래 소포가 유의하게 감소되어 있음을 확인하였다 (정상인 vs 당뇨병환자: 0.46% vs 0.04%; fold change: 0.10; p<0.000001) (도 9 참조).In the method of Example 2, blood was extracted from the vesicles in the normal control group matched with sex and age of 73 diabetic patients and the blood was extracted from the vesicles in the blood, followed by a metagenome analysis, and then the bacterium-derived vesicles of Bacillus spp. The distribution of was evaluated. As a result, it was confirmed that bacterium-derived vesicles of Bacillus in blood of diabetic patients were significantly reduced compared to normal blood (normal vs diabetic patients: 0.46% vs 0.04%; fold change: 0.10; p <0.000001) (FIG. 9) Reference).
실시예Example
10. 간경화증환자 혈액 세균 유래 소포 10. Blood Bacteria Derived from Liver Cirrhosis Patients
메타게놈Metagenome
분석을 통한 Through analysis
바실러스Bacillus
속 세균 유래 소포의 감소 확인 Reduction of Bacterial-Derived Vesicles in Genus
실시예 2의 방법으로 간경화증환자 73명의 혈액과 성별과 연령을 매칭한 정상대조군 146명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 바실러스 속 세균유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 간경화증환자의 혈액에 바실러스 속 세균유래 소포가 유의하게 감소되어 있음을 확인하였다 (정상인 vs 간경화증환자: 0.54% vs 0.25%; fold change: 0.47; p=0.003) (도 10 참조).73 genes of liver cirrhosis patients and 146 blood of normal control group matched with sex and age were extracted from the vesicles in the blood and subjected to metagenomic analysis. The distribution of was evaluated. As a result, the bacterium-derived vesicles of Bacillus were significantly reduced in the blood of cirrhosis patients compared to normal blood (normal vs liver cirrhosis patients: 0.54% vs 0.25%; fold change: 0.47; p = 0.003) (Fig. 10). Reference).
실시예Example
11. 당뇨병환자 혈액 세균 유래 소포 11. Blood-derived vesicles from diabetic patients
메타게놈Metagenome
분석을 통한 Escherichia 속 세균 유래 소포의 증가 확인 Analysis of Escherichia Genus Bacteria-Derived Vesicles
실시예 2의 방법으로 당뇨병환자 73명의 혈액과 성별과 연령을 매칭한 정상대조군 146명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, Escheriachia 속 세균유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 당뇨병환자의 혈액에 Escheriachia 속 세균유래 소포가 현저히 증가되어 있음을 확인하였다 (정상인 vs 당뇨병환자: 0.008% vs 0.8.165%; fold change: 947.3; p<0.000001) (도 11 참조).In the method of Example 2, blood was extracted from the vesicles in the normal control group of 73 diabetic patients and 146 blood of the matched sex and age group, and then subjected to metagenomic analysis after extracting genes from the vesicles in the blood. The distribution of was evaluated. As a result, it was confirmed that the bacteria-derived vesicles of Escheriachia were significantly increased in the blood of diabetic patients compared to the normal blood (normal vs diabetic patients: 0.008% vs 0.8.165%; fold change: 947.3; p <0.000001) (Fig. 11).
실시예 12. 고초균(Bacillus subtilis) 배양액에서 소포 분리 및 특성 평가Example 12 Isolation and Characterization of Vesicles in Bacillus subtilis Culture
바실러스 속 세균 유래 소포의 특성을 평가하기 위하여 고초균 표준균주의 배양액에서 소포를 분리하여 특성을 평가하였는 바, 소포를 분리하기 위해 고초균을 멸균된(autoclaved) Brain heart infusion broth(BD 237500) 2 L에 접종하고 72시간 동안 흡광도(O.D) 값이 1.5가 되도록 챔버에서 배양한 후, 배양액을 20분간 고속 원심분리(10,000×g)하여 균 침전 펠렛을 제외한 상층액을 얻었다. 상층액은 차례로 0.45 ㎛ 필터와 0.22 ㎛ 필터를 이용해 여과한 후 Quixstand benchtop system을 이용하여 약 14배 정도 농축된 배양액을 얻었다. 상기 배양액에 대하여 다시 150,000×g, 4℃에서 2시간 동안 초고속 원심분리를 수행하여 펠렛을 얻은 후 멸균 생리식염수(PBS)로 녹여 단백질을 정량하였다. To evaluate the characteristics of vesicles derived from Bacillus bacteria, vesicles were isolated from the culture medium of Bacillus subtilis strains. To isolate the vesicles, 2 L of autoclaved brain heart infusion broth (BD 237500) was isolated. After inoculation and incubation in a chamber so that the absorbance (OD) value was 1.5 for 72 hours, the culture solution was centrifuged at high speed (10,000 × g) for 20 minutes to obtain a supernatant except for the bacterial precipitation pellets. The supernatant was sequentially filtered using a 0.45 μm filter and a 0.22 μm filter, and then cultured about 14-fold concentrated using a Quixstand benchtop system. The culture medium was again subjected to ultrafast centrifugation for 2 hours at 150,000 × g, 4 ° C. to obtain pellets, and then dissolved in sterile saline (PBS) to quantify the protein.
상기 방법에 따라 고초균 배양액으로부터 수득한 고초균 유래 소포의 형태 및 크기를 관찰 및 측정하였다. 먼저 소포의 형태를 평가하기 위하여, 단백질 정량에 의해 50 ㎍/㎖의 샘플을 얻어 JEM 1011 전자현미경(Jeol, Japan)으로 관찰한 결과, 도 12a에 나타난 바와 같이, 고초균 유래 소포가 구형임을 확인하였다. 다음으로, 50 ㎍/㎖의 샘플을 가지고 zetasizer nano ZS (Malverk, UK)를 통해 동적광산란법(dynamic light scattering; DLS)으로 고초균 유래 소포의 크기를 측정한 결과, 도 12b에 나타난 바와 같이, 고초균 유래 소포의 평균 직경이 40~100nm의 범위임을 알 수 있었다.According to the above method, the form and size of Bacillus subtilis obtained from Bacillus subculture were observed and measured. First, in order to evaluate the morphology of the vesicles, 50 μg / ml sample was obtained by protein quantification and observed with a JEM 1011 electron microscope (Jeol, Japan). As shown in FIG. 12A, it was confirmed that the vesicle-derived vesicles were spherical. . Next, the size of Bacillus subtilis derived from Bacillus subtilis was measured by dynamic light scattering (DLS) through zetasizer nano ZS (Malverk, UK) with a sample of 50 µg / ml, as shown in FIG. 12B. It was found that the average diameter of the derived vesicles was in the range of 40 to 100 nm.
실시예Example
13. 고초균 유래 소포와 대장균 유래 소포에 의한 염증 유발성 차이 비교 13. Comparison of Inflammation-induced Difference between Bacillus subtilis and Escherichia coli Derivatives
상기 실시예 12의 방법에 따라 분리한 고초균 유래 소포의 염증 유발성을 평가하기 위해 양성 대조군으로 병원성 원인인자인 대장균 유래 소포를 이용해 염증세포인 대식세포주(Raw 264.7)에서 염증 유발성을 비교하고자 하였다. 이를 위해, 상기 대식세포주에 대장균 유래 소포(E. coli EV, 1 ㎍/㎖), 또는 다양한 농도의 고초균 표준균주 유래 소포(Bacillus subtilis EV, 0.1. 1, 10 ㎍/㎖) 및 고초균 낫토아형 균주 유래 소포(Bacillus subtilis natto EV, 0.1. 1, 10 ㎍/㎖)를 처리하고 12시간 후에 염증성 사이토카인인 IL-6 및 TNF-α의 농도를 ELISA법으로 측정하여 염증유발 능력을 비교하였다. 이때, 음성대조군(NC)으로는 인산완충액(PBS)를 처리하였다. In order to evaluate the inflammation induction of Bacillus subtilis derived from the Bacillus subtilis according to the method of Example 12, we tried to compare the induction of inflammation in macrophage lines (Raw 264.7) inflammatory cells using E. coli-derived vesicles, a pathogenic causative agent, as a positive control. . To this end, Escherichia coli-derived vesicles (E. coli EV, 1 µg / ml), or Bacillus subtilis EV, 0.1. 1, 10 µg / ml, and Bacillus subtilis natto-type strains on the macrophage line. Twelve hours after treatment with the derived vesicles (Bacillus subtilis natto EV, 0.1. 1, 10 ug / ml), the concentrations of inflammatory cytokines IL-6 and TNF-α were measured by ELISA to compare the proinflammatory ability. At this time, the negative control group (NC) was treated with phosphate buffer (PBS).
그 결과, 도 13에 나타낸 바와 같이, 대장균 유래 소포를 처리한 경우에는 IL-6 분비가 유도되었으나, 고초균 표준균주 유래 소포 또는 고초균 낫토균주(낫토균) 유래 소포를 처리한 경우에는 처리 농도에 관계없이 IL-6 분비가 유도되지 않은 것을 확인하였다(도 13a 참조). 또한, TNF-α 분비와 관련하여서는 동일한 농도에서 대장균 유래 소포에 비하여 고초균 표준균주 유래 소포 또는 낫토균 유래 소포에 의한 TNF-α 분비능이 현저히 낮은 것을 확인하였다(도 13b 참조). As a result, as shown in Fig. 13, when treated with E. coli-derived vesicles, IL-6 secretion was induced, but when treated with vesicle-derived standard strains or vesicles derived from Natto strains (natto bacteria), it was related to the treatment concentration. It was confirmed that no IL-6 secretion was induced (see FIG. 13A). In addition, it was confirmed that the TNF-α secretion ability was significantly lower than the E. coli-derived vesicle-derived or Natto-derived vesicles in comparison with E. coli-derived vesicles at the same concentration (see FIG. 13B).
실시예Example
14. 대장균 유래 소포에 의한 염증의 발생에서 고초균 유래 소포에 의한 항염증 효과 확인 14. Confirmation of anti-inflammatory effect caused by Bacillus subtilis vesicles in the development of inflammation caused by E. coli-derived vesicle
Escherichia 속 세균 중에서 대표적인 세균인 대장균(Eschericahia
coli)은 대장뿐만 아니라 주변 환경에 서식하는 주요 세균으로서, 항생제 내성을 잘 일으킨다. 또한, 대장균 유래 소포가 천식, 만성폐쇄성폐질환(COPD) 등과 같은 만성폐쇄성 기도질환의 주요 원인인자임이 밝혀지면서, 만성염증질환의 주요 원인인자로 알려지게 되었다. 따라서 본 발명에서는 대장균 유래 소포에 의한 염증의 발생에 고초균 유래 소포의 항염증 효과를 평가하고자 하였다. 이를 위해, 대식세포주(Raw 264.7)에 고초균 표준균주 유래 소포(B. subtilis EV, 0.1, 1, 10 ㎍/㎖), 혹은 고초균 낫토균주 유래 소포(B. subtilis natto EV, 0.1, 1, 10 ㎍/㎖)를 처리하고 12시간 후에 대장균 소포(1 ㎍/㎖)를 대식세포에 처리하여 염증성 사이토카인인 IL-6 및 TNF-α의 농도를 ELISA법으로 측정하였다. 이때, 음성대조군(NC)으로는 상기 실시예 13과 동일하게 PBS를 처리하였고, 양성대조군(PC)으로는 대장균 유래 소포(1 ㎍/㎖)만 처리하였으며, 비교군으로는 항염증 효과가 있다고 알려진 락토바실러스 플란타룸 유래 소포(Lactobacillus plantarum EV, 1 ㎍/㎖)를 고초균 유래 소포와 동일한 조건으로 전처리하였다. Escherichia coli ( Eschericahia) coli ) is a major bacterium that lives not only in the large intestine but also in the surrounding environment, and is highly resistant to antibiotics. In addition, as E. coli-derived vesicles are found to be a major cause of chronic obstructive airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), it has become known as a major cause of chronic inflammatory diseases. Therefore, the present invention was intended to evaluate the anti-inflammatory effect of Bacillus subtilis-derived vesicles on the occurrence of inflammation caused by E. coli-derived vesicles. To this end, B. subtilis EV, 0.1, 1, 10 μg / ml, or B. subtilis natto EV, 0.1, 1, 10 μg, in the macrophage line (Raw 264.7). / Ml) and E. coli vesicles (1 μg / ml) were treated with macrophages to determine the concentration of inflammatory cytokines IL-6 and TNF-α by ELISA method. At this time, the negative control group (NC) was treated with PBS in the same manner as in Example 13, the positive control group (PC) was treated only with E. coli-derived vesicles (1 ㎍ / ㎖), there is an anti-inflammatory effect in the comparison group Known Lactobacillus plantarum vesicles (Lactobacillus plantarum EV, 1 μg / ml) were pretreated under the same conditions as Bacillus subtilis.
그 결과, 도 14에 나타낸 바와 같이, 대장균 유래 소포 자극에 의한 IL-6의 분비가 고초균 표준균주 유래 소포의 전처리에 락토바실러스 플란타룸 유래 소포와 비슷한 정도로 억제되었고, 고초균 낫토균주 유래 소포의 전처리에 의해선 락토바실러스 플란타룸 유래 소포에 비해 더욱 완벽히 억제되었다(도 14a 참조). 또한, 락토바실러스 플란타룸 유래 소포인 경우에는 TNF-α의 분비를 억제되지 않았으나, 고초균 표준균주 및 낫토균주 유래 소포에 의해선 용량의존적으로 TNF-α의 분비를 억제함을 관찰하였다(도 14b 참조). 상기 결과는 IL-6 및 TNF-α 등의 염증매개체의해 발생하는 염증을 고초균 유래 소포가 효율적으로 억제할 수 있음을 의미하는 것이다. As a result, as shown in Fig. 14, the secretion of IL-6 by E. coli-derived vesicle stimulation was suppressed to a degree similar to that of Lactobacillus plantarum-derived vesicles in the pretreatment of the standard strain derived from Bacillus subtilis. Was more completely inhibited compared to Lactobacillus plantarum derived vesicles (see FIG. 14A). In addition, in the case of Lactobacillus plantarum-derived vesicles, the secretion of TNF-α was not inhibited, but it was observed that the vesicles derived from Bacillus subtilis and natto strains inhibited TNF-α secretion (FIG. 14B). Reference). The above results mean that vesicle-derived vesicles can efficiently inhibit inflammation caused by inflammatory mediators such as IL-6 and TNF-α.
상기 진술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The description of the present invention set forth above is for illustrative purposes, and one of ordinary skill in the art may understand that the present invention may be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.
본 발명자들은 장내 세균인 경우에는 체내에 흡수되지 않지만, 세균유래 소포인 경우에는 체내에 흡수되어, 전신적으로 분포하고, 콩팥, 간, 폐를 통해 체외로 배설됨을 확인하였고, 환자 혈액, 소변, 대변 등에 존재하는 세균유래 소포 메타게놈 분석을 통해 간암, 방광암, 유방암, 난소암 등의 고형암, 천식, 아토피피부염 등의 만성염증질환, 당뇨병, 간경화 등의 대사질환 환자의 혈액에 존재하는 바실러스 속 세균유래 소포가 정상인에 비하여 유의하게 감소되어 있음을 확인하였다. The present inventors confirmed that the intestinal bacteria are not absorbed into the body, but the bacteria-derived vesicles are absorbed into the body and distributed systemically and excreted externally through the kidneys, liver, and lungs. Bacterial-derived bacteria of Bacillus present in the blood of patients with metabolic diseases such as solid cancers such as liver cancer, bladder cancer, breast cancer, ovarian cancer, asthma, atopic dermatitis, diabetes, liver cirrhosis, etc. It was confirmed that the vesicles were significantly reduced compared to the normal persons.
또한, 프로테우스 속 세균의 한 종인 고초균 표준균주 및 고초균의 아형인 낫토균을 체외에서 배양하여 소포를 분리하여, 체외에서 염증세포에 투여하였을 때, 병원성 소포에 의한 염증매개체 분비를 유의하게 억제함을 관찰하였는 바, 본 발명에 따른 바실러스 속 세균 유래 소포는 암, 만성염증질환, 또는 대사질환의 진단 또는 예측방법, 및 식품, 흡입제, 화장품, 혹은 약물 등의 예방용 혹은 치료용 조성물에 유용하게 이용될 수 있을 것이다. In addition, when the vesicles were isolated by culturing in vitro and incubated with a standard strain of Bacillus subtilis, a subtilis of Bacillus subtilis and Natto, a subtype of Bacillus subtilis, it significantly inhibited the secretion of inflammatory mediators caused by pathogenic vesicles. As observed, the bacterium-derived vesicles according to the present invention are useful for the diagnosis or prediction method of cancer, chronic inflammatory disease, or metabolic disease, and for the prevention or treatment of food, inhalants, cosmetics, or drugs. Could be.
Claims (33)
- 하기의 단계를 포함하는, 암, 염증질환, 또는 대사질환의 진단을 위한 정보제공방법:Method for providing information for the diagnosis of cancer, inflammatory disease, or metabolic disease, comprising the following steps:(a) 정상인 및 피검자 유래 샘플에서 분리한 소포로부터 DNA를 추출하는 단계;(a) extracting DNA from vesicles isolated from normal and subject derived samples;(b) 상기 추출한 DNA에 대하여 16S rDNA 서열에 존재하는 바실러스 속 세균 유래 소포 검출 프라이머 쌍을 이용하여 PCR을 수행하여 각각의 PCR 산물을 수득하는 단계; 및(b) performing PCR on the extracted DNA using a Bacillus bacteria-derived vesicle detection primer pair present in the 16S rDNA sequence to obtain respective PCR products; And(c) 상기 PCR 산물의 정량분석을 통하여 정상인에 비하여 바실러스 속 세균 유래 소포의 함량이 낮을 경우 암, 염증질환, 또는 대사질환으로 판정하는 단계.(c) determining the cancer, inflammatory disease, or metabolic disease when the content of the bacterium-derived vesicles in Bacillus is lower than that of the normal person through quantitative analysis of the PCR product.
- 제1항에 있어서,The method of claim 1,상기 암은 간암, 방광암, 유방암, 또는 난소암인 것을 특징으로 하는, 정보제공방법.The cancer is liver cancer, bladder cancer, breast cancer, or ovarian cancer, characterized in that the information providing method.
- 제1항에 있어서,The method of claim 1,상기 염증질환은 천식, 만성간염, 또는 아토피피부염인 것을 특징으로 하는, 정보제공방법.The inflammatory disease is asthma, chronic hepatitis, or atopic dermatitis, characterized in that the information providing method.
- 제1항에 있어서,The method of claim 1,상기 대사질환은 당뇨병, 또는 간경화증인 것을 특징으로 하는, 정보제공방법.The metabolic disease is diabetes, or cirrhosis, characterized in that the information providing method.
- 제1항에 있어서,The method of claim 1,상기 환자 유래 샘플은 혈액, 소변, 또는 대변인 것을 특징으로 하는, 정보제공방법.The patient-derived sample is characterized in that the blood, urine, or stool.
- 하기의 단계를 포함하는, 당뇨병의 진단을 위한 정보제공방법:Method for providing information for the diagnosis of diabetes, comprising the following steps:(a) 정상인 및 피검자 유래 샘플에서 분리한 소포로부터 DNA를 추출하는 단계;(a) extracting DNA from vesicles isolated from normal and subject derived samples;(b) 상기 추출한 DNA에 대하여 16S rDNA 서열에 존재하는 Escherichia 속 세균 유래 소포 검출 프라이머 쌍을 이용하여 PCR을 수행하여 각각의 PCR 산물을 수득하는 단계; 및(b) performing PCR on the extracted DNA using a pair of Escherichia bacteria-derived vesicle detection primers present in the 16S rDNA sequence to obtain respective PCR products; And(c) 상기 PCR 산물의 정량분석을 통하여 정상인에 비하여 Escherichia 속 세균 유래 소포의 함량이 높을 경우 당뇨병으로 판정하는 단계.(c) Determining diabetes mellitus when the content of the vesicles derived from the genus Escherichia is higher than the normal person through quantitative analysis of the PCR product.
- 제6항에 있어서,The method of claim 6,상기 환자 유래 샘플은 혈액, 소변, 또는 대변인 것을 특징으로 하는, 정보제공방법.The patient-derived sample is characterized in that the blood, urine, or stool.
- 바실러스 속 세균 유래 소포를 유효성분으로 포함하는, 암, 염증질환, 또는 대사질환 예방 또는 치료용 약학적 조성물.Bacillus bacteria-derived vesicles as an active ingredient, cancer, inflammatory diseases, or metabolic diseases preventing or treating pharmaceutical composition.
- 제8항에 있어서,The method of claim 8,상기 암은 간암, 방광암, 유방암, 또는 난소암인 것을 특징으로 하는, 약학적 조성물.The cancer is characterized in that the liver cancer, bladder cancer, breast cancer, or ovarian cancer.
- 제8항에 있어서,The method of claim 8,상기 염증질환은 천식, 만성간염, 탈모, 또는 아토피피부염인 것을 특징으로 하는, 약학적 조성물.The inflammatory disease is asthma, chronic hepatitis, hair loss, or atopic dermatitis, characterized in that the pharmaceutical composition.
- 제8항에 있어서,The method of claim 8,상기 대사질환은 당뇨병 또는 간경화증인 것을 특징으로 하는, 약학적 조성물.The metabolic disease is characterized in that diabetes or cirrhosis of the pharmaceutical composition.
- 제8항에 있어서,The method of claim 8,상기 염증질환은 IL-6 또는 TNF-α에 의해 매개되는 질환인 것을 특징으로 하는, 약학적 조성물.The inflammatory disease is characterized in that the disease mediated by IL-6 or TNF-α, pharmaceutical composition.
- 제12항에 있어서,The method of claim 12,상기 TNF-α에 의해 매개되는 염증질환은 탈모, 류마티스관절염, 또는 염증성장염인 것을 특징으로 하는, 약학적 조성물.The inflammatory disease mediated by TNF-α is characterized in that the hair loss, rheumatoid arthritis, or inflammatory growth inflammation, pharmaceutical composition.
- 제8항에 있어서,The method of claim 8,상기 바실러스 속 세균은 고초균(Bacillus subtilis)인 것을 특징으로 하는, 약학적 조성물.The Bacillus genus bacteria is Bacillus subtilis, characterized in that the pharmaceutical composition.
- 제14항에 있어서,The method of claim 14,상기 고초균(Bacillus subtilis)은 낫토균(Bacillus subtilis natto)인 것을 특징으로 하는, 약학적 조성물.The Bacillus subtilis is characterized in that the Bacillus subtilis natto, pharmaceutical composition.
- 제8항에 있어서,The method of claim 8,상기 소포는 바실러스 속 세균의 배양액에서 분리되는 것을 특징으로 하는, 약학적 조성물. The vesicles are characterized in that separated from the culture medium of bacteria of the genus Bacillus, pharmaceutical composition.
- 제8항에 있어서,The method of claim 8,상기 소포는 바실러스 속 세균을 첨가하여 배양한 식품에서 분리한 것을 특징으로 하는, 약학적 조성물.The vesicles are separated from food cultured by adding bacteria of the genus Bacillus, pharmaceutical composition.
- 제8항에 있어서,The method of claim 8,상기 소포는 평균 직경이 10 내지 1000 nm인 것을 특징으로 하는, 약학적 조성물.The vesicles are characterized in that the average diameter of 10 to 1000 nm, pharmaceutical composition.
- 바실러스 속 세균 유래 소포를 유효성분으로 포함하는, 암, 염증질환, 또는 대사질환 개선용 건강기능성 식품 조성물.A functional food composition for improving cancer, inflammatory disease, or metabolic disease, comprising the bacterium-derived vesicles as an active ingredient.
- 제19항에 있어서,The method of claim 19,상기 암은 간암, 방광암, 유방암, 및 난소암으로 이루어지는 군으로부터 선택되고,; The cancer is selected from the group consisting of liver cancer, bladder cancer, breast cancer, and ovarian cancer;상기 염증질환은 천식, 만성간염, 탈모, 류마티스관절염, 염증성장염, 및 아토피피부염으로 이루어지는 군으로부터 선택되며; The inflammatory disease is selected from the group consisting of asthma, chronic hepatitis, hair loss, rheumatoid arthritis, inflammatory growth inflammation, and atopic dermatitis;상기 대사질환은 당뇨병 또는 간경화증인 것을 특징으로 하는, 건강기능성 식품 조성물.The metabolic disease is characterized in that diabetes or cirrhosis, health functional food composition.
- 제19항에 있어서,The method of claim 19,상기 소포는 바실러스 속 세균의 배양액에서 분리되는 것을 특징으로 하는, 건강기능성 식품 조성물.The vesicles are characterized in that separated from the culture medium of bacteria of the genus Bacillus, health functional food composition.
- 제19항에 있어서,The method of claim 19,상기 소포는 바실러스 속 세균을 첨가하여 배양한 식품에서 분리한 것을 특징으로 하는, 건강기능성 식품 조성물.The vesicles are separated from the food cultured by the addition of bacteria of Bacillus, health functional food composition.
- 제19항에 있어서,The method of claim 19,상기 바실러스 속 세균은 고초균(Bacillus subtilis)인 것을 특징으로 하는, 건강기능성 식품 조성물.The Bacillus genus bacteria is Bacillus subtilis, characterized in that the health functional food composition.
- 제23항에 있어서,The method of claim 23,상기 고초균(Bacillus subtilis)은 고초균 낫토아형 (Bacillus subtilis natto)인 것을 특징으로 하는, 건강기능성 식품 조성물.The Bacillus subtilis is a Bacillus subtilis natto, characterized in that the health functional food composition.
- 바실러스 속 세균 유래 소포를 유효성분으로 포함하는, 염증질환 예방 또는 치료용 흡입제 조성물.Bacillus bacteria-derived vesicles comprising as an active ingredient, inflammatory diseases prevention or treatment inhalation composition.
- 제25항에 있어서,The method of claim 25,상기 바실러스 속 세균은 고초균(Bacillus subtilis)인 것을 특징으로 하는, 흡입제 조성물.The bacterium of the genus Bacillus is characterized in that Bacillus subtilis, inhalant composition.
- 제26항에 있어서,The method of claim 26,상기 고초균(Bacillus subtilis)은 낫토균 (Bacillus subtilis natto)인 것을 특징으로 하는, 흡입제 조성물.The Bacillus subtilis is characterized in that the natto (Bacillus subtilis natto), inhalant composition.
- 바실러스 속 세균 유래 소포를 유효성분으로 포함하는, 염증질환 개선용 화장료 조성물.Bacillus bacteria-derived vesicles comprising as an active ingredient, inflammatory disease cosmetic composition for improving.
- 제28항에 있어서,The method of claim 28,상기 염증질환은 아토피피부염, 탈모, 건선, 지루성피부염, 및 여드름으로 이루어지는 군으로부터 선택되는 질환인 것을 특징으로 하는, 화장료 조성물.The inflammatory disease is characterized in that the disease is selected from the group consisting of atopic dermatitis, hair loss, psoriasis, seborrheic dermatitis, and acne.
- 제28항에 있어서,The method of claim 28,상기 바실러스 속 세균은 고초균(Bacillus subtilis)인 것을 특징으로 하는, 화장료 조성물.The Bacillus genus bacteria is Bacillus subtilis, characterized in that the cosmetic composition.
- 제30항에 있어서,The method of claim 30,상기 고초균(Bacillus subtilis)은 낫토균(Bacillus subtilis natto)인 것을 특징으로 하는, 화장료 조성물.The Bacillus subtilis is characterized in that the Bacillus subtilis natto, cosmetic composition.
- 바실러스 속 세균 유래 소포를 유효성분으로 포함하는 약학적 조성물을 개체에 투여하는 단계를 포함하는 것을 특징으로 하는 암, 염증질환, 또는 대사질환 예방 또는 치료방법.A method for preventing or treating cancer, inflammatory disease, or metabolic disease, comprising administering to a subject a pharmaceutical composition comprising a bacterium-derived bacterium-derived vesicle as an active ingredient.
- 바실러스 속 세균 유래 소포의 암, 염증질환, 또는 대사질환 예방 또는 치료 용도.Use for the prevention or treatment of cancer, inflammatory diseases, or metabolic diseases of vesicles derived from bacteria of Bacillus.
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| EP17839750.1A EP3498285A4 (en) | 2016-08-12 | 2017-08-07 | Nanovesicles derived from genus bacillus bacteria and use thereof |
| US16/325,023 US12312636B2 (en) | 2016-08-12 | 2017-08-07 | Nanovesicles derived from genus Bacillus bacteria and use thereof |
| CN201780049499.7A CN109789173B (en) | 2016-08-12 | 2017-08-07 | Nanovesicles derived from bacillus bacteria and uses thereof |
| JP2019507291A JP6839265B2 (en) | 2016-08-12 | 2017-08-07 | Nanovesicles derived from Bacillus bacteria and their uses |
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| KR20160102650 | 2016-08-12 | ||
| KR10-2016-0102650 | 2016-08-12 | ||
| KR1020170098860A KR102085787B1 (en) | 2016-08-12 | 2017-08-04 | Nanovesicles derived from Bacillus bacteria and Use thereof |
| KR10-2017-0098860 | 2017-08-04 |
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