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WO2004073731A1 - Proteines 14-3-3 pour la prevention et le traitement de troubles fibroproliferatifs - Google Patents

Proteines 14-3-3 pour la prevention et le traitement de troubles fibroproliferatifs Download PDF

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Publication number
WO2004073731A1
WO2004073731A1 PCT/CA2004/000245 CA2004000245W WO2004073731A1 WO 2004073731 A1 WO2004073731 A1 WO 2004073731A1 CA 2004000245 W CA2004000245 W CA 2004000245W WO 2004073731 A1 WO2004073731 A1 WO 2004073731A1
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protein
nucleic acid
treatment
acid encoding
collagen
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PCT/CA2004/000245
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WO2004073731A8 (fr
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Aziz Ghahary
Edward E. Tredget
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Aziz Ghahary
Tredget Edward E
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Priority to EP04712453A priority Critical patent/EP1603586A1/fr
Publication of WO2004073731A1 publication Critical patent/WO2004073731A1/fr
Publication of WO2004073731A8 publication Critical patent/WO2004073731A8/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like

Definitions

  • the present invention relates generally to methods for the prevention and/or treatment of fibroproliferative disorders in a mammal. More particularly, this invention relates to proteins which mediate collagenase expression and content of the tissues and organs of a mammal. It also relates to a method of increasing the collagen content of
  • the skin of a mammal Furthermore it relates to a method for the prevention, alleviation and/or treatment of skin having a low collagen content.
  • the invention also relates to a method of screening for compounds that are capable of increasing the collagen content of the skin, and to compounds identified by said method.
  • the invention also discloses
  • Fibrosis which is a
  • Fibroproliferative disorders are believed to be caused by trauma, surgery, infection environmental pollutants, alcohol and other types of toxins. There are various examples of fibroproliferative disorders, including the formation of scar tissue after a heart attack, which scar tissue impairs the ability of the
  • Fibroproliferative disorders can be acute or chronic, but they always share a common characteristic of excessive collagen accumulation and an associated loss of function when normal tissue is replaced with scar tissue.
  • the acute form of fibrosis is concern, it occurs as a common response to various forms of trauma including burns, surgery severe injuries, infections and radiation treatment.
  • the chronic form is concerned this is believed to be caused by diabetes, hypertension, various viral infections like viral hepatitis which induce a progressive fibrosis which causes a continuous loss of tissue • function.
  • liver, kidney and lung are most affected.
  • the skin has a protective role and provides a barrier between our organs and tissues, giving support, balancing fluid metabolism, regulating temperature etc.
  • the skin's functions are manifold, and therefore an intact integument is necessary so that these functions can be fulfilled.
  • burn injuries demand medical care, and approximately three quarters of a million people suffer every year from diabetic leg ulcers, thus creating a large market for replacement skin grafts.
  • the skin comprises dermal and epidermal layers each of them being derived from different stem cells, with the former being formed by fibroblasts and the latter being a product of the differentiation of keratinocytes.
  • fibroblasts produce collagen and elastin that serve as a foundation upon which keratinocytes differentiate into the epidermal layer.
  • the epidermis is the thin outer layer of the skin, which itself consists of the following three parts; the stratum corneum which consists of fully mature keratinocytes which contain fibrous proteins (keratins). The outermost layer is continuously shed. The stratum corneum prevents the entry of most foreign substances as well as the loss of fluid from the body.
  • the layer of keratinocytes (squamous cells), which, as the name implies, contains living keratinocytes (squamous cells), which mature and form the stratum corneum.
  • the deepest layer of the epidermis is the basal layer which contains basal cells. Basal cells continually divide, forming new keratinocytes, replacing the old ones that are shed from the skin's surface.
  • the epidermis also contains melanocytes which are usually evenly dispersed and which are cells that produce melanin.
  • the dermis which is the middle layer of the skin.
  • the dermis contains blood vessels, fibroblasts, hair follicles, sweat glands, lymph vessels, collagen bundles and nerves.
  • the dermis is held together by collagen which in turn is produced by fibroblasts.
  • the dermis also contains pain and contact receptors.
  • the subcutis consists of a network of collagen and fat cells and it helps to conserve the body's heat. Furthermore it protects the body from injury by acting as a "shock absorber". In a, human population there are both genetic and cultural differences as to the thickness of the subcutis layer.
  • Keloids are dermal fibrotic lesions that are a variation of the normal wound healing process. They usually occur during the healing of a deep skin wound.
  • hypertrophic scars and keloids are included along a spectrum of the above mentioned fibroproliferative disorders. These abnormal scars appear to result from the loss of the control mechanisms that normally regulate the fine balance of tissue repair and regeneration. The excessive proliferation of normal tissue healing processes results in both hypertrophic scars and keloids. The production of extracellular matrix proteins, collagen, elastin and proteoglycanes is believed to be due to a prolonged inflammatory process in the wound. Hypertrophic scars are raised, erythematous, fibrotic lesions, that usually remain confined within the borders of the original wound. These scars occur within months of the initial trauma and have a tendency to remain stable or regressive with time.
  • Keloid formation can occur within a year after injury, and keloids enlarge well beyond the original scar margin.
  • the most frequently involved sites of keloids are areas of the body that constantly are subjected to high skin tension. Wounds on the anterior chest, shoulders, flexor surfaces of the extremities, anterior neck and wounds that cross skin tension lines are more susceptible to abnormal scar formation.
  • HLA human leukocyte antigen
  • collagen synthesis in keloids is three times greater than in hypertrophic scars and believed to be twenty times greater than in normal scars.
  • Type III collagen, chondroitin-4-sulfate and glycosaminoglycan contents is higher in keloids than in both hypertrophic and normal scars.
  • Collagen cross linking is greater in normal scars, while keloids have immature cross links that do not form normal scar stability.
  • the increased number of fibroblasts, which are recruited to the site of tissue damage, synthesize an overabundance of fibronectin, and receptor expression is increased in keloids.
  • Mast cell population within keloid scars is also increased and subsequently an increase in histamin production occurs. Growth factors and cytokines are involved intimately in a cycle of wound healing.
  • IFN-alpha, IFN-beta and IFN-gamma reduce fibroblasts synthesis of collagen types I, III and possibly VI.
  • a relationship between irnmunoglobulines and keloid formation appears to exist, while levels of immunoglobulin IgG and IgM are normal in a serum of patients with keloids, the concentration of IgG in the scar tissues elevated in comparison to hypertrophic and normal scar tissue.
  • Scars contain few cells and blood vessels, but high amounts of connective tissue. Scars not only present a severe cosmetic problem, but also affect the function of the skin: the elasticity of scars is greatly reduced compared to healthy skin due to the minor quality of collagen fibers; also appendages like sweat glands, hair follicles and melanocytes are missing. Therefore the scarred skin cannot fulfill its normal function as a barrier for both fluid metabolism and heat loss. This leads to severe problems of the affected individuals when the affected skin is exposed for example to mechanical stress or extreme temperatures. Currently, no therapy exists which reliably prevents or treats hypertrophic scar formation and keloids. Usually scars are treated mechanically by excision or by laser or cooling techniques; however these techniques again lead to wounds and eventually scars.
  • collagen or hyaluronic acid may be injected into scar tissue to make the scar surface more even.
  • the material is reabsorbed within months, and the treatment has to be repeated every 6- 12 months.
  • this is mainly a cosmetic treatment ameliorating scar appearance.
  • No single therapy is rated experimentally to be the most effective form of treating keloid scars.
  • keloids have been treated by occlusive dressings, including silicone gel sheets and silicone occlusive dressings which can be worn for as long as 24 hours per day for up to one year. The silicone does not appear to enter the skin, and the antikeloid effect appears to be secondary to both occlusion and hydration.
  • keloids have been treated by mechanical compression dressings, in particular in relation to ear lobe keloids.
  • keloids have been treated by intralesional steroid injections which apparently act by diminishing collagen synthesis, decreasing mucinous ground substance. There are the usual adverse side effects of prolonged treatment with steroids, including atrophy of the skin or subcutaneous tissue, hypopigmentation, necrosis, ulceration, etc. Keloids have also been treated by additional excision surgery which is usually combined with other kinds of therapy like external radiation, steroid injection and/or pressure therapy.
  • Cryosurgery has also been applied, using liquid nitrogen to cause cell damage and to affect the microvasculature which causes subsequent stasis, thrombosis and transudation of fluid that result in cells anoxia.
  • keloids have also been treated by using intralesional injections of IFN-alpha, IFN-beta and IFN-gamma.
  • Stratifin which is also called 14-3-3 sigma, is a 28kDa protein which is known to be involved in cell cycle progression and cell cycle control as an intracellular protein (see for example Prasad et al., 1992, Cell Growth
  • the present invention reduces the difficulties and disadvantages of the prior art by providing a method for the prevention and/or treatment of fibroproliferative disorders in a mammal, comprising administering an effective amount of a 14-3-3 protein, an active fragment thereof, or a functional analogue thereof; or of a nucleic acid encoding for said protein in order to increase collagenase.
  • the present invention also provides a method for increasing the collagen content in the skin by reducing the collagenase content in a tissue or organ.
  • a method for the prevention and/or treatment of a fibroproliferative disorder in a mammal comprising administering an effective amount of a 14-3-3 protein, any active 14-3-3 fragment thereof, or a functional analogue thereof, or of a nucleic acid encoding for said protein to said mammal.
  • a method of increasing collagen content of the skin and/or for the prevention, alleviation and/or treatment of skin having a low collagen content comprising administering an antibody or binding fragment thereof, wherein the antibody or fragment specifically binds to a 14-3-3 protein, its receptors, or its fragments, or administering a nucleic acid that is antisense to a nucleic acid encoding for said mammalian 14-3-3 protein or administering siRNA directed towards a nucleic acid encoding for a 14-3- 3- protein.
  • a method of screening for compounds that are capable of increasing the collagen content of the skin comprising:
  • a mammalian 14-3-3 protein in particular a mammalian 14-3-3 protein as represented by SEQ ID NO: 1 or 2, or an active fragment thereof, or a functional analogue thereof,
  • an active compound being a compound that reduces/inhibits expression or secretion of said protein, or a compound that reduces/inhibits expression of collagenase.
  • an active compound being a compound that reduces/inhibits expression or secretion of said protein, or a compound that reduces/inhibits expression of collagenase.
  • an antibody or a binding fragment thereof which can specifically bind to a mammalian 14-3-3 protein, its fragments, or its receptors, or of a siRNA directed towards a nucleic acid encoding for a mammalian 14-3-3 protein, or a nucleic acid that is antisense to a nucleic acid encoding for a mammalian 14-3-3 protein to increase the collagen content of a mammal's skin.
  • a 14-3-3 protein any active fragment thereof, or a functional analogue thereof; or a nucleic acid encoding for a 14-3-3 protein in the manufacture of a medicament for the treatment of fibroproliferative disorders.
  • an antibody or a binding fragment thereof which can specifically bind to a mammalian 14-3-3 protein, or of a siRNA directed towards a nucleic acid encoding for a mammalian 14-3-3 protein, or a nucleic acid that is antisense to a nucleic acid encoding for a mammalian 14-3-3 protein, in the manufacture of a medicament for the treatment of a condition associated with a decreased level of collagen.
  • the invention provides the use of a mixture of a pharmaceutically acceptable weak acid or a derivative thereof, an example of which is acetylsalicylic acid and a 14-3-3 protein, any active fragment thereof, or a functional analogue thereof; or a nucleic acid encoding for a 14-3-3 protein in the manufacture of a medicament for the treatment of fibroproliferative disorder.
  • the invention provides a pharmaceutically acceptable weak acid or a derivative thereof, an example of which is acetylsalicylic acid and a 14-3-3 protein, any active fragment thereof, or a functional analogue thereof; or a nucleic acid encoding for a 14-3-3 protein together with a pharmaceutically acceptable carrier therefor.
  • a pharmaceutically acceptable weak acid or a derivative thereof an example of which is acetylsalicylic acid and a 14-3-3 protein, any active fragment thereof, or a functional analogue thereof; or a nucleic acid encoding for a 14-3-3 protein together with a pharmaceutically acceptable carrier therefor.
  • Derivatives of the weak acids may encompass any pharmaceutically acceptable ester or salt or other suitable derivative.
  • the ratio of a pharmaceutically acceptable weak acid or a derivative thereof, an example of which is acetylsalicylic acid and a 14-3-3 protein, any active fragment thereof, or a functional analogue thereof; or a nucleic acid encoding for a 14-3-3 protein may range from 1 :1 to 1 :1000 on a dry weight basis mixed in cream or solution. A mixture in a 1 :1 ratio has been found to be effective when the weak acid is acetylsalicylic acid.
  • the mixture provides complimentary benefits for the proposed treatment since it not only suppresses the expression of collagen, but also significantly increases the expression of collagenase.
  • the 14-3-3 protein, any active fragment thereof, or a functional analogue thereof; or a nucleic acid encoding for a 14-3-3 protein may be used in implantable medical devices, such as, for example stents used in angioplasty or for other therapeutic purposes to prevent fibroproliferative disorders in patients. Combinations with the weak acids mentioned above are also envisaged for such purposes.
  • the protein, etc. may be coated onto the implant or otherwise incorporated into it in a suitable manner as would be apparent to a person skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS Having thus generally described the invention, reference with now be made to the accompanying drawings, showing by way of illustration preferred embodiments thereof.
  • Fig. 1 illustrates the expression of collagenase by fibroblasts co- cultured with human dermal keratinocyles.
  • Fig. 2 illustrates the expression of collagenase mRNA and 18S ribosomal RNA by dermal fibroblasts which received various percentages of keratinocyte conditioned medium.
  • Fig. 3 illustrates the expression of collagenase and 18 S ribosomal RNA by dermal fibroblasts which have received keratinocyte condition medium from keratinocytes grown in test medium at the indicated time intervals.
  • Fig. 4 illustrates the results of an activity test of protein that is present in keratinocyte conditioned medium and that has been precipitated by ammonium sulfate.
  • Fig. 5 illustrates a section of a dermal/epidermal skin substitute stained with H Si E (panel A); panel B shows the expression of collagenase and 18 S ribosomal RNA from fibroblasts in a collagen matrix, keratinocytes in the epidermal layer and fibroblasts alone on plastic.
  • Fig. 6 illustrates collagenase activity in keratinocyte conditioned medium.
  • Fig. 7 shows an SDS-PAGE gel showing the purification of stratifin.
  • Fig. 8 illustrates the effects of various doses of stratifin on the collagenase mRNA expression in fibroblasts.
  • Fig. 9 illustrates that the KDAF (Stratifin) collagenase stimulatory effect is not permanent.
  • Fig. 10 illustrates that Keratinocyte conditioned medium contains a collagenase stimulating factor that increases the expression of collagenase in a time dependent fashion.
  • Fig. 11 illustrates that that ⁇ / ⁇ form of protein 14-3-3 has also collagenase stimulatory effect in fibroblasts.
  • Fig. 12 illustrates collagenase stimulatory effects of RNA by Northern Analysis.
  • Fig. 13A illustrates an analysis of KDAF protein fragments on native gel (upper panel) and an analysis of KDAF protein fragments on SDS-Page (lower panel).
  • Fig. 13B illustrates that deletion of up to 75 amino acids is not critical to the KDAF collagenase stimulatory effect.
  • Figs. 14A and B illustrate Northern Analysis using cDNA probe for collagenase (top panel) or 185 ribosomal RNA (bottom panel).
  • Fig. 15 illustrates evaluation of proteins if fractions were run in a
  • Fig. 17 illustrates the histological appearance of KDAF in treated and untreated healing wounds in rats.
  • Fig. 18 illustrates how the combination of KDAF and acetylsalicylic acid suppresses the expression of collagen and increases the expression of collagenase.
  • 14-3-3 protein is meant to designate any protein belonging to the family of 14-3-3 proteins. These proteins in general have a core region encoding an amphipathic groove that binds a multitude of client proteins that have conserved 14-3-3 recognition sequences. The amino and carboxy termini of 14-3-3 proteins are much more diverse than the core region and may interact with isoform-specific client proteins and/or confer specialized sub-cellular and tissue localisation.
  • 14-3-3 proteins have so far only been found in eukaryotes, in different isoforms including, beta/alpha, gamma, epsilon, eta, sigma, tau/theta and zeta/delta (Yaffe MB, FEBS Letters 513: 53-57, 2002). They also have been found in yeasts, Drosophila and plants (Martens GJM, Piosik PA, Danen EHJ, Evolutionary Conservation of the 14-3-3 protein, Biochem. Biophy. Res. Comm. 184:1456-1459, 1992).
  • the term "functional analogue" of a protein is meant to designate a protein that carries out the same essential function as a 14- 3-3 protein without necessarily having the same sequence. This includes active fragments and variants of a 14-3-3 protein. Variants include deletion and/or substitution of at least one amino acid of the protein, e.g., by manipulation of the nucleic acid encoding the protein or by altering the protein itself. An active fragment or portion of the protein means a stretch of amino acid residues of sufficient length or having amino acids deleted therein, while retaining its essential function.
  • the present invention provides methods for the prevention and/or treatment of fibroproliferative disorders in a mammal. More particularly, this invention provides proteins, nucleic acids, and antibodies, which mediate collagen expression and content of the tissues and organs of a mammal. It also relates to a method of increasing the collagen content of the skin of a mammal. Furthermore it provides a method for the prevention, alleviation and/or treatment of skin having a low collagen content, such as those of non-healing wounds. The invention also provides a method of screening for compounds that are capable of increasing the collagen content of the skin, and to compounds identified by said method.
  • the 14-3-3 protein stratifin, which can also be referred to as "KDAF” or "Keratinocyte Derived Anti-fibrogenic Factor”
  • KDAF Keratinocyte Derived Anti-fibrogenic Factor
  • Stratifin is naturally released from human skin cells and its potency is more than 20 fold higher than a well known anti-fibrogenic cytokine, such as IFN-alpha2b.
  • Stratifin is thus an anti-fibrogenic factor for treatment of fibroproliferative disorders, especially fibrosis disorders, e.g.
  • KDAF is a natural Ketatinocyte releasable protein whose collagenase stimulating effects on dermal fibroblasts has been confirmed in a dose and time dependent fashion, and as such is believed to function as a wound healing stopping signal after wound re-epithelealgation.
  • a method for the prevention and/or treatment of a fibroproliferative disorder in a mammal comprising administering an effective amount of a 14-3-3 protein or a functional analogue thereof, or an active fragment thereof, or a nucleic acid encoding for such a protein.
  • a 14-3-3 protein or a functional analogue thereof, or an active fragment thereof, or a nucleic acid encoding for such a protein is likewise provided.
  • the 14-3-3 protein is a mammalian protein, preferably of human origin.
  • the protein is an isoforrn of 14-3-3 sigma protein, including (see page 12, lines 18 and 19) isoforms found in human, as well as other forms found in plants, yeast and fungi as a monomer, homodimer, heterodimer.
  • the protein is any other form that can function as a collagenase stimulating factor as can be determined by methods as set out in the examples below.
  • the protein comprises a fragment of a 14-3-3 protein, or an isoforrn thereof, that functions as a collagenase stimulating factor as can be determined by methods as set out in the examples below.
  • proteins and fragments can be naturally or synthetically produced using conventional methods well known to those skilled in the art. Synthetic production of the proteins or fragments thereof can provide an inexpensive, safe and simple method of producing the desired protein or fragment thereof . Fragments may be administered via typical routes, including topically and systemically.
  • the 14-3-3 protein is a sigma 14-3-3 protein having a sequence as represented by SEQ ID NO: 1 as follows: SEQ ID NO: 1
  • the mammalian 14-3-3 protein is an ⁇ / ⁇ 14-3-3 protein having a sequence as represented by SEQ ID No: 2 as follows:
  • nucleic acid encoding for said sigma 14-3-3 protein has a sequence as represented by SEQ ID NO: 3 as follows:
  • nucleic acid encoding for said 14-3-3 alpha/beta protein has a sequence as represented by SEQ ID NO: 4 as follows:
  • Seq. ID NO. 4 14-3-3 protein beta/alpha
  • the fibroproliferative disorder is associated with an increased deposition of collagen and/or other extracellular matrix proteins.
  • the fibroproliferative disorder is selected from the group comprising fibrosis disorders, in particular fibrosis of liver, kidney, lung, bowel, heart, pancreas, peritoneum, skin, and keloid formation and hypertrophic scarring.
  • the fibroproliferative disorder is selected from the group comprising hypertrophic scarring and keloid formation, wherein more preferably, the hypertropic scarring is due to surgery, thermal injury and deep trauma.
  • the disorder is a thickening of airways/airway fibrosis seen in asthma patients that is associated with accumulation of ECM (namely collagen).
  • a 14-3-3 protein, derivatives and fragments thereof, and a cDNA in a form of sense and anti-sense in treating asthma patients to reduce airway fibrosis.
  • the extracellular matrix proteins may be selected from the group comprising fibrin, elastin, proteoglycans, keratin, collagens, decorin, fibronectin, and MM PS).
  • the mammalian 14-3-3 protein or its functional analogues is recombinantly produced or isolated from tissue and/or body fluids.
  • the recombinantly produced mammalian 14-3-3 protein is a fusion protein comprising 14-3-3 protein and a fusion peptide.
  • the recombinantly produced mammalian 14-3-3 protein is 14-3-3 protein devoid of any fusion peptide.
  • administering occurs prior to the fusion peptide has been cleaved off, or administering occurs without cleaving off the fusion peptide.
  • the fusion peptide may have a sequence comprising RGD.
  • administering occurs by topical application of the protein, topical and/or systemic injection of the protein and/or expression of the protein from the nucleic acid encoding for it, wherein preferably, for expression of the protein from the nucleic acid encoding for it, the nucleic acid is brought into a cell by a process selected from the group comprising transfection, transformation, electroporation, and DMA- injection.
  • the topical injection is a subcutaneous injection, in particular an intradermal injection.
  • administering occurs by application of specific cells expressing and secreting a mammalian 14-3-3 protein, in particular a mammalian 14-3-3 protein as represented by SEQ ID No: 1 or 2, and/or its functional analogues.
  • the mammal may be a human being.
  • a method of increasing collagen content of the skin and/or for the prevention, alleviation and/or treatment of skin having a low collagen content comprising administering an antibody or binding fragment thereof, wherein the antibody or fragment specifically binds to a 14-3-3 protein, its receptor, or an active fragment thereof, or administering a nucleic acid that is antisense to a nucleic acid encoding for said 14-3-3 protein or administering siRNA directed towards a nucleic acid encoding for a 14-3- 3- protein.
  • an antibody or a binding fragment thereof which can specifically bind to a mammalian 14-3-3 protein, or of a siRNA directed towards a nucleic acid encoding for a mammalian 14-3-3 protein, or a nucleic acid that is antisense to a nucleic acid encoding for a mammalian 14-3-3 protein to increase the collagen content of a mammal's skin is likewise provided.
  • KDAF neutralizing/blocking compounds are likewise useful in the treatment of collagenase-induced non-healing wounds, collagenase induced nerve degenerative disorders and neuronal damaging in the brain and spinal chord.
  • the mammalian 14-3-3 protein is selected from the group comprising 14-3-3 beta/alpha protein, 14-3-3 epsilon protein, 14- 3-3 eta protein, 14-3-3 sigma protein, 14-3-3 tau/theta protein and 14-3-3 zeta/delta protein.
  • the mammalian 14-3-3 protein is a human 14-3-3 sigma protein.
  • the mammalian 14-3-3 protein has a sequence as represented by SEQ ID NO: 1.
  • the 14-3-3 protein has a sequence as represented by SEQ ID. NO. 2.
  • nucleic acid encoding for said mammalian 14-3-3 protein has a sequence as represented by SEQ ID NO: 3. In another embodiment, the nucleic acid encoding for said 14-3-3 protein has a sequence as represented by SEQ ID. No. 4.
  • the skin is ageing and/or wrinkled skin, for example caused by radiation treatment.
  • the mammal is a human being. It has also surprisingly been found that cells, which express or which express and secrete a mammalian 14-3-3 protein can be used for screening methods directed at identifying compounds that are capable of increasing the collagen content of the skin. To this end, these cells are incubated with a potentially active candidate compound whereafter the expression and/or secretion of the 14-3-3 protein is measured.
  • a 14-3-3 protein is incubated together with cells expressing collagenase, in combination with a potentially active compound, whereafter the expression of collagenase is measured.
  • an active compound "active” meaning it will increase the collagen content of the skin, reduces/inhibits the expression, or secretion of the 14-3-3 protein whereby the induction of collagenase expression is reduced.
  • a potentially active compound is incubated together with a 14-3-3 protein and cells expressing collagenase, whereupon the expression of collagenase is measured.
  • the potentially compound is an active compound it will interfere with the stimulating effect that the 14-3-3 protein has on the expression of collagenase.
  • the result will be that such a compound has the effect that the collagen content of an association of cells, for example the skin, will be increased.
  • a method of screening for compounds that are capable of increasing the collagen content of the skin comprising: - providing cells expressing or expressing and secreting a mammalian 14-3-3 protein, in particular a mammalian 14-3-3 protein as represented by SEQ ID NO: 1 , or SEQ ID. NO. 2, or a functional analogue thereof, or an active fragment thereof,
  • a mammalian 14-3-3 protein in particular a mammalian 14-3-3 protein as represented by SEQ ID. NO: 1 , or SEQ ID. NO. 2, or a functional analogue thereof, or an active fragment thereof, - incubating said protein with cells expressing collagenase, together with a potentially active compound,
  • an active compound being a compound that reduces/inhibits expression or secretion of said protein, or a compound that reduces/inhibits expression of collagenase.
  • the cells expressing or expressing and secreting a mammalian 14-3-3 protein, or a functional analogue thereof are keratinocytes and epithelial cells.
  • the cells expressing collagenase may be fibroblasts.
  • 14-3-3 proteins and their analogues and nucleic acid sequences can be used for the manufacture of a medicament for the treatment of fibroproliferative disorders, in particular fibrosis of various internal organs, including liver, kidney, lung, bowel, heart, pancreas, fibrosis of vessels and airways, peritoneum, skin, and for the treatment of kiloid formation and hypertrophic scaring.
  • the 14- 3-3 protein is topically or systemically administered for example by injection into the skin, preferably by subcutaneous injection, even more preferably by intradermal injection.
  • the 14-3-3 protein can be recombinantly produced or it can be isolated from tissue and/or body fluids.
  • recombinantly expressed it can be expressed in eukaryotes and/or prokaryotic cells, in particular in yeast and/or E. coli.
  • recombinantly expressed it can be expressed as a fusion protein, for example a GST-fusion protein, wherein the fusion peptide has the function of facilitating the purification protocol and, after purification, is cleaved off.
  • the fusion peptide may serve the purpose of targeting the 14- 3-3 protein to which it is fused to its particular site of action.
  • a fusion peptide, containing the sequence of an active fragment of the 14-3- 3 protein may serve the purpose of facilitating the binding and/or uptake by fibroblasts.
  • an antibody or a binding fragment thereof which can specifically bind to a mammalian 14-3-3 protein, or of a siRNA directed towards a nucleic acid encoding for a mammalian 14-3-3 protein, or a nucleic acid that is antisense to a nucleic acid encoding for a mammalian 14-3-3 protein, can be utilized in the manufacture of a medicament for the treatment of a condition associated with a decreased level of collagen.
  • fetal foreskin or normal skin punch biopsies obtained from adult patients undergoing elective reconstructive surgery, were collected individually and washed three times in sterile Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, New York) supplemented with an antibiotic-antimycotic preparation (100 ⁇ g/ml penicillin, 100 ⁇ g/ml streptomycin, 0.25 ⁇ g/ml amphotericin B) (Gibco).
  • DMEM Dulbecco's modified Eagle's medium
  • an antibiotic-antimycotic preparation 100 ⁇ g/ml penicillin, 100 ⁇ g/ml streptomycin, 0.25 ⁇ g/ml amphotericin B
  • the cells Upon reaching confluence, the cells were released by trypsinization, split for subculture at a ratio of 1 : ⁇ , and reseeded into 75-crn ⁇ flasks. Fibroblasts at passages 3-7 were used in fibroblasts/keratinocyte co- culture system.
  • GITC guanidinium thiocyanate
  • Collagenase Activity To determine the effects of KDAF on collagenase activity, conditioned medium from either keratinocytes alone, fibroblasts alone or fibroblasts co-cultured with keratinocytes was replaced with serum-free medium supplemented with 25 u/ml conconavalin A (to increase enzyme production) and 0.5%(w/v) lactalbumin hydrolysate (Dibco). After 2 days the conditioned medium from each experimental condition was collected, centrifuged at 1000X g for 10 min, and stored at 4°C. Collagenase assay was carried out as described elsewhere (Reference 5) with slight modifications.
  • Proteins in conditioned medium were precipitated by ammonium sulphate and the precipitates collected by centrifugation, dissolved in assay buffer (0.05M Tris, 0.2M NaCI, 0.005M CaCI 2 , 0.02% sodium azide, pH 7.4) and then dialyzed overnight against 4 litters of the same buffer. The final volume of each sample was adjusted according to the cell number.
  • Procollagenase was activated proteolytically with trypsin (10 ⁇ g/ml) and soybean trypsin inhibitor (100 ⁇ g/ml) was used to inactivate the trypsin.
  • Acetic acid soluble collagen 50 ⁇ g in 25 ul
  • bovine skin was incubated with the activated enzyme solution in the presence of 1 M glucose for 15-24 hrs.
  • Keratinocyte Derived-Anti- Fibrogenic Factor As the factor of interest present in the KCM possesses a collagenase stimulating activity in fibroblasts, which favours resolution of fibrogenic condition such as hypertrophic scarring, we also refer to this protein as a Keratinocyte Derived Anti-fibrogenic Factor ("KDAF").
  • KDAF Keratinocyte Derived Anti-fibrogenic Factor
  • keratinocyte/fibroblast co-culture system in which keratinocytes and fibroblasts are grown in the upper and lower chambers of this system, respectively.
  • fibroblasts in the lower chamber can be exposed to any soluble factor, which may release from keratinocytes.
  • total RNA was extracted from fibroblasts grown in the lower chamber and the expression of collagenase mRNA was evaluated by Northern analysis.
  • FIG. 1 illustrates the expression of collagenase by fibroblast with human dermal keratinocytes.
  • Lane 1 RNA from keratinocytes is shown.
  • Lane 2 RNA from fibroblasts co-cultured with keratinocytes is indicated.
  • Lane 3 RNA from fibroblasts cultured alone is shown.
  • collagenase mRNA expression is significantly increased in fibroblasts grown in this system relative to fibroblasts grown either alone or in fibroblast/fibroblast co-culture system.
  • keratinocyte conditioned medium (KCM) at different time interval following an addition of test medium (49% serum free medium + 49% DMEM and 2% Fetal bovine serum) was also evaluated.
  • Fig. 3 illustrates the expression of collagenase and 18S ribosomal RNA (control) by dermal fibroblasts received keratinocyte conditioned media from keratinocytes grown in test media at the indicated time intervals. The results shown in Fig. 3 indicate that test medium induced keratinocyte differentiation increases the expression of KDAF at the later time points of differentiation. This activity was also found in both 65 and 95% ammonium sulphate precipitable proteins present in keratinocyte conditioned medium as is illustrated in Fig. 4.
  • FIG. 4 illustrates that KDAF present in keratinocyte-conditioned medium can be precipitated by ammonium sulfate.
  • KCM was precipitated by either 65 or 95% ammonium sulfate and protein concentration was determined.
  • Dermal fibroblasts were then treated with KCM (Lane 1 , positive control), non-conditioned medium (NCM) (Lane 2, negative control), either 10 (Lanes 3 and 5) or 100 (Lanes 4 and 6) ⁇ g of precipitated proteins for 24 h.
  • Total RNA was then extracted individually and subjected for dot blot analysis. The blots were initially hybridized with collagenase cDNA and subsequently with cDNA specific for 18 S ribosomal RNA used as a control for RNA loading.
  • Fig. 5 shows a dermal-epidermal skin substitute prepared from keratinocyte/fibroblasts collagen-GAG gel.
  • Panel A shows a section of dermal-epidermal skin substitute stained with H&E .
  • Panel B shows expression of collagenase and 18 S ribosomal RNA from fibroblasts in collagen matrix (lane 1 ), keratinocytes in epidermal layer (lane B) and fibroblasts alone on plastic (lane 3). This finding shows that KDAF is functional on fibroblasts grown in a three dimensional environment. The collagenase expression in fibroblasts populated collagen gel in the presence and absence of epidermis was then evaluated. As shown in Fig 5, the expression of collagenase mRNA significantly increased only in those fibroblasts that were exposed to keratinocytes as an epidermal layer.
  • KADF significantly breaks down type I collagen to its 1/4 and 3/4 fragments of the ⁇ ( ⁇ 1.1 and ⁇ 1.2) and ⁇ ( ⁇ 1.1 and ⁇ 1.2) chains of type I collagen which was used as an index for collagenase activity.
  • Collagenase activity in conditioned medium obtained either from keratinocytes alone (K), fibroblasts alone (F) or keratinocyte/fibroblast (K/F) co-culture was evaluated in triplicate using type I collagen as a substrate.
  • Type I collagen with no treatment (collagen) was also included as a control.
  • KCM was passed through 50 and 30 kDa cut-off filters of the Centricon Tubes (Amicon) and portions of unfiltered material, filtrate and retentate were individually analyzed.
  • 50 kDa cut-off filters were used, collagenase stimulating activity was found in the unfiltered KCM, retentate and filtrate treated fibroblasts relative to the corresponding samples obtained from non-conditioned medium (Data not shown).
  • Collagenase stimulatory effect of KDAF was not due to RNA loading as the pattern of 18S ribosomal RNA was relatively the same in all samples (data not shown).
  • Matrix assisted laser adsorption mass spectrometry (MALDI MS) and matrix assisted laser adsorption/ionization post source decay mass spectrometry (MALDI PSD MS) were performed on a Voyager Elite MALDI MS instrument (Voyager Elite, PerSeptive Biosystem, Inc., Framingham, MA) equipped with a delayed extraction (DE) device.
  • MALDI MS Matrix assisted laser adsorption mass spectrometry
  • MALDI PSD MS matrix assisted laser adsorption/ionization post source decay mass spectrometry
  • a two-layer method was used for MALDI MS analysis in which 1 to 2 ul of first layer solution [10 mg/ml of 4-hydroxy-alpha-cyanocinnamic acid (HCCA) per mL of 20% methanol/acetone (v/v)] was deposited onto a probe tip, and evaporated to form a thin matrix layer, and then 0.5 - 1 uL of gel extract from 50% acetonitrile or 40% methanol saturated by HCCA was deposited onto the first layer, allowed to air dry, and washed three times with water. The PSD spectra were recorded in the PSD mode of the Voyager Elite instrument.
  • HCCA 4-hydroxy-alpha-cyanocinnamic acid
  • Nanoelectrospray (NanoES) ion trap MS was performed on an Esquire-LC ion trap spectrometer (Hewlett-Packard, Reno, NV) with NanoES interface. Spectra were acquired over the mass range 200 to 2200 Da.
  • Samples from candidate protein bands were prepared according to ACB (Alberta Cancer Board) Proteomics Resource Laboratory's protocols (Karimi-Busheri et al, J. Cell. Biochem, 64:258-272, 1997]. In brief, these protocols include washing, reduction, alkylation, tryptic digestion and extraction of tryptic peptides from the gel spots.
  • Peptide extracts were analyzed on a Bruker REFLEX III (Bremen/Leipzig, Germany, Serial#: FM 2413) time of flight mass spectrometer using MALDI in positive ion mode. Obtained peptide maps were used for database searching to identify proteins. Furthermore, for each sample 1-3 selected peptides were fragmented using MALDI MS/MS analysis done on a PE Sciex API-QSTAR pulsar (MDS-Sciex, Toronto, Ontario, Canada, Serial# K0940105). The obtained partial sequence information for each peptide was used to either confirm or correct the previously obtained results from the peptide map search.
  • KDAF sigma 14-3- 3 protein sigma isoforrn
  • total RNA was prepared by the acid- guanidium-phenol-chloroform method from either human keratinocytes (for KDAF sigma) or fibroblasts (for 14-3-3 protein ⁇ / ⁇ which we now refer as KDAF ⁇ / ⁇ ).
  • KDAF ⁇ / ⁇ human keratinocytes
  • fibroblasts for 14-3-3 protein ⁇ / ⁇ which we now refer as KDAF ⁇ / ⁇
  • Each cDNA was then synthesized with oligo (dt) primer and MMLV reverse transcriptase (Gibco-BRL)). Samples were then incubated at 42°C for 60 minutes, and the reaction was terminated by heating at 70°C for 15 minutes and followed by rapid chilling on ice.
  • PCR reaction was carried out with either KDAF- ⁇ primer sense: 5'-
  • DNA in agarose gel was purified with QIAEX II gel extraction kit according to manufacturer's instructions (Qiagen). Purified DNA was then digested with either ECoRI/Xhol (for KDAF- ⁇ ) or BamHI/Xhol (for KDAF ⁇ / ⁇ ) for 2 hours at 37°C. The digested products were separated by electrophoresis on 1 % agarose gel and specific DNA band related to either KDAF- ⁇ or KDAF- ⁇ / ⁇ was purified by QIAEX II gel extraction kit. Finally, the purified DNA was ligated into a pGEX-6P-1 expressing vector using GST protein (Amersham/Pharmacia Biotech).
  • the ligated products were transformed to competent XL0blue-1 cells with regular heat shock transformation method. Positive clones were identified by the size of restriction enzyme digested products. DNA sequence was confirmed by fluorescence dNTP sequence analysis. The plasmid DNA containing either KDAF- ⁇ or KDAF- ⁇ / ⁇ was transformed into protein expressing bacteria BL-21 (DE3) (Novagene).
  • single positive clone was grown in 100 ml of LB medium containing 50 ⁇ g/ml of ampicillin for 4-6 hours at 29°C until an OD ⁇ oonm of 0.4-0.6 was reached. Bacteria were then diluted to 1 :10 with fresh LB medium grown in the presence of 0.1 mM of IPTG for 24 hrs.
  • Lane 7A shows pre-stained SDS-PAGE standards (low range); Lane 1 shows GST-sigma KDAF expressed in BL-21 -DE3 cells; Lane 2 shows extraction of GST-KDAF expressed in BL-21 -DE3 cells; Lane 3 shows purified GST-KDAF fusion protein by Glutathione affinity purification; Lane 4 shows purified KDAF.
  • Lane M shows pre- stained SDS-PAGE standards (low range); Lane 1 shows GST-KDAF expressed in BL-21 -DE3 cells; Lane 2 shows extraction of GST-KDAF expressed in BL-21 -DE3 cells; Lane 3 shows purified GST- ⁇ / ⁇ KDAF fusion protein by Glutathione affinity purification; Lane 4 shows purified ⁇ / ⁇ KDAF Example 9
  • fibroblasts were treated with keratinocyte conditioned medium (KCM), DMEM +2%FBS (C), or 0.01 , 0.1 , 0.5, 1.0, 2.5, 5.0 (lanes 1-6) ⁇ g of purified recombinant KDAF for 24 hrs.
  • KCM keratinocyte conditioned medium
  • C DMEM +2%FBS
  • Total RNA was extracted and collagenase mRNA was detected by Northern analysis.
  • KDAF increased the expression of collagenase mRNA more than 10 fold relative to that of control.
  • time dependent keratinocyte conditioned medium induced the expression of collagenase mRNA.
  • Dermal fibroblasts were treated with conditioned medium collected from keratinocyte grown in 50/50 medium and cells were harvested at indicated time intervals and collagenase mRNA was evaluated by Northern analysis. Pattern of 18 S shows that this increase is not due to RNA loading.
  • KCM increased the collagenase mRNA expression in fibroblast as early as 12 hr post treatment and its level reached plateau within 24 hrs.
  • Fig. 11 using the same validation assay, it was also found the KDAF ⁇ / ⁇ increases the expression of collagenase mRNA.
  • a dose dependent ⁇ / ⁇ form of KDAF induced the expression of collagenase (A) but not type I collagen (B) mRNA.
  • Fibroblasts were treated with indicated concentrations of ⁇ / ⁇ form of KDAF for 24 hr. Cells were then harvested and total RNA was evaluated for collagenase and type I collagen expression by Northern analysis.
  • Example 10 Preparation of Active fragments of 14-3-3 protein.
  • Purified KDAF protein 14-3-3 sigma
  • the samples were then cleaned by hpl ziptip.
  • the fractions were collected every minute and dried in the speed vac. with a liquid nitrogen trap installed.
  • the fractions were individually added to cultured fibroblasts and total RNA was extracted after 24 hrs.
  • the expression of collagenase mRNA was then evaluated as an index for KDAF activity. From 70 fractions collected as above, fraction numbers 12, 40 and 50 were identified to be active.
  • Peptides contained in any of the fractions can be synthetically produced using conventional techniques well known to those skilled in the art.
  • a peptide comprising 8 amino acids was isolated from a fraction and was found to be active upon testing with the methods described herein.
  • Example 11 Preparation of antibodies.
  • Rabbit anti-human polyclonal antibody that specifically reacts with protein 14-3-3 sigma were raised using conventional methods well known to those skilled in the art.
  • poly or monoclonal anti-bodies will raised and used as an anti-KDAF factor in both in vitro and in vivo.
  • the lower panel shows the pattern of 18 and 28 S ribosomal RNA for the same blots and is used as a total RNA loading control in Northern analysis.
  • Monomeric form of KDAF possesses collagenase stimulatory effects in dermal fibroblasts.
  • a serial deletion of KDAF cDNA sequence was performed so that the KDAF protein became 15 (d15), 26 (d26), 50 (d50), 75 (d75), and 100 (d100) amino acids shorter at their N-terminal.
  • the corresponding cDNA for each of these peptides was then cloned and expressed in E.coli. As shown in Fig.
  • the purified d15, 26, 50, 75 and d100 amino acid deleted KDAF proteins were then analysed on native gel (Upper panel) and SDS-PAGE (lower panel) for confirmation.
  • the protein standard (lane 1 ) was also included, trypsin inhibitor with apparent MW of 21 kDa (lane 2) and bovine serum albumin (BSA) with apparent MW of 66 kDa (lane 3) in our samples run on a native gel.
  • the purified proteins corresponding to each of these deleted constructs were then individually added to fibroblasts and the levels of MMP-1 protein were evaluated. Results shown in Fig. 13 B, clearly indicate that deletion of up to 75 amino acids is not critical to the KDAF collagenase stimulatory effect.
  • KDAF is Present in Sera Collected from Rat and Bovine and Functions as a Circulating Collagenase Stimulating Factor.
  • sera from rat, bovine and human were collected and evaluated for the presence of KDAF protein by western analysis.
  • rat pituitary extract was included as a possible source of circulating KDAF.
  • human dermal fibroblasts were treated with various concentrations (0, 0.05, 0.1 , 0.5, 1.0, 5.0 %) of FBS and bovine pituitary extract (BPE) in DMEM for 24 hr.
  • KDAF protein was visualized by Western blotting using our recently raised anti-KDAF antibody. Two samples of our recently purified recombinant KDAF were also loaded as positive controls.
  • FBS proteins (3 ⁇ g/lane) were run on SDS-PAGE and fractionated proteins were identified according to their MW in reference to MW marker. As the size of KDAF is determined to be 28-30 kDa, protein fractions containing KDAF with apparent MW of 28-32 kDa (K+) and those without KDAF with MW of 45-55 kDa (K-) were excised and recovered by electroelution. As shown in Fig.
  • proteins of these fractions were either run in a SDS-PAGE using un-fractionated FBS sample as a control (panel A) or used to treat dermal fibroblast to evaluate its collagenase activity (Panel B). Proteins in panel A were then blotted and evaluated for the presence of KDAF by Western blot analysis. Note, KDAF antibody identified a band with apparent MW of 30 kDa only in whole FBS sample and K+ fraction (Panel A).
  • Panel B shows the pattern of collagenase mRNA expression and 18 S ribosomal RNA (loading control) in fibroblasts treated with either nothing (C), recombinant KDAF (K), electroelusion buffer (B), various concentrations of proteins in K- fraction (K-) or K+ fraction (K+). Please note that collagenase mRNA expression is markedly increased only in response to either rKDAF or various concentrations of proteins in K+ fraction.
  • Panel C demonstrates the pattern of western blot analysis showing the presence and quantities of KDAF in sera collected from normal, sham operated or hypophysectomized rats. Note that KDAF level is remarkably increased upon removal of pituitary gland (Hypo) relative to that of controls. This indicates that circulating KDAF is regulated by hormones but not released from pituitary gland.
  • Human serum contains relatively high levels of KDAF.
  • KDAF human serum contains a relatively high level of KDAF protein.
  • rat and bovine sera KDAF in human serum may function as a circulating collagenase stimulating factor.
  • a M ' mtur® ⁇ f t ⁇ D F and Aspirin possesses both a collagenase stimulatory and collagen inhibitory effects in dermal fibroblasts.
  • an effective anti-fibrogenic factor for treatment of fibroproliferative disorders such as hypertrophic scarring should possess either a collagenase stimulatory or a collagen (the main ECM component in skin) inhibitory effect for fibroblasts.
  • any factor with a combination of both should be even more effective than either of these factors used individually.

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Abstract

L'invention porte d'une manière générale sur des procédés de prévention et/ou de traitement de troubles fibroprolifératifs chez les mammifères, et plus particulièrement sur les protéines 14-3-3 qui médient l'expression de la collagénase et sa teneur dans les tissus et organes de mammifères; elle porte également: sur un procédé d'accroissement de la teneur en collagène de la peau de mammifères; sur la prévention l'allégement et ou le traitement de peaux à faible teneur en collagène; sur une méthode de criblage de composés susceptibles d'accroître la teneur en collagènes de la peau; et sur les composés identifiés par cette méthode. L'invention porte en outre sur des combinaisons de protéines peu acides dont les 14-3-3 qui permettent une thérapie fibroproliférative complémentaire.
PCT/CA2004/000245 2003-02-19 2004-02-19 Proteines 14-3-3 pour la prevention et le traitement de troubles fibroproliferatifs WO2004073731A1 (fr)

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Cited By (5)

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Publication number Priority date Publication date Assignee Title
WO2006020230A3 (fr) * 2004-07-21 2006-06-08 Medtronic Inc Methode de reduction ou prevention des fibroses localisees, utilisant la technologie l'arnsi
WO2007098198A3 (fr) * 2006-02-17 2008-03-13 Wyeth Corp Modulation de formation d'os
US7767652B2 (en) 2004-07-21 2010-08-03 Medtronic, Inc. Medical devices and methods for reducing localized fibrosis
WO2020102623A1 (fr) * 2018-11-15 2020-05-22 The University Of Toledo Matériels et méthodes pour la prévention de la polyarthrite rhumatoïde
CN117899196A (zh) * 2024-03-18 2024-04-19 中国人民解放军总医院第一医学中心 14-3-3zeta蛋白或YWHAZ基因在角膜损伤治疗中的应用

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YAFFE M B: "How do 14-3-3 proteins work? - Gatekeeper phosphorylation and the molecular anvil hypothesis", FEBS LETTERS, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 513, no. 1, 20 February 2002 (2002-02-20), pages 53 - 57, XP004344935, ISSN: 0014-5793 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006020230A3 (fr) * 2004-07-21 2006-06-08 Medtronic Inc Methode de reduction ou prevention des fibroses localisees, utilisant la technologie l'arnsi
US7767652B2 (en) 2004-07-21 2010-08-03 Medtronic, Inc. Medical devices and methods for reducing localized fibrosis
WO2007098198A3 (fr) * 2006-02-17 2008-03-13 Wyeth Corp Modulation de formation d'os
WO2020102623A1 (fr) * 2018-11-15 2020-05-22 The University Of Toledo Matériels et méthodes pour la prévention de la polyarthrite rhumatoïde
CN117899196A (zh) * 2024-03-18 2024-04-19 中国人民解放军总医院第一医学中心 14-3-3zeta蛋白或YWHAZ基因在角膜损伤治疗中的应用
CN117899196B (zh) * 2024-03-18 2024-06-04 中国人民解放军总医院第一医学中心 14-3-3zeta蛋白或YWHAZ基因在角膜损伤治疗中的应用

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