CN118685365B - Construction and application of miR-423-5p high-abundance engineering vesicle - Google Patents
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- 230000009885 systemic effect Effects 0.000 description 1
- 229940126703 systemic medication Drugs 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
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Abstract
本发明公开了一种miR‑423‑5p高丰度工程化囊泡的构建及应用，属于生物医药技术领域。本发明构建的工程化囊泡具备和天然囊泡一样的双层膜结构，相近的粒径和电位值以及相似的标志蛋白表达，并且本发明构建的工程化囊泡内的miR‑423‑5p表达水平为天然囊泡的10万倍，同时能通过被牙周膜干细胞内化，将自身携带的miR‑423‑5p递送至牙周膜干细胞，使细胞内的miR‑423‑5p含量上调338倍，并能在胞内存留至少14 d。此外，本发明构建的miR‑423‑5p高丰度工程化囊泡能有效增强天然囊泡的成骨效能，显著促进牙周膜干细胞成骨向分化。
The present invention discloses the construction and application of a miR-423-5p high-abundance engineered vesicle, belonging to the field of biomedical technology. The engineered vesicle constructed by the present invention has the same double-layer membrane structure as natural vesicles, similar particle size and potential value, and similar marker protein expression, and the miR-423-5p expression level in the engineered vesicle constructed by the present invention is 100,000 times that of natural vesicles, and can be internalized by periodontal ligament stem cells, and the miR-423-5p carried by itself can be delivered to periodontal ligament stem cells, so that the miR-423-5p content in the cell is increased by 338 times, and can remain in the cell for at least 14 d. In addition, the miR-423-5p high-abundance engineered vesicle constructed by the present invention can effectively enhance the osteogenic efficacy of natural vesicles and significantly promote the osteogenic differentiation of periodontal ligament stem cells.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to construction and application of miR-423-5p high-abundance engineering vesicles.
Background
Periodontitis is one of six common inflammatory diseases worldwide, and severe periodontitis can result in tooth loss, and is closely related to a variety of systemic diseases. At present, the clinical treatment mainly uses the combination of plaque removal and operation treatment and is assisted by local or systemic medication, and although inflammation can be controlled to a certain extent, periodontal tissue structural and functional regeneration is difficult to realize.
Periodontal ligament stem cells (periodontal LIGAMENT STEM CELLS, PDLSCs) are an important class of undifferentiated mesenchymal stem cells in periodontal tissues, and their good proliferation and differentiation potential are of great significance for periodontal ligament tissue and alveolar bone formation. However, under chronic inflammatory infiltration for a long period, PDLSCs functions are impaired, and damaged PDLSCs further promotes the formation of periodontitis niches, forming a malignant negative regulation closed loop, which is a main cause of persistent and non-healing periodontitis and progressive damage of periodontal tissues. Thus, modulating the regenerating microenvironment, restoring impaired PDLSCs function and maintaining its dryness is of great importance for repairing and regenerating periodontal tissue. The odontoblast stem cells (dental follicle STEM CELLS, DFSCs) are closely related to PDLSCs as precursor cells for various periodontal tissues.
The small extracellular vesicles (small extracellular vesicles, sEVs) are novel cell-free technology, can mediate intercellular communication, have high physical and chemical stability and high biocompatibility, communicate among cells through carrying various biological information structures, further regulate the microenvironment of organisms, maintain the functions of local resident host cells, and are beneficial to repairing and regenerating various soft and hard tissues of the organisms, so that the small extracellular vesicles can be used as excellent carriers of various bioactive substances. Studies have shown that natural sEVs from tumor cells or immune cells can elicit anti-tumor activity, with potential for becoming a cancer vaccine. However, natural sEVs still has some problems in cancer treatment: natural sEVs is prone to be trapped in non-specific tissues (especially liver and lung), resulting in insufficient targeting in vivo; the heterogeneity and compositional complexity of natural sEVs can reduce the therapeutic effect and even present safety concerns. Because of the lack of processes such as efficient separation, industrialized production, accurate monitoring of drug loading, and the like, the natural sEVs has hidden danger in clinical use. Thus, the engineered sEVs becomes a new therapeutic strategy and is expected to be another option for sEVs-based therapy.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide construction and application of miR-423-5p high-abundance engineering vesicles so as to solve the problem that periodontal tissue structural and functional regeneration is difficult to realize in the existing periodontitis treatment method.
The technical scheme for solving the technical problems is as follows: the miR-423-5p high-abundance engineering vesicle comprises a small extracellular vesicle and miR-423-5p, wherein the positive strand nucleotide sequence of the miR-423-5p is as follows: UGAGGGGCAGAGAGCGAGACUUU the reverse nucleotide sequence is: AAAGUCUCGCUCUCUGCCCCUCA; the small extracellular vesicles are small extracellular vesicles secreted by the dental follicle stem cells; the miR-423-5p high-abundance engineering vesicle is prepared by the following method:
(1) Uniformly mixing miR-423-5p with a nucleic acid transfection reagent, and then adding small extracellular vesicles for incubation;
(2) Putting the product obtained in the step (1) into an adsorption column for centrifugation to obtain miR-423-5p small extracellular vesicles;
(3) Adding miR-423-5p small extracellular vesicles into a ultrafiltration tube for purification and concentration, and reserving upper-layer liquid to obtain the miR-423-5p small extracellular vesicles.
Further, the incubation temperature in the step (1) is 35-38 ℃ and the incubation time is 0.5-2 h.
Further, in the step (2), the centrifugal speed is 1500-2500 rpm, and the centrifugal time is 30-60 s.
Further, the molecular weight cut-off of the ultrafiltration tube in step (3) is 25-35 kD.
The invention also provides application of the miR-423-5p high-abundance engineering vesicle in preparation of periodontitis anti-inflammatory drugs.
The invention has the following beneficial effects: the inventor researches and discovers that small extracellular vesicles (DFSCs-sEVs) secreted by the dental follicle stem cells exhibit certain periodontal tissue regeneration capacity by promoting PDLSCs proliferation, migration and osteogenic differentiation. High-throughput sequencing and experimental verification prove that miR-423-5p serving as high-abundance miRNAs in DFSCs-sEVs has strong bone-promoting capacity. The miR-423-5p high-abundance engineering vesicle constructed by the invention has a double-layer membrane structure which is the same as that of a natural vesicle, similar particle size and potential value and similar marker protein expression, and the expression level of miR-423-5p in the engineering vesicle is 10 ten thousand times that of the natural vesicle, so that miR-423-5p carried by the engineering vesicle can be delivered to periodontal ligament stem cells through internalization by the periodontal ligament stem cells, the content of miR-423-5p in the cells is up-regulated by 338 times, and meanwhile, the miR-423-5p in the cells can stay in the cells for at least 14 days. In addition, the miR-423-5p high-abundance engineering vesicle constructed by the invention has higher osteogenic efficacy, and can obviously promote the osteogenic differentiation of periodontal ligament stem cells.
Drawings
FIG. 1 is a heat map showing the biological processes and associated signaling pathways involved in differentially expressing miR-423-5 p;
FIG. 2 is a pie chart showing the distribution of the expression level of differentially expressed miR-423-5p in sEVs;
FIG. 3 is a schematic diagram of a method of constructing an engineered vesicle;
FIG. 4 is an identification map of engineered vesicles; wherein, the A diagram is a transmission electron microscope diagram of sEVs and sEVs miR-423-5p; panel B is a particle size plot of sEVs and sEVs miR-423-5p; panel C shows Western blot results of sEVs and sEVs miR-423-5p; plot D is the Zeta potential of sEVs and sEVs miR-423-5p; e is the expression level of miR-423-5p in sEVs and sEVs miR-423-5p;
FIG. 5 is a diagram of sEVs miR-423-5p targeted delivery of miR-423-5p through PDLSCs; wherein, panel A is a fluorescence confocal plot of sEVs and sEVs miR -423-5p targeted delivery of miR-423-5p through PDLSCs; panel B shows the expression levels of miR-423-5p, miR-100-5p, miR-125B-5p, miR-26a-5p and miR-24-3p in PDLSCs after PDLSCs ingests sEVs and sEVs miR-423-5p;
FIG. 6 is a comparison of sEVs miR-423-5p and liposome delivery patterns; wherein, the A graph is a degradation fluorescence confocal graph of miR-423-5p which is delivered in two ways and is in PDLSCs; panel B shows the variation of the expression level of miR-423-5p in PDLSCs delivered in two ways;
FIG. 7 is a graph showing the effect of CCK-8 assay sEVs miR-423-5p on PDLSCs proliferation potency;
FIG. 8 is a graph showing the effect sEVs miR-423-5p on the osteoblast differentiation capacity of periodontal ligament stem cells; wherein panel A is an alkaline phosphatase staining of PDLSCs after sEVs and sEVs miR-423-5p intervention culture; panel B is a alizarin red staining chart of PDLSCs after sEVs and sEVs miR-423-5p intervention culture; panel C shows the expression level of osteogenic related proteins of PDLSCs after sEVs and sEVs miR-423-5p intervention culture; FIG. D shows the quantitative analysis of the expression level of the osteogenic related protein after PDLSCs has been subjected to sEVs and sEVs miR-423-5p intervention culture; FIG. E shows quantitative analysis of the expression level of the osteogenic related gene of PDLSCs after the intervention culture of sEVs and sEVs miR-423-5p.
Detailed Description
The examples given below are only intended to illustrate the invention and are not intended to limit the scope thereof. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In order to find sEVs miRNAs with good bone promoting effect, the invention cultures and collects DFSCs and sEVs of six different human sources, adds a proper amount of Trizol to lyse cells and vesicles 20 min on ice, collects the lysed samples in an EP tube, marks DFSCs-1 to DFSCs-6 and sEVs-1 to sEVs-6 respectively, carries out high throughput sequencing on the marked samples to obtain gene sequences of the 12 samples, compares the gene sequences of the 12 samples with a reference genome through a comparison system HISAT, and then carries out statistical annotation and comparison results. The regenerating effector miRNAs in sEVs were found by performing a belief analysis of the differential miRNAs in DFSCs and sEVs. Based on high-throughput sequencing data and bioinformatics analysis, hsa-miR-423-5p is found to be one of the important bone effect-promoting miRNAs of sEVs, and is named miR-423-5p. The heat map shows biological process and related signal path involved in differential expression miR-423-5p, including TGF-beta, hippo, MAPK signal path related to cell growth and differentiation (see figure 1, in which the right side is marked ：hsa-miR-409-3p|Tarbase、hsa-miR-100-5p|Tarbase、hsa-miR-143-3p|Tarbase、hsa-miR-1246|Tarbase、hsa-miR-423-3p|Tarbase、hsa-miR-199a-3p|Tarbase、hsa-miR-423-5p|Tarbase、hsa-miR-125b-5p|Tarbase、hsa-miR-222-3p|Tarbase、hsa-miR-221-3p|Tarbase、hsa-miR-320a|Tarbase、hsa-miR-148a-3p|Tarbase、hsa-let-7a-5p|Tarbase、hsa-let-7f-5p|Tarbase、hsa-let-7i-5p|Tarbase、hsa-let-7b-5p|Tarbase、hsa-miR-92a-3p|Tarbase、hsa-miR-21-5p|Tarbase、hsa-miR-26a-5p|Tarbase、hsa-miR-24-3p|Tarbase; from top to bottom, and the left to right is marked FATTY ACID metabolism (fatty acid metabolism), FATTY ACID biosynthesis (fatty acid biosynthesis), and the like, Steroid biosynthesis (steroid biosynthesis), prion diseases (Prion diseases), huntington's disease, ECM-receptor interaction (extracellular matrix receptor interaction), glycosaminoglycan biosynthesis-keratan sulfate (glycosaminoglycan biosynthesis-keratin sulfate), other types of O-glycan biosynthesis (Other types of O-glycan biosynthesis), Viral carcinogenesis (viral carcinogenesis), P53 SIGNALING PATHWAY (P53 signaling pathway), oocyte meiosis (oocyte meiosis), glioma (glioma), lysine degradation (lysine degradation), proteoglycans in cancer (proteoglycan in cancer), CELL CYCLE (cell cycle), blader cancer, hippo signaling pathwayHippo (signaling pathway), and, arrhythmogenic right ventricular cardiomyopathy (ARVC, arrhythmogenic right ventricular cardiomyopathy), protein processing in endoplasmic reticulum (protein processing in the endoplasmic reticulum), RNA transport (RNA transport), epstein-Barr virus infection (EB virus infection), ubiquitin mediated proteolysis (ubiquitin-mediated proteolysis), HTLV-l infection (HTLV-l infection), melanoma (melanoma), SMALL CELL lung cancer (small cell lung carcinoma), HEPATITIS B (hepatitis B), chronic myeloid leukemia (chronic myelogenous leukemia), colorectal cancer (colorectal cancer), shigellosis (Shigella disease), thyroid cancer (thyroid cancer), estrogen SIGNALING PATHWAY (estrogen signaling pathway), and, Adherens junction (adhesion linkage), bacterialinvasion of EPITHELIAL CELLS (bacterial invasion of epithelial cells), RENAL CELL carpinoma (renal cell carcinoma), endocytosis (endocytosis), MAPK SIGNALING PATHWAY (MAPK signaling pathway), thyroid hormone SIGNALING PATHWAY (thyroid hormone signaling pathway), endometrial cancer (endometrial carcinoma), Transcriptional misregulation in cancer (transcriptional dysregulation in cancer), PATHWAYS IN CANCER (cancer pathway), prostate cancer (Prostate cancer), SIGNALING PATHWAYS regulating pluripotency of STEM CELLS (signaling pathway regulating stem cell pluripotency), TGF-beta SIGNALING PATHWAY (TGF-beta signaling pathway), FoxO SIGNALING PATHWAY (FoxO signaling pathway)), the pie graph shows the distribution of the expression level of differentially expressed miR-423-5p within sEVs (see fig. 2).
Example 1
An engineered vesicle (sEVs miR-423-5p) with high abundance of miR-423-5p comprises a small extracellular vesicle and miR-423-5p, wherein the positive strand nucleotide sequence of miR-423-5p is as follows: UGAGGGGCAGAGAGCGAGACUUU the reverse nucleotide sequence is: AAAGUCUCGCUCUCUGCCCCUCA.
SEVs miR-423-5p was constructed using Exo-Fect ™ siRNA/miRNA Transfection Kit (SBI) as shown in FIG. 3, comprising the steps of:
(1) Taking 100 mu L of transfection Buffer, 4 mu L of transfection reagent and 1.25 mu L of miR-423-5p mimic (miR-423-5 p mic,20 mu M), carrying out instantaneous centrifugation and mixing after light blowing by a pipetting gun, and standing for 15 min at room temperature;
(2) Then adding 100 mu L of small extracellular vesicles (sEVs) into the product obtained in the step (1), lightly blowing and uniformly mixing, and incubating at 37 ℃ for 1h to prepare transfection suspension;
(3) Taking out a purifying column (Clean-up column), slightly screwing down a screw cap below the column, placing the column aside for standby, placing the column into a 2 mL collecting pipe, placing the column into a centrifugal machine, and centrifuging for 30s at 2000 rpm to remove storage liquid; then 500. Mu.L Buffer (Column Buffer) was added, the mixture was put into a centrifuge, centrifuged at 2000 rpm for 30s to wash the Column, and the procedure was repeated once;
(4) Tightly fastening the screw cap removed in the step (3) below the column and screwing to prevent liquid from flowing out, sucking 205.25 mu L of the transfection suspension in the step (2) into the column, incubating at room temperature for 15min, then putting into a centrifuge, and centrifuging for 30 s at 2000 rpm to obtain sEVs miR-423-5p vesicles;
(5) Adding sEVs miR-423-5p vesicles obtained in the step (4) into a30 kD ultrafiltration tube for concentration and purification to remove toxicity of the transfection reagent, wherein the concentrated supernatant is the purified sEVs miR-423-5p vesicles.
Example 2 identification of sEVs miR-423-5p vesicles
(1) Transmission electron microscope observation sEVs miR-423-5p vesicle morphology
And (3) dripping 10 mu L sEVs and sEVs miR-423-5p on a copper net to precipitate 1 min, dripping uranyl acetate to dye 30 s-1 min, sucking the floating liquid by filter paper, drying at room temperature, and observing and collecting images by a transmission electron microscope under the voltage of 100 kv. The transmission electron microscopy results showed sEVs and sEVs miR-423-5p to be both in a bilayer membrane tea tray-like structure (see panel a in fig. 4).
(2) Diameter and potential value detection of sEVs miR-423-5p vesicles
And measuring the number and the size of particles in a sample by adopting a NanoFCM instrument, performing instrument performance qualification detection by using a standard substance, loading samples after diluting sEVs and sEVs miR-423-5p vesicles by PBS, and obtaining the particle sizes and the concentrations of sEVs and sEVs miR-423-5p after detection. As can be seen from panel B in FIG. 4, the particle size kurtosis of sEVs and sEVs miR-423-5p vesicles was 81.22, nm and 82.93 nm, respectively. In addition, the potential values were measured by a Zeta View PMX110 nm Particle size tracer analyzer (Particle Metrix, germany), and Zeta potential results showed that the potential means of sEVs and sEVs miR-423-5p were-35.77 mV, -35.24 mV, respectively, which were similar, suggesting that engineering modifications did not reduce the structural stability of native sEVs (FIG. 4, panel D).
(3) Marker protein for identifying sEVs miR-423-5p vesicles by Western blot
① Collection of protein samples: DFSCs, svvs and sEVs miR-423-5p were removed, cells were washed by centrifugation in PBS after conventional digestion DFSCs, 100 μl of lysis solution (RIPA: pmsf=100:1) was added dropwise after blotting the PBS, and lysed on ice, during which time the cell and vesicle samples were shaken or blown. 15-20 min, collecting the sample in a centrifuge tube, putting the centrifuge tube into a 12000 rpm and centrifuging the sample in the centrifuge tube for 25 min, obtaining supernatant which is the total protein of the sample, collecting the supernatant and measuring the protein concentration.
② BCA assay for protein concentration:
A. preparing and drawing a standard curve: a 96-well elisa plate was taken and the following reagents were added according to table 1:
TABLE 1 BCA Standard Curve
B. preparing BCA working solution according to the number of samples, and reversing and uniformly mixing;
C. fully and uniformly mixing a sample to be tested, sucking 2 mu L of the sample to be tested, sucking 18 mu L of a standard substance diluent to complement the sample to be tested to 20 mu L (10 times of dilution), and setting three compound holes as repetition;
D. Adding 200 mu L of BCA working solution to each sample to be tested and each standard protein hole, incubating for 30 min at 37 ℃ in a dark place, then placing on an enzyme-labeled instrument, measuring and recording the absorbance value of each hole at 562: 562 nm, and calculating and manufacturing a standard curve;
E. Substituting the absorbance value of the measured sample into a standard curve formula to calculate the protein concentration, and aligning the concentration of each sample by using the lysate.
① Protein denaturation: diluting 5 XSDS loading buffer to 1×, adding into protein sample, shaking by a vortex mixer, mixing, and denaturing at 100deg.C for 5 min, wherein the denatured protein sample can be directly subjected to subsequent experiment or temporarily stored in a refrigerator at-80deg.C.
② SDS-PAGE electrophoresis: two long and short clean glass plates without damage are taken and mounted on the glue making device. Taking Omni-Easy ™ one-step method PAGE gel rapid preparation kit (10%) as an example, preparing separating gel and concentrated gel according to table 2, fully mixing, injecting the separating gel into a glass plate, slowly injecting the concentrated gel above the separating gel, rapidly inserting a matched sample feeding comb, and standing at room temperature for 15: 15 min; taking down the glass plate after the concentrated gel and the separation gel are completely solidified, flushing excessive gel on the outer side of the glass plate by distilled water, then fixing the glass plate in an electrophoresis tank, adding 1 x electrophoresis buffer solution until the glass plate is completely soaked, slightly pulling off a sample feeding comb, feeding each protein sample with the mass of 15 mu g protein/hole, and adding 4 mu L protein pre-dyeing markers into the hole on the left side of the first sample to indicate the position of each molecular weight protein; connecting a power supply, and concentrating according to the concentration glue: 80 V,20 min, seperate gum: 120 V,50 min, and stopping electrophoresis when bromophenol blue reaches the gel bottom.
Table 2 elegance enzyme gum formulation
① Transferring: and taking out the glass plate after electrophoresis is finished, washing the plate with distilled water to remove residual electrophoresis liquid on the plate, gently prying the plate along one corner of the glass plate, cutting off concentrated glue, reserving a complete separation glue part of a sample, soaking glue, filter paper and sponge in precooling film transferring liquid, cutting a proper PVDF film according to the size of the glue, soaking the PVDF film in film activating liquid for 1 min, and transferring the PVDF film into the film transferring liquid. The film transfer device is installed according to the following sequence: the cathode plate, the sponge, the 3 layers of filter paper, the glue, the PVDF film, the 3 layers of filter paper, the sponge and the anode plate are placed into an electrophoresis tank after being installed, a film transferring buffer solution is poured into the electrophoresis tank, the film is transferred to the membrane at a constant pressure of 20V and 60min, and the film transferring time is correspondingly regulated according to the molecular weight of the target protein if the molecular weight of the target protein to be detected is too large or too small.
② Closing: and after the film transfer is finished, TBST washes PVDF film 1 min to remove residual film transfer liquid on the film surface, and then the film is soaked in sealing liquid to be sealed at room temperature for 30 min.
③ Incubation resistance: the anti-dilution ratios are respectively: CD9 (1:2000), CD63 (1:2000), HSP90 (1:1000), TSG101 (1:1000) and Calnexin (1:1000), membranes were incubated in the corresponding primary antibody solutions, respectively, according to the protein of interest detected, on a shaker at room temperature for 5min and then transferred to a refrigerator at 4℃overnight.
④ Secondary antibody incubation: recovering the primary antibody solution, and washing the membrane by shaking the membrane for three times, 10min each time, soaking the membrane in the secondary antibody solution, wherein the dilution ratio is as follows: rabbit antibody (1:5000), murine antibody (1:5000), were incubated on a shaker for 90 min at room temperature, washed three times with TBST, 15 min each time.
⑤ Developing: preparing a developing reaction liquid under the dark condition, sucking redundant TBST liquid on the PVDF film by filter paper, dripping the developing liquid on the film, and developing on a machine.
Western blot results show that sEVs and sEVs miR-423-5p all positively express exosome marker proteins CD9, CD63, TSG101 and HSP90 and negatively express endoplasmic reticulum protein Calnexin (see C diagram in FIG. 4).
(4) MiR-423-5p expression level
① MiRNA extraction: sEVs and sEVs miR-423-5p vesicles were added to the cell lysate for lysis, allowed to stand at room temperature for 5 min, vigorously shaken for 15 s after 200. Mu.L of chloroform was added, and allowed to stand at room temperature for 5 min; putting into a centrifuge, centrifuging at 12000 rpm at 4deg.C for 15: 15 min, transferring the upper colorless water phase into a new tube, adding anhydrous ethanol with volume 0.43 times of the transfer liquid, mixing, transferring into an adsorption column, centrifuging at 12000 and rpm for 30: 30 s, and retaining the lower filtrate; adding 0.75 times of absolute ethyl alcohol with the volume of the filtrate, uniformly mixing, transferring into an adsorption column, centrifuging by a centrifuge 12000 rpm for 30 s, and reserving an upper adsorption column; adding 500 mu L of MRD deproteinized solution into an adsorption column, standing at room temperature for 2 min, centrifuging with a centrifuge 12000 rpm for 30 s, and discarding the filtrate; subsequently, 500. Mu.L RW rinse solution was added, allowed to stand at room temperature for 2 min hours, centrifuged at 12000/rpm for 30/s hours, the filtrate was discarded, and the procedure was repeated once; resetting an adsorption column in a collecting pipe, centrifuging 1 min by a centrifuge 12000 rpm, removing residual liquid, transferring the adsorption column to a new 1.5 mL RNase-free EP pipe, adding 20 mu L RNASE FREE DDH 2 O, standing 2 min at room temperature, centrifuging 2 min by a centrifuge 12000 rpm, and collecting miRNA; after fully mixing, 2 mu L of samples are taken, and a Quawell Q trace ultraviolet spectrophotometer is used for measuring the miRNA concentration.
② Reverse transcription reaction: according to the specification of Mir-X MIRNA FIRST-STRAND SYNTHESIS KIT. The RNASE FREE 0.2 mL reaction tube was placed on ice and the reagents according to Table 3 were added to 10. Mu.L.
TABLE 3 mRQ Enzyme reagents and amounts used for the reverse transcription reaction
Collecting and mixing the prepared reaction liquid by instantaneous centrifugation, and putting the reaction liquid into a gradient PCR instrument for reverse transcription reaction under the reaction conditions: 37. terminating the reaction at 85 ℃ for 5 min at 4 ℃ after 1h at the temperature of 10 times, and directly using the reaction liquid obtained by transcription in PCR experiments or temporarily storing at-80 ℃.
③ Real-time quantitative PCR: the PCR reaction solution was prepared in accordance with Table 4 on ice. The components in the table were added to 96-well plates, and the reaction solution was collected and mixed by instantaneous centrifugation 1 min and reacted under QuantStudio TM Real-TIME PCR SYSTEM according to the following procedure: 95. denaturation at 3-5 s, annealing at 55deg.C for 10s, extension at 72deg.C for 34 s,40 cycles; after the reaction procedure is completed, CT values of the genes are recorded, and the relative expression quantity of the genes is calculated according to the formula x=2-fatin Ct, wherein x is the multiple of the target genes of each group relative to the control group after being corrected by U6.
As shown in the graph E of FIG. 4, the expression level of miR-423-5p contained in sEVs miR-423-5p was 10 ten thousand times that of sEVs, which suggests that the sEVs miR-423-5p vesicle was successfully constructed.
TABLE 4 TB Green Advantage PCR reagents required for the reactions and amounts used
EXAMPLE 3 sEVs miR-423-5p Targeted delivery of miR-423-5p to PDLSCs
(1) PKH26 labeling sEVs (red dot fluorescence), green fluorescence labeling miR-423-5p, cellmask ™ ACTIN TRACKING STAINS labeling PDLSCs cytoskeleton (red sheet fluorescence), and then sEVs and sEVs miR -423-5p were found to be both taken up by PDLSCs into the perinuclear and cytosol after 24-h co-culture with PDLSCs, respectively (FIG. 5A). PDLSCs upon uptake sEVs miR-423-5p, not only was structural integrity sEVs miR-423-5p (orange fluorescence, indicated by the yellow arrow in FIG. 5, of the overlapping red-green fluorescence) but sEVs (red fluorescence, indicated by the red arrow in FIG. 5, of the A panel) and miR-423-5p (green fluorescence, indicated by the green arrow in FIG. 5, of the A panel) were observed at the perinuclear.
(2) And (3) co-culturing sEVs and sEVs miR-423-5p with PDLSCs of 10 mug/mL respectively, extracting miRNA in cells after 48 hours, and quantitatively detecting the expression level of miR-423-5p and other miRNAs (miR-100-5 p, miR-125b-5p, miR-26a-5p and miR-24-3 p) in PDLSCs by a PCR experiment.
PCR quantitative analysis shows that after PDLSCs ingests sEVs, the expression levels of various miRNAs such as miR-423-5p, miR-100-5p, miR-125B-5p, miR-26a-5p and miR-24-3p are all increased, however, after PDLSCs ingests sEVs miR-423-5p, the expression level of the miR-423-5p contained in the miRNAs is obviously increased and is 338.38 times that of the original miRNAs, and the expression levels of the other four miRNAs are not obviously changed, so that the result indicates that the engineering vesicle of the construction method can target and accurately deliver the miR-423-5p on the basis of not influencing the expression levels of the other miRNAs in target cells (see a diagram B in fig. 5).
Example 4 sEVs miR-423-5p comparison with Liposome delivery modes
After the fact that sEVs miR-423-5p can be taken up by PDLSCs and miR-423-5p is delivered in a targeted manner is confirmed, the degradation rate of miR-423-5p delivered by sEVs miR-423-5p in cells is further observed by comparing with a common liposome delivery mode, after co-culturing for 1 d under a confocal microscope, a large amount of miR-423-5p mimics with green fluorescence are observed in cytoplasm of PDLSCs in a miR-423-5p mimic group, a large amount of red fluorescence is observed in cytoplasm and perinuclear of PDLSCs in a sEVs miR-423-5p group, sEVs and miR-423-5p mimics with green fluorescence are observed in a nuclear cycle of PDLSCs, the fluorescence quantity of the two groups is gradually reduced along with the extension of an observation time point, no fluorescence is observed in a miR-423-5p mimic group at 14 d under a microscopic view, and a small amount of green fluorescence and a large amount of red fluorescence are still visible in a nuclear cycle and cytoplasm of PDLSCs in a sEVs miR-423-5p group (A diagram in FIG. 6); in addition, cellular miRNAs were extracted on days 1,3, 7 and 14 of miR-423-5p mimic or sEVs miR-423-5p -intervening culture PDLSCs, and the change in miR-423-5p expression in PDLSCs was quantitatively detected by PCR experiments.
PCR quantitative analysis shows that miR-423-5p content in miR-423-5p mimic group PDLSCs is sharply reduced after 3d, and miR-423-5p content in sEVs miR-423-5p group PDLSCs is slowly decreased in 1 d-14 d (B diagram in FIG. 6), which suggests that sEVs miR-423-5p plays a role in protecting and slowly releasing wrapped miR-423-5 p.
Example 5 CCK-8 experiment to examine the effect of sEVs miR-423-5p on the proliferation potency of PDLSCs
Inoculating P3 generation PDLSCs into 96-well plate at a density of 3000/well/100 μl, changing culture medium to DMEM/F-12 culture medium without FBS after cells adhere to wall and form is stretched, and starving cells overnight; the next day, except the day 0 plates, the other plates are replaced with each group of culture medium according to the grouping requirement, the day 0 plates discard old culture solution, 90 mu L of DMEM/F-12 culture medium and 10 mu L of CCK-8 solution are added into each hole, after incubation at 37 ℃ in the dark for 2h, OD value is measured mechanically, after 1-7 d later, one plate is taken at the same time every day to incubate the CCK-8 solution and OD value is measured mechanically, and the other plates are cultured conventionally according to the grouping replacement of culture solution once every two days until the plate detection of 7 d is completed.
The results are shown in fig. 7, which shows that sEVs and sEVs miR-423-5p have no obvious effect on the proliferation level of PDLSCs in the early stage, and the proliferation of PDLSCs is obviously promoted in the later 3-7 d; while the pro-proliferative capacity of sEVs miR-423-5p was lower than sEVs at 4d and 5 d, the proliferation levels at groups 6 d and 7 d sEVs miR-423-5p were gradually leveled to the natural vesicle group, indicating that sEVs miR-423-5p retained the pro-proliferative properties of sEVs.
Example 6 sEVs miR-423-5p Effect on periodontal ligament Stem cell osteogenic differentiation Capacity
In order to clearly determine the influence of sEVs miR-423-5p on the osteogenic differentiation potential of PDLSCs, sEVs and sEVs miR-423-5p are used for intervening in culture PDLSCs under mineralization induction conditions, alkaline phosphatase staining is performed after intervening in 5d, the expression level of osteogenic related indexes (ALP, OSX, runx and COL 1) is detected after intervening in 7 d, alizarin red staining is performed after intervening in 10 d, and the specific steps are as follows:
(1) Alkaline phosphatase staining: after mineralization induction of 5 d, the old culture solution was washed with PBS, cells were fixed, alkaline phosphatase staining working solution was prepared according to Table 5 and added dropwise to the cells to stain them in the dark for 10min, staining was stopped with distilled water and the cells were rinsed 3 times, and each group of alkaline phosphatase staining was observed under an inverted phase contrast microscope under wet conditions and mapped.
TABLE 5 alkaline phosphatase working solution
The alkaline phosphatase staining results are shown in FIG. 8, panel A, and both sEVs and sEVs miR-423-5p showed that both promote PDLSCs alkaline phosphatase production, wherein sEVs miR-423-5p treated PDLSCs had significantly deeper alkaline phosphatase staining than group sEVs.
(2) Western blot and PCR detection of osteogenic protein expression levels: after induction for 7 days, old culture solution is discarded, cell samples of each group are collected, total protein is extracted, protein concentration is measured, western blot is used for detecting the expression level of osteogenic related proteins ALP (1:1000), OSX (1:1000), runx2 (1:1000) and COL1 (1:1000), and the steps are as follows:
Extraction of total RNA of cells: taking a mineralization-induced sample of No. 7 d, extracting total RNA according to the operation manual of TaKaRa MiniBEST Universal RNA Extraction Kit, and specifically comprising the following steps:
① Absorbing and discarding old culture medium of each group of cells, washing the cells for 2 times by PBS, discarding the PBS, adding 300 mu L of pre-prepared Buffer RL (prepared according to the ratio of Buffer RL:50×DTT solution=50:1) into each hole of a six-hole plate, and shaking the hole plate to enable lysate to uniformly contact the cells, horizontally placing 20 min to fully lyse the cells, and stripping the cells which are firmly attached and difficult to fall off by using cell scraping;
② Cell lysates in each set of well plates were collected well and transferred to gDNA-ERASER SPIN Column, centrifuged at 12000: 12000 rpm for 1:1 min, gDNA-ERASER SPIN Column was discarded, and filtrate in the lower collection tube was retained;
③ Measuring the volume of the filtrate, adding 70% ethanol (RNASE FREE DH 2 O) with equal volume, fully and uniformly mixing, transferring the mixed solution to RNA Spin Column, centrifuging 1min under a centrifuge 12000 rpm, and discarding the lower filtrate;
④ Adding 500 mu L Buffer RWA,12000 rpm into RNA Spin Column upper layer centrifugal Column, centrifuging for 30 s, and discarding lower layer filtrate;
⑤ Adding 600 mu L Buffer RWB (absolute ethyl alcohol is added in advance) into an RNA Spin Column upper layer centrifugal Column, centrifuging for 30 s under a centrifugal machine 12000 rpm, and discarding lower layer filtrate;
⑥ DNase I digestion:
A. Preparing a certain amount of DNase I reaction solution according to the sample quantity in the following proportion: 10 XDNase I Buffer: recombinant DNase I: RNASE FREE DH 2 o=5:4:41, and mixing the reaction solution by instantaneous centrifugation;
B. Dropwise adding 50 mu L of DNase I reaction solution into the center of the membrane of the RNA Spin Column upper layer Column, and standing at room temperature for 15 min;
C. adding 350 μL Buffer RWB into RNA Spin Column upper Column, centrifuging under 12000 rpm for 30 s, and discarding lower filtrate;
⑦ Repeating step ⑤;
⑧ Resetting RNA Spin Column in the lower collecting pipe, centrifuging under a centrifuge 12000 rpm for 2 min to completely remove residual liquid in the upper Column;
⑨ Reset RNA Spin Column to new EP tube of 1.5 mL RNase Free, add 50. Mu.L RNASE FREE DH 2 O to the center of the membrane of the upper Column, stand 5min at room temperature, centrifuge 12000 rpm and centrifuge 2 min to elute RNA;
⑩ After the extracted total RNA was thoroughly mixed, 2. Mu.L was extracted, and the concentration was measured by using Quawell Q.about.3000 micro ultraviolet spectrophotometer.
Reverse transcription reaction: the reverse transcription reaction was performed according to PRIMESCRIPT TM RT Master Mix (PERFECT REAL TIME) instructions, the specific steps are as follows:
① The RNASE FREE, 0.2 mL tube was placed on an ice box and the reagents were added separately to a total volume of 10 μl as required in table 6:
TABLE 6 PrimeScript TM reverse transcription reaction
② Collecting and uniformly mixing the prepared mixed solution through instantaneous centrifugation, and placing the mixed solution into a gradient PCR instrument for reverse transcription reaction under the reaction conditions: 37. terminating the reaction at-80deg.C for 15min at 85deg.C for 5 s at 4deg.C, and diluting the transcribed reaction solution 10 times for direct use in PCR experiment or temporary storage at-80deg.C.
Real-time quantitative PCR: the experimental steps are carried out according to the operation requirement of the TaKaRa kit, and the specific steps are as follows:
① Primer design: the sequences of the primer (ALP, OSX, runx, COL1, GAPDH) genes required for this experiment are shown in Table 7;
TABLE 7 primer sequences
② Preparing PCR reaction liquid on ice according to the requirement of the dosage of each component in table 8:
Table 8 TB Green Premix Ex TaqII PCR reaction
③ The reaction solution is added into a 96-hole PCR reaction plate after instantaneous centrifugation, and is evenly mixed by centrifugal 1 min of a flighted plate machine, and the reaction is carried out under QuantStudio TM Real-TIME PCR SYSTEM according to the following procedures: stage1:1 cycle, 95℃30 s; stage2:40 cycles, denaturation at 95℃for 5s, annealing at 60℃for 34 s;
④ After the PCR reaction is completed, the CT values of each gene are recorded, and the relative expression of the genes is calculated according to the formula x=2-father CT, where x is the multiple of each group of genes of interest after GAPDH correction relative to the control group.
The WB and PCR results are shown in fig. 8, C-E, and WB shows that sEVs up-regulates the expression level of osteogenic related proteins in PDLSCs, whereas sEVs miR-423-5p significantly up-regulates the protein expression levels of Runx2 and COL1 (see fig. 8, C), and the gene levels of ALP and Runx2 (see fig. 8, D), wherein COL1 gene levels were lower than sEVs but still significantly higher than control (see fig. 8, E), suggesting that sEVs miR-423-5p enhances the up-regulation of the osteogenic index of sEVs on the intracellular portion of the target cells.
(3) Alizarin red staining: inducing 10 d, discarding old culture solution, washing cells with PBS, absorbing PBS, fixing cells with paraformaldehyde for 10 min, washing with PBS for 3 times, dripping alizarin red dye solution to submerge cells, dyeing for 5min in dark place, discarding dye solution, washing with PBS to obtain floating color, observing mineralized nodule formation condition of each group under wet condition, and collecting images.
The alizarin red staining results are shown in a diagram B in fig. 8, and the results show that sEVs has no obvious effect on the formation of PDLSCs mineralized nodules, while sEVs miR-423-5p remarkably promotes the formation of PDLSCs mineralized nodules, which suggests that sEVs miR-423-5p remarkably enhances the bone differentiation promoting performance of sEVs.
The above results indicate that the engineered vesicles of the inventive construction method significantly enhance bone-promoting properties of natural vesicles.
The forward and reverse nucleotide sequences and the primer sequences of miR-423-5p in the invention are respectively as follows:
(1) Positive chain: UGAGGGGCAGAGAGCGAGACUUU when the nucleotide sequence table is manufactured, changing 'U' into 'T', and manufacturing the nucleotide sequence table, specifically TGAGGGGCAGAGAGCGAGACTTT (SEQ ID NO. 1);
(2) Reverse chain: AAAGUCUCGCUCUCUGCCCCUCA when the nucleotide sequence table is manufactured, changing 'U' into 'T', and manufacturing the nucleotide sequence table, specifically AAAGTCTCGCTCTCTGCCCCTCA (SEQ ID NO. 2);
（3）ALP：F：5'-TAAGGACATCGCCTACCAGCTC-3'（SEQ ID NO.3）；R：5'-TCTTCCAGGTGTCAACGAGGT-3'（SEQ ID NO.4）；
（4）OSX：F：5'-AGGTGTATGGCAAGGCTTCG-3'（SEQ ID NO.5）；R：5'-GCAGGCAGGTGAACTTCTTCT-3'（SEQ ID NO.6）；
（5）RUNX2：F：5'-CTTTACTTACACCCCGCCAGTC-3'（SEQ ID NO.7）；R：5'-AGAGATATGGAGTGCTGGTC-3'（SEQ ID NO.8）；
（6）COL1：F：5'-AACATGGAGACTGGTGAGACCT-3'（SEQ ID NO.9）；R：5'-CGCCATACTCGAACTGGAATC-3'（SEQ ID NO.10）；
（7）GAPDH：F：5'-CTTTGGTATCGTGGAAGGACTC-3'（SEQ ID NO.11）；R：5'-GTAGAGGCAGGGATGATGTTCT-3'（SEQ ID NO.12）。
Claims (4)
1. The construction method of the miR-423-5p high-abundance engineering vesicle is characterized in that the miR-423-5p high-abundance engineering vesicle comprises a small extracellular vesicle and miR-423-5p, and the positive strand nucleotide sequence of the miR-423-5p is as follows: UGAGGGGCAGAGAGCGAGACUUU the reverse nucleotide sequence is: AAAGUCUCGCUCUCUGCCCCUCA;
The small extracellular vesicles are small extracellular vesicles secreted by dental follicle stem cells;
the construction method of the miR-423-5p high-abundance engineering vesicle comprises the following steps:
(1) Uniformly mixing miR-423-5p with a nucleic acid transfection reagent, and then adding small extracellular vesicles for incubation;
(2) Putting the product obtained in the step (1) into an adsorption column for centrifugation to obtain miR-423-5p small extracellular vesicles;
(3) Adding miR-423-5p small extracellular vesicles into a ultrafiltration tube for purification and concentration, and reserving upper-layer liquid to obtain the miR-423-5p small extracellular vesicles.
2. The method for constructing the miR-423-5p high-abundance engineering vesicle according to claim 1, characterized in that the incubation temperature in the step (1) is 35-38 ℃ and the incubation time is 0.5-2 h.
3. The method for constructing the miR-423-5p high-abundance engineering vesicle according to claim 1, characterized in that in the step (2), the centrifugal speed is 1500-2500 rpm, and the centrifugal time is 30-60 s.
4. The method for constructing the miR-423-5p high-abundance engineering vesicle according to claim 1, characterized in that the molecular weight cut-off of the ultrafiltration tube in the step (3) is 25-35 kD.
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