CN107243012B

CN107243012B - Application of exosomes-loaded miR-93-5p in treatment of rheumatoid arthritis

Info

Publication number: CN107243012B
Application number: CN201710379029.9A
Authority: CN
Inventors: 王胜军; 朱栋炜; 田洁; 王运刚; 马洁; 吴昕雨; 许化溪
Original assignee: Jiangsu University
Current assignee: Jiangsu University
Priority date: 2017-05-25
Filing date: 2017-05-25
Publication date: 2020-01-24
Anticipated expiration: 2037-05-25
Also published as: CN107243012A

Abstract

The invention provides application of exosomes loaded miR-93-5p in treatment of rheumatoid arthritis, belonging to the technical field of biomedicine; the method firstly prepares G-exosomes for over-expressing or blocking miR-93-5p, and in-vitro experiments prove that the G-exosomes for over-expressing miR-93-5p more obviously inhibits the differentiation of Th17 cells; by researching the effect of the G-exosomes over-expressing and blocking miR-93-5p on the pathogenesis of a CIA model mouse, the protection effect of the G-exosomes over-expressing miR-93-5p on the CIA model mouse is found to be stronger; g-exosomes blocking miR-93-5p do not have the effect; the result shows that the G-exosomes over-expressing miR-93-5p can be used for treating rheumatoid arthritis.

Description

Application of exosomes-loaded miR-93-5p in treatment of rheumatoid arthritis

Technical Field

The invention belongs to the technical field of biomedicine, and relates to application of miR-93-5p loaded by exosomes in treatment of rheumatoid arthritis, in particular to application of miR-93-5p loaded by granulocyte-like myelogenous suppressor cell-derived exosomes in treatment of rheumatoid arthritis.

Background

Rheumatoid Arthritis (RA) is a chronic progressive autoimmune disease with unknown etiology and involves multiple systems of the whole body, and the pathological characteristic is mostly chronic synovial inflammation with symmetry of small joints of hands and feet, which causes physical harm and seriously affects human production and life. The exact pathogenesis of RA is unknown, and immunological and epidemic studies suggest that it is associated with genetic, hormonal, environmental, viral infections, and other factors. The main aim of the current treatment is to reduce the inflammation of the joints, inhibit the development of the disease, protect the joints as much as possible and reduce the mobility of the disease. The clinical treatment for RA mainly includes drug therapy and immunological therapy. The drug therapy mainly comprises non-steroidal drugs, antirheumatic drugs, glucocorticoid and the like, but the drugs bring more toxic and side effects, influence the metabolism function of liver and kidney and easily generate drug dependence, and besides, hormone drugs can cause gastrointestinal adverse reactions, central obesity, low immunity, osteoporosis and the like of patients. The immunological therapy mainly comprises immune purification and biological targeting drug therapy, wherein the immune purification can remove autoantibodies, immune complexes, immunoglobulin and the like in blood of patients with rheumatoid arthritis, but the treatment cost is high, the operation is complex and the wound is large. The biological targeting drugs mainly comprise TNF-alpha antagonist and biological preparation aiming at RA inflammation molecules, and the drugs have the effects of quickly inhibiting inflammation reaction and repairing injured joints, but are very expensive, and have the risks of causing tuberculosis recrudescence, liver injury, tumor development and the like of patients with latent tuberculosis infection. Other biological agents directed against RA-inflammatory molecules include IL-1 receptor antagonists and IL-6 receptor antagonists, among others. The traditional drug therapy brings great toxic and side effects, the existing immunological therapy method is relatively single and expensive, and a lot of research and improvement are still needed for the treatment of RA.

The present investigators have discovered a new class of CD4⁺Helper T cells, called helper T cell type 17 (Th 17) by secreting IL-17 specifically from this cell subset, can also secrete cytokines such as IL-6, TNF-alpha, I L-17 and Th17 cells play an important role in the pathogenesis of RA and its animal models.

In recent years, relevant researches show that MDSCs have great potential in the aspect of treatment of autoimmune diseases, and experiments have been carried out to treat collagen-induced arthritis (CIA) of mice by using MDSCs and obtain certain curative effect; however, the disadvantages of insufficient source of MDSCs, inconvenient storage and complex components limit the application of the MDSCs. Myeloid Derived Suppressor Cells (MDSCs) are heterogeneous populations of myeloid derived cells, and in mice, MDSCs are cells that are double positive for Gr-1 (consisting of Ly6G and Ly 6C) and CD11b, and are divided into two subtypes according to cell morphology and the expression level of Ly6G and Ly 6C: CD11b⁺Ly6G⁺Ly6C^lowGranulocyte-like MDSC (G-MDSC) and CD11b⁺Ly6G^-Ly6C^hiMonocyte-like MDSCs (M-MDSCs), wherein G-MDSCs inhibit T cell activation and proliferation primarily through arginase 1(Arg 1, Arg-1) and Reactive Oxygen Species (ROS), and M-MDSCs exert inhibitory effects primarily through Arg-1 and nitric oxide.

Research shows that G-MDSC derived exosomes (G-exosomes) have a protective effect on both inflammatory bowel disease model mice and CIA model mice, the exosomes are vesicles with biological activity generated by an endocytosis system, and various lipids, proteins and nucleic acids are encapsulated in the exosomes and can transport substances carried by the exosomes to receptor cells to play a biological role. At present, applications of exosomes mainly include drug carriers and disease diagnosis, and exosomes are used as drug delivery carriers, and have the advantages of low immunogenicity, high transportation efficiency, good stability, strong targeting and capability of crossing blood brain barriers compared with current liposome carriers and polymer carriers. Since the exosomes secreted by different cells under different diseases contain different nucleic acid and protein components, analysis of the components contained in the exosomes isolated from the patient's body fluid can be used as a basis for disease diagnosis. More and more studies have shown that exosomes play an important role in the development of inflammation in autoimmune diseases, but the composition of G-MDSC-derived exosomes is complex and its specific mechanism of action is still unclear.

More studies have shown that exosomes-carried mirnas have the effect of inhibiting T-cell proliferative differentiation, e.g. Treg cell-derived exosomes-carried Let-7d can inhibit Th1 cell-mediated inflammatory responses and reduce the disease severity in vivo in a mouse model of inflammatory bowel disease (Okoye IS, Coomes SM, Pelly VS, Czieso S, papayannoopoulos V, tolmechora T, et al. Micro RNA (microRNA, miRNA) is a series of small non-coding RNAs with the length of 18-22 nucleotides, and the small non-coding RNAs are mainly specifically combined with a 3 'untranslated region (3' -UTR) of a target gene to inhibit the translation of the target gene or induce the degradation of mRNA of the target gene, so that the expression of a target protein is inhibited.

According to the structural and functional characteristics of the G-exosomes, the miRNA loaded by the G-exosomes is used for treating RA, so that the aims of high safety and strong targeting property can be achieved, the clinical application prospect is good, the active effect on maintaining the immune balance of the organism is expected to be played, and a brand-new treatment way is provided for treating RA. The invention carries out artificial intervention on miRNA in G-exosomes, and can obviously improve the application effect after overexpression so as to obtain the G-exosomes with better treatment effect.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a preparation method of G-exosomes over-expressing miR-93-5p and application of the G-exosomes in preparation of a medicine for treating rheumatoid arthritis.

In order to achieve the purpose, the technical means adopted by the invention are as follows:

the invention firstly provides application of miR-93-5p in preparation of a medicament for treating rheumatoid arthritis.

The invention also provides a screening method of the miR-93-5 p.

The invention also provides a preparation method of the G-exosomes over-expressing the miR-93-5 p.

The invention also provides a medicament for treating rheumatoid arthritis, which comprises G-exosomes over-expressing miR-93-5 p.

Specifically, the technical scheme adopted is as follows:

according to the invention, M-MDSC and G-MDSC are firstly separated and extracted, then exosomes (G-exosomes and M-exosomes) secreted by the two cells are extracted and identified, the influence of the G-exosomes and the M-exosomes on the pathogenesis of a CIA model mouse is verified through experiments, and the result shows that the G-exosomes have the effect of inhibiting the pathogenesis of the CIA model mouse, and the Th17 cells are treated by the G-exosomes and the M-exosomes, so that the G-exosomes can inhibit the differentiation of the Th17 cells, further the pathogenesis of the mouse is reduced, and the M-exosomes have no obvious inhibition effect.

Carrying out extraction sequencing analysis on miRNA (micro ribonucleic acid) on G-exosomes and M-exosomes, and screening out the first twenty miRNA with larger content difference absolute value in the G-exosomes and the M-exosomes; further screening according to the structure and functional characteristics of miRNA, screening out 9 miRNA which are likely to act on RA, respectively: miR-16-5p, miR-29a-3p, miR-93-5p, miR-22-3p, miR-26b-5p, Let-7f-5p, Let7g-5p, miR-221-3p and miR-92a-3 p.

And designing primers according to the screened 9 miRNA sequences, detecting the change of the expression quantity of the 9 miRNAs in the Th17 cells after the G-exosomes treatment by qRT-PCR, and screening 3 miRNAs with the maximum increase of the expression quantity in the Th17 cells after the G-exosomes treatment as miR-16-5p, miR-29a-3p and miR-93-5 p.

And (3) transfecting initial T cells with mimics and mimics-negative controls corresponding to the screened 3 miRNAs (miR-16-5p, miR-29a-3p and miR-93-5p), inducing and differentiating Th17 cells, and detecting through qRT-PCR (quantitative reverse transcription-polymerase chain reaction), wherein results show that miR-93-5p can inhibit the differentiation of the initial T cells to Th17 cells, and also show that G-exosomes can inhibit the differentiation of Th17 cells through the loaded and carried miR-93-5 p.

G-exosomes for over-expressing or blocking miR-93-5p is further prepared by transfecting G-MDSC of mimics and inhibors, and in-vitro experiments prove that the G-exosomes for over-expressing miR-93-5p more obviously inhibits the differentiation of Th17 cells compared with the original G-exosomes; and G-exosomes blocking miR-93-5p lose the inhibition effect of the original G-exosomes on Th17 cell differentiation. The result shows that miR-93-5p carried by G-exosomes inhibits the differentiation of Th17 in vitro, and the G-exosomes over-expressing miR-93-5p prepared by the invention has stronger inhibition capability on the differentiation of Th17 cells compared with the original G-exosomes.

According to the invention, by researching the effect of the G-exosomes over-expressing and blocking miR-93-5p on the pathogenesis of CIA model mice, the G-exosomes over-expressing miR-93-5p is found to have stronger protective effect on the CIA mice than the original G-exosomes, and the G-exosomes blocking miR-93-5p loses the inhibitory effect of the original G-exosomes on the pathogenesis of the CIA mice. Results show that miR-93-5p carried by G-exosomes reduces the morbidity of a CIA model mouse, and the G-exosomes over-expressing miR-93-5p prepared by the invention has stronger protective effect on the CIA model mouse compared with the original G-exosomes.

The invention discovers that miR-93-5p loaded on G-exosomes can inhibit the expression of a corresponding receptor cell target protein STAT3 in vivo and in vitro, so that the inhibition effect on Th17 cell differentiation is exerted.

THE ADVANTAGES OF THE PRESENT INVENTION

(1) The invention discovers for the first time that miR-93-5p carried by G-exosomes can reduce the morbidity of a CIA model mouse by inhibiting the differentiation of Th17 cells, and provides a new method for treating RA.

(2) The method for preparing the G-exosomes over-expressing miR-93-5p has the advantages of short time consumption, convenient operation, stable activity of the contained components, and long preservation time of more than 1 year at-80 ℃; the structure and active ingredients are stable at room temperature, and the clinical application is facilitated.

(3) Compared with the common G-exosomes, the G-exosomes over-expressing miR-93-5p prepared in the invention have stronger effects of inhibiting Th17 cell differentiation and relieving the severity of diseases of a CIA model mouse.

(4) The miR-93-5p loaded by G-exosomes in the invention can inhibit the expression of corresponding receptor cell target protein STAT3 in vivo and in vitro, and the action target is clear.

(5) The exosomes as the drug transport carrier have the advantages of low immunogenicity, high transport efficiency, good stability, quick absorption and strong targeting property, compared with the existing drugs for treating RA, the G-exosomes over-expressing miR-93-5p prepared by the invention has the advantages of no toxic or side effect, no influence on normal liver and kidney metabolism function, simple and convenient preparation process and low required cost, and in addition, the G-exosomes over-expressing miR-93-5p prepared by the invention has clear drug action mechanism and is beneficial to monitoring of drug curative effect.

Drawings

FIG. 1 shows the purity identification results of the G-MDSC and M-MDSC prepared by sorting according to the present invention, wherein A is G-MDSC and B is M-MDSC.

FIG. 2 is a transmission electron micrograph of G-exosomes and M-exosomes prepared by sorting according to the present invention, wherein A is G-exosomes and B is M-exosomes.

FIG. 3 is a graph showing the particle size frequency distribution of G-exosomes and M-exosomes prepared by sorting according to the present invention, wherein A is G-exosomes and B is M-exosomes.

FIG. 4 shows the results of Westem blot detection of the expression of marker proteins in G-exosomes and M-exosomes.

FIG. 5 shows the effect of G-exosomes and M-exosomes on mean arthritis index during pathogenesis in CIA model mice.

FIG. 6 shows the effect of G-exosomes and M-exosomes on the degree of toe swelling in CIA model mice.

FIG. 7 shows the effect of G-exosomes and M-exosomes on the differentiation of Th17 cells in vitro, where A is the ratio of Th17 cells in phosphate buffer control; b is the proportion of Th17 cells in the group treated by N-exosomes; c is the proportion of Th17 cells in the M-exosomes treated group; d is the proportion of Th17 cells in the G-exosomes treated group.

FIG. 8 shows the miRNA at the first 20 th position of the absolute value of the difference between the miRNA content loaded by G-exosomes and M-exosomes.

FIG. 9 shows the results of changes in the expression level of miRNA in G-exosomes-treated Th17 cells.

FIG. 10 shows the results of the changes in the expression levels of miR-93-5p, miR-16-5p and miR-29a-3p in the induced-differentiation Th17 cells after transfection of corresponding mimics into the naive T cells.

FIG. 11 shows the effect of the transfection of corresponding mimics of miR-93-5p, miR-16-5p and miR-29a-3p into the primary T cells on induced differentiation of Th17 cells, wherein A is the proportion of the mimics to the Th17 cells of the negative control group; b is the proportion of Th17 cells in the miR-16-5p-mimics treatment group; c is the proportion of Th17 cells in the miR-29a-3p-mimics treatment group; d is the proportion of Th17 cells in the miR-93-5p-mimics treatment group.

FIG. 12 is a graph showing the effect of transfecting mimics or inhibitors of miR-93-5p into G-MDSC and then expressing miR-93-5 p.

FIG. 13 is a graph showing the expression amount of miR-93-5p expressed by G-exosomes after transfection of a mimics or inhibitors of miR-93-5p into G-MDSC.

FIG. 14 shows the effect of G-exosomes over-expressing or blocking miR-93-5p on Th17 cell differentiation, wherein A is the proportion of negative control group Th17 cells, B is the proportion of phosphate buffer control group Th17 cells, C is the proportion of G-exosomes treatment group Th17 cells, D is the proportion of mimics-NC treatment group Th17 cells, E is the proportion of mimic treatment group Th17 cells, F is the proportion of inhibitor-NC treatment group Th17 cells, and G is the proportion of inhibitor treatment group Th17 cells.

FIG. 15 shows the expression amount of miR-93-5p in cells in a Th17 induction system by G-exosomes overexpressing or blocking miR-93-5 p.

FIG. 16 shows the degree of effect of G-exosomes overexpressing or blocking miR-93-5p on the arthritis index of a mouse model of CIA.

FIG. 17 is a visual image of the degree of influence of G-exosomes over-expressing or blocking miR-93-5p on toe swelling of a CIA model mouse.

FIG. 18 shows the results of HE stained sections of hind toes of CIA model mice treated with G-exosomes overexpressing or blocking miR-93-5p, wherein A is a PBS buffer group, B is a G-exosomes group, C is a mimics-NC-exosomes group, D is a mimics-exosomes group, E is an inhibos-NC-exosomes group, and F is an inhibos-exosomes group.

FIG. 19 is a flow chart of the proportion of Th17 cells in popliteal lymph node cells after treatment of CIA model mice with G-exosomes overexpressing or blocking miR-93-5 p.

FIG. 20 is a graph of G-exosomes overexpressing or blocking miR-93-5p against CIA model mouse draining lymph node CD4⁺Results of miR-93-5p expression level in T cells.

FIG. 21 is a graph of possible binding sites for miR-93-5p and STAT 3.

FIG. 22 shows the expression of total cell STATs 3 in a Th17 induction system by G-exosomes overexpressing or blocking miR-93-5p, wherein A is a Western blot detection result, and B is a statistical analysis chart.

FIG. 23 shows G-exosomes overexpressing or blocking miR-93-5p to CIA model mouse draining lymph node CD4⁺And (3) expression quantity of T cell total STAT3, wherein A is a Western blot detection result, and B is a statistical analysis chart.

Detailed Description

The technical solutions of the present invention will be further described with reference to specific examples, but the present invention is not limited to these examples.

Example 1: extraction and identification of G-MDSC and M-MDSC

(1) Constructing a tumor-bearing mouse model: the culture was carried out in a DMEM medium (Gibco, USA) containing 10% calf serum (Gibco, USA) and pH 7.3 at 37 ℃ and 5% CO₂Mouse Lewis lung adenocarcinoma cell lines (purchased from Shanghai Life sciences) were cultured under the conditions. The morphology, adherence, density and color change of the culture solution of the cells are closely observed in the culture process, and when the cells grow to reach the density of about 85% of the bottom of the culture dish, 0.25% of pancreatin (purchased from doctor de bioengineering, Ltd.) is used for digestion and passage. Taking Lewis cells in logarithmic growth phase, injecting 3.0 × 10 subcutaneous injection into right abdomen of each mouse⁶The individual cell method is to construct a transplantation tumor of a male C57BL/6 mouse (purchased from the center of experimental animals of Jiangsu university) with the age of 6-8 weeks, and the living state and the tumor size of the mouse are closely observed in the process of model construction.

(2) Isolation of splenocytes from tumor-bearing mice: on day 30 after injection of Lewis cells into mice, the mice were sacrificed by taking blood from the eyeballs and dislocation of the neck, and after soaking the mice in 75% alcohol for 5min, the mice were dissected in an ultra-clean bench to obtain spleens; filtering the ground spleen with a 0.22 μm mesh screen, washing with PBS (phosphate buffer solution), collecting the suspension, centrifuging at 4 deg.C and 500g for 5min, and discarding the supernatant; adding 5mL of ACK erythrocyte lysate (ACK) (Shanghai Rui Valley Biotechnology Co., Ltd.) into the precipitated cells, mixing, and lysing erythrocytes for 5 min; adding 10% calf serum-containing RPMI-1640 culture solution (Gibco, USA) to 10mL, mixing, centrifuging at 4 deg.C and 500g for 5min, and discarding the supernatant; a small amount of cells were counted under a microscope while the cells were resuspended to 10 mL.

(3) Magnetic bead sorting G-MDSC: every 10 th⁸The splenocytes were resuspended in 350. mu.L PBE, 50. mu.L of FcR Blockingreagent (America, Edward, Germany) was added, mixed well and incubated on ice for 10 min; every 10 th⁸Adding 40 μ L of anti-Ly-6G Biotin (America and whirlwind, Germany), mixing, incubating on ice for 30min, and mixing once every 10 min; adding 10mL PBE, mixing uniformly, centrifuging the suspension at 4 ℃ and 500g for 5min, and removing the supernatant; every 10 th⁸50 μ L of anti-Ly-6G-Biotin Beads (Mei Germany) was added to each splenocyteTian and whirly company), mixing, placing on ice, incubating for 30min, and mixing once every 10 min; adding 10mL PBE, mixing uniformly, centrifuging the suspension for 5min at 4 ℃ under 500g, and removing the supernatant; adding 500 μ L PBE, and mixing; loading the MACS sorting column on a VarioMACS sorter (America whirlwind, Germany), adding 3mL of PBE for rinsing for 1 time, adding the spleen cell suspension, after the last drop of cell suspension flows out, washing the sorting column with 3mL of PBE, and repeating for 3 times; and (3) removing the sorting column from the sorter, placing the sorting column on a 10mL centrifuge tube, adding 5mL precooled PBE, pushing a piston, pressing out the cells in the separation column, repeating for 1 time to obtain 10mL cell suspension, namely G-MDSC, and counting under a microscope.

(4) Magnetic bead sorting of M-MDSC: collecting cell suspension flowing out of a sorting column during sorting of G-MDSC, centrifuging for 5min at 4 ℃ under 500G, and discarding supernatant; every 10 th⁸Adding 40 μ L of anti-Gr-1Biotin (America and whirlwind, Germany) into each splenocyte, mixing, placing on ice, incubating for 30min, and mixing once every 10 min; adding 10mL PBE, mixing uniformly, centrifuging at 4 deg.C and 500g for 5min, and removing supernatant; every 10 th⁸Adding 50 μ L of anti-Gr-1-Biotin Beads (America and whirlwind, Germany) into each splenocyte, mixing, incubating on ice for 30min, and mixing every 10 min; adding 10mL PBE, mixing uniformly, centrifuging for 5min at 4 ℃ under 500g, and removing supernatant; adding 500 mu L of PBE, and uniformly mixing; loading a MACS sorting column on a sorter (America, Gentle company), adding 3mL of PBE for rinsing for 1 time, then adding the cell suspension to be sorted, adding 3mL of PBE for washing the sorting column after the last drop is drained, and repeating for 3 times; and (3) removing the sorting column, placing the sorting column on a 10mL centrifuge tube, adding 5mL of PBE, pushing a piston, pressing out cells in the column, repeating for 1 time to obtain 10mL of cell suspension, namely M-MDSC, and taking a small amount of the M-MDSC under a microscope for counting.

(5) Purity identification of G-MDSC and M-MDSC: taking the G-MDSC 1 x 10 obtained in the step (4) and the step (5) respectively⁶M-MDSC 1X 10⁶Resuspend 1mL of PBS in an EP tube, centrifuge at 500g for 5min at 4 deg.C, discard the supernatant and leave about 100. mu.L of PBS; after mixing, 0.5. mu.L of an anti-Ly-6G antibody (E bioscience, USA) and 0.5. mu.L of an anti-CD 11b antibody (E bioscience, USA) were added to the EP tube for G-MDSC, and 0.5. mu.L of an anti-CD 11b antibody (E bioscience, USA) was added to the EP tube for M-MDSCanti-Ly-6C antibody (eBioscience, usa) and 0.5 μ L of anti-CD 11b antibody (eBioscience, usa); incubating at 4 deg.C for 30 min; after resuspending 1mL of PBS, the mixture was centrifuged at 500g for 5min at 4 ℃ and the supernatant was discarded, and 200. mu.L of PBS was added for resuspension and detection by flow cytometry (BD Co., USA). As shown in FIG. 1, the purity of G-MDSC obtained in the present invention was 97.6%, and the purity of M-MDSC was 95.3%.

Example 2: extraction of G-exosomes and M-exosomes and determination of protein concentration

(1) The sorted G-MDSC and M-MDSC were resuspended in RPMI-1640 culture medium (Gibco, USA) containing 10% calf serum (serum was centrifuged at 4 ℃ and 100000G for 16h, and the upper layer of serum was left 2/3 to remove exosomes from serum) at 1.5X 10 per well⁶One cell, and a volume of 1mL per well seeded in 24-well plates; at 37 deg.C, 5% CO₂After culturing under the conditions of (1) for 24 hours, a culture supernatant was collected.

(2) Centrifuging the collected culture supernatants of G-MDSC and M-MDSC for 20min at 4 deg.C and 300G, and collecting the supernatant; centrifuging the supernatant at 4 deg.C for 30min at 1000g, and collecting the supernatant; centrifuging the supernatant at 4 deg.C and 10000g for 30min, and collecting the supernatant; the supernatant was put into a 100kDa ultrafiltration tube (Milipore, Germany), centrifuged at 1000g for 30min at 4 ℃ and the concentrated solution in the tube was collected.

(3) By ExoQuick-TC^TMExosomes kit (SBI corporation, usa) extracts exosomes: mixing the concentrated solution with ExoQuick-TC^TMMixing the Exosome reagents according to the volume ratio of 5:1, slightly mixing the mixed reagents, and standing the mixture in a refrigerator at the temperature of 4 ℃ for more than 16 hours; centrifuging at 4 deg.C and 1000G for 30min, discarding supernatant, and collecting precipitate to obtain G-exosomes or M-exosomes. The exosomes obtained by the preparation were dissolved in PBS and dispensed into EP tubes and stored at-80 ℃.

(4) Mixing exosomes dissolved solution with equal volume of lysis solution (RIPA: PMSF: 250:1), standing on ice for 1 hr while shaking on a shaker for 1min every 10 min; centrifuging at 12000g for 15min at 4 deg.C, and collecting supernatant. The protein concentration in the exosomes lysis supernatant was measured according to the method of the BCA miniprotein quantification kit (beijing kang, century limited) instructions to determine the content of the extracted exosomes. The results show that the inventionEach of the G-MDSC or M-MDSC obtained in Ming was extracted at about 3X 10 after 24 hours of culture^-6μ g of exosomes.

Example 3: identification of G-exosomes and M-exosomes

(1) The morphology of G-exosomes was observed by transmission electron microscopy: dripping 20 mu L G-exosomes or M-exosomes suspension on a sample-carrying copper net with the diameter of 3mm, and standing for 2min at room temperature; gently sucking the liquid with filter paper, dripping 2% phosphotungstic acid solution (Shanghai Junrui science and technology Co., Ltd.) with pH of 6.8 on a copper net, and re-dyeing lmin; the filter paper absorbs the dye solution, the dye solution is baked under an incandescent lamp, and the result is observed under a transmission electron microscope (Holland Philips company), the result is shown in figure 2, G-exosomes and M-exosomes are in a round or oval microcapsule structure, and have complete envelopes, and low-electron-density components are in a cavity; FIG. 3 shows the particle size frequency distributions of G-exosomes and M-exosomes, respectively, showing that the particle sizes of G-exosomes and M-exosomes are mainly distributed between 20-120 nm.

(2) Western blot detection of protein molecules CD9, CD63 contained in G-exosomes and M-exosomes and Calnexin molecule contained in mitochondria: preparing 5% concentrated glue and 12% separation glue, cracking G-exosomes and M-exosomes by RIPA protein cracking solution (Shanghai Bin Yuntan biotechnology, Ltd.), loading the cracked G-exosomes according to the total amount of 250 mu G protein, and performing 100V constant-pressure electrophoresis; after 90min of constant current rotation by 350mA, sealing the PVDF membrane lh by 5% skimmed milk; incubating overnight at 4 ℃ with anti-CD 63 monoclonal antibody (eBioscience, USA) and anti-Calnexin monoclonal antibody (eBioscience, USA); after being taken out, the membrane was washed with TBS/T (Shanghai Jing chemical science and technology Co., Ltd.) for 10min × 3 times, and incubated with a secondary antibody labeled with horseradish peroxidase (E Bioscience, USA) at 37 ℃ for 30 min; taking out, washing with TBS/T (Shanghai Jing chemical science and technology Co., Ltd.) for 10min × 3 times; ImageQuant LAS 4000 gel imaging system (GE corporation, usa) was exposed to light for color development, and as shown in fig. 4, G-exosomes and M-exosomes specifically highly express CD9 and CD63 molecules, but G-exosomes and M-exosomes do not contain calnexin localized on the endoplasmic reticulum membrane of the cell. In view of the morphology, size and protein molecule expression results of the extracted G-exosomes and M-exosomes, the G-exosomes and M-exosomes were successfully extracted in the present example.

Example 4: experiments prove that the immunosuppressive functions of G-exosomes and M-exosomes

(1) Constructing a CIA model mouse:

first immunization: mixing 2mg/mL bovine type II collagen (Sigma, USA) and 2mg/mL Freund's complete adjuvant (Sammerfei, USA) in equal volume, and grinding on ice to water-in-oil state; subcutaneously injecting 0.1mL of the above mixture into the tail root of each DBA/1 mouse (Shanghai Slek laboratory animals, Ltd.);

and (3) second immunization: the mixture was mixed in equal volume with 2mg/mL bovine type II collagen (Sigma, USA) and 2mg/mL Freund's incomplete adjuvant (Sermer fly, USA), ground to water-in-oil state on ice, and injected subcutaneously with 0.1mL of the above mixture into the tail root of each DBA/1 mouse (Shanghai Si Rick laboratory animals, Ltd.) on day 21 after the first immunization.

(2) Preparation of mouse mature neutrophil-derived exosomes (N-exosomes):

male C57BL/6 mice (Experimental animals center of Jiangsu university) of 6-8 weeks old were bled by removing eyeballs and dropped into the mice containing EDTA-Na₂In the anticoagulated blood collection tube (Suzhou Lingjie medical instruments Co., Ltd.), the eyeball blood was centrifuged at 1000rpm for 10min, the plasma was aspirated, and the blood cells were resuspended in 500. mu.L of PBS. 1mL of a single nuclear lymphocyte separation medium (Tianjin tertiary products science and technology Co., Ltd.) was placed in a 10mL glass tube, and the blood cell suspension was slowly dropped along the tube wall into the glass tube, and centrifuged at 2000rpm for 15min at 4 ℃ using a horizontal centrifuge. Slowly sucking the cell layer except the red blood cell layer from top to bottom with a suction pipe, discarding, adding 5mL of ACK (Shanghai Rui Gu Biotech Co., Ltd.), blowing, mixing, standing at room temperature for 5min, adding 5mL of RPMI1640 culture solution (Gibico, USA) containing 10% calf serum to terminate the lysis, mixing, and centrifuging at 4 deg.C for 5min at 500 g. The supernatant was discarded and washed 2 times with 10ml of PPBE to obtain neutrophils. N-exosomes were then extracted as in example 2.

(3) Grouping experiments:

PBS control group: each mouse was injected intravenously with 100 μ L of PBS at day 18 and 24 after the first immunization;

group of N-exosomes: 100 μ L of PBS-solubilized N-exosomes were injected intravenously into each mouse tail at 18 and 24 days after the first immunization, respectively, wherein 100 μ L of LPBS solubilized 100 μ g of N-exosomes;

group of G-exosomes: 100 μ L of PBS-solubilized G-exosomes were injected intravenously into each mouse tail at 18 days and 24 days after the first immunization, respectively, wherein 100 μ L of LPBS solubilized 100 μ G of G-exosomes;

group of M-exosomes: 100 μ L PBS-solubilized M-exosomes were injected intravenously into each mouse tail vein at day 18 and 24 after the first immunization, respectively, wherein 100 μ L PBS solubilized 100 μ g M-exosomes.

(4) The toes of each group of mice were monitored every 3 days after the second immunization, and the Arthritis Score (AS) of each mouse was recorded, AS representing the sum of all the scores of the diseased joints of each mouse, and the score of each joint lesion was scored at 5 points, with no redness and swelling scored at0 points, redness and swelling of joints scored at 1 points, mild redness and swelling of joints scored at 2 points, moderate redness and swelling of joints scored at3 points, and severe redness and swelling of joints accompanied by dysfunction scored at 4 points. The Mean Arthritis Index (MAI) was calculated for each group of mice, the MAI being the sum of AS of each group of mice divided by the number of mice in each group. As shown in FIG. 5, the mean arthritis index of the mice in the G-exosomes-treated group was reduced compared to that in the N-exosomes-treated group, while there was no significant difference between the PBS buffer, N-exosomes, M-exosomes-treated groups; FIG. 6 shows that the toes of the G-exosomes-treated mice had only mild redness and normal joint movement; however, most of the toes of the mice treated with PBS buffer, N-exosomes and M-exosomes were red and swollen significantly, and the joint movement was limited, and these results indicate that G-exosomes have the effect of inhibiting the onset of the diseases in the CIA model mice, while M-exosomes have no significant effect of inhibiting the onset of the diseases in the CIA model mice.

(5) Establishing a Th17 cell induced differentiation system: 24-well round bottom plates were coated with 200. mu.L of a coating solution containing 2. mu.g/mL of an anti-mouse CD3 monoclonal antibody (eBioscience, USA) overnight at 4 ℃; the coating solution was aspirated from each well, washed 1 time with PBS, and primary T cells were sorted by primary T cell sorting kit from David, America, and whirlwind, 1 per well.5×10⁶Density of individual cells primary T cells were seeded in 24-well plates with a total volume per well of 1 mL; anti-mouse CD28 monoclonal antibody (E bioscience, USA) with a final concentration of 2 μ g/mL, mouse recombinant TGF- β (E bioscience, USA) with a final concentration of 5ng/mL, IL-6 (E bioscience, USA) with a final concentration of 30ng/mL, IL-23 (E bioscience, USA) with a final concentration of 30ng/mL, anti-IL-4 (E bioscience, USA) with a final concentration of 5 μ g/mL, and anti-IFN- γ (E bioscience, USA) with a final concentration of 5 μ g/mL are added to each well; the cells were cultured in RPMI-1640 cell culture medium (Gibico, USA) containing 15% fetal bovine serum (Gibico, USA, treated with 100000g centrifugation for 16 h) and pH 7.2-7.4 at 37 deg.C and 5% CO₂Culturing for 72h under the condition.

(6) Grouping experiments: control group: 100 μ L of PBS was added as a control; n-exosomes, G-exosomes and M-exosomes were added to each well of the remaining groups at a final concentration of 60. mu.g/mL, as shown in FIG. 7, the proportion of Th17 cells was reduced in the G-exosomes treated group compared to the N-exosomes treated group, whereas there was no significant difference between PBS buffer, N-exosomes, M-exosomes treated groups. These results indicate that G-exosomes act to reduce mouse morbidity in CIA model mice, inhibit differentiation of Th17 cells in vitro, and exhibit a greater immunosuppressive function than M-exosomes.

Example 5: sequencing results and analysis of miRNA in G-exosomes and M-exosomes

(1) The extraction sequencing analysis of miRNA carried in G-exosomes and M-exosomes is entrusted to Sharp corporation in Guangzhou, and the skilled person in the art knows that G-exosomes and M-exosomes contain many specific miRNA and that there is a certain difference in expression amount. Determining the type of miRNA according to the analysis of the sequencing result and the information of related miRNA on the TargetScan website; as shown in FIG. 8, the first twenty bits of absolute values of the difference in the content of related miRNAs contained in G-exosomes and M-exosomes are miR-148a-3p (MIMAT0000516), miR-340-5p (MIMAT0004651), miR-16-5p (MIMAT0000527), miR-22-3p (MIMAT0000531), miR-27a-3p (MIMAT0000537), miR-92a-3p (MIMAT0000539), miR-140-3p (MIMAT0000152), miR-27b-3p (MIMAT0000126), miR-24-3p (MIMAT0000219), miR-26b-5p (MIMAT0000534), miR-423-3p (MIMAT0000516), Let-7f-5p (MIMAT0000525), miR-29a-3p (MIMAT0000535), miR-7a-5p (MIMAT 0000000000007), miR-670000140, miR-7 f-5p (MIMAT0000 7G), miR-29 a-0000535), miR-7 a-0000-7 p (miR-0000140), and miR-7 p (MIMAT 00008978), miR-26a-5p (MIMAT0000533), miR-221-3p (MIMAT0000669), miR-93-5p (MIMAT0000540) and miR-148b-3p (MIMAT0000580), and the number in brackets is the number information corresponding to the analysis of the MIRBASE website, and the number information is used as the miRNA to be screened.

(2) The miRNA to be screened is further screened, and miR-16-5p, miR-29a-3p, miR-93-5p and Let7g-5p are related to development of osteoarthritis and proliferation, differentiation and migration of osteoblasts; miR-22-3p and miR-26b-5p are involved in the pathogenesis of RA; miR-29a-3p, miR-93-5p and miR-221-3p are related to T cell proliferation, differentiation, immune response and the like; smad7, Smad6, FOS, PIK3R1, STAT3, EMOS, PIK3R1 and the like which are potential targets of miR-16-5p, miR-29a-3p, Let-7f-5p and miR-92a-3p are closely related to the proliferation and differentiation of T cells. Therefore, miR-16-5p, miR-29a-3p, miR-93-5p, miR-22-3p, miR-26b-5p, Let-7f-5p, Let7g-5p, miR-221-3p and miR-92a-3p are further screened out to serve as candidate miRNAs.

(3) Detecting the fold change of the expression amount of the candidate miRNA in Th17 induced cells after the treatment of G-exosomes: the control group and G-exosome-treated Th 17-induced cells in step (6) of example 4 were collected, and 1X 10 cells were cultured using 1mL of Trizol (Invitrogen, USA)⁶Inducing cell lysis by Th17, fully beating and uniformly mixing by using a pipette, adding 200 mu L of precooled chloroform (Shanghai chemical reagent company), immediately shaking for 15-30 s, and standing for 10min at normal temperature; centrifuging at 12000g for 15min at 4 ℃; liquid in the EP tube is layered, an upper water phase layer (containing RNA) is sucked into a new clean EP tube, 500 mu L of isopropanol is added, a pipettor is used for gently mixing the materials uniformly, and the mixture is kept stand at normal temperature for 5-10 min; centrifuging at 12000g for 15min at 4 deg.C, wherein RNA is deposited at the bottom of the tube and is semitransparent; the supernatant was discarded, and 1mL of 75% ethanol (Shanghai chemical Co., Ltd.) was added thereto for washing; centrifuging at 7800g for 5min at 4 deg.C; drying in a superclean bench at room temperature until RNA is semitransparent; dissolving with 5 μ L DEPC (Shanghai Biyuntian Co., Ltd.) water; the concentration of RNA was measured using a American Saimer flight tester.

(4) Reverse transcription is carried out on the RNA extracted in the step (3) according to the requirement of a Japanese Takara reverse transcription kit, primers are designed according to miRNA sequences, then the Guangzhou Sharpo company is entrusted to synthesize forward and backward primers of miR-16-5p, miR-29a-3p, miR-93-5p, miR-22-3p, miR-26b-5p, Let-7f-5p, Let7G-5p, miR-221-3p and miR-92a-3p, and expression quantity change of candidate miRNA in Th17 induced cells after G-exosomes treatment is detected by a qRT-PCR kit purchased from Shanghai BIO-RAD company, and the results are shown in figure 9, and the expression quantity of the candidate miRNA is increased most in the Th17 induced differentiated cells after G-exosomes treatment, namely miR-16-5p, miR-29a-3p and miR-93-5 p.

(5) Mimics and mimics-negative control (Shaw, Engreen Co., Ltd.) of miR-16-5p, miR-29a-3p and miR-93-5p are transfected to the primary T cells sorted in the step (5) in the example 4 by an Entrance TM-R transfection reagent (Beijing Engreen Co., Ltd.), and then induced to differentiate into Th17 cells according to the method in the example 4, and the results are shown in FIG. 10 by qRT-PCR detection, and the expression levels of miR-16-5p, miR-29a-3p and miR-93-5p in the Mimics transfected Th17 cells are correspondingly increased; as shown in FIG. 11, the ratio of differentiation from the initial T cells transfected with the mimics of miR-93-5p to Th17 cells was significantly lower than that of the initial T cells transfected with the mimics-negative control, but the initial T cells transfected with the mimics corresponding to miR-16-5p and miR-29a-3p had no significant change. These results indicate that exogenous miR-93-5p can inhibit the differentiation of primary T cells to Th17 cells, which suggests that G-exosomes may inhibit the differentiation of Th17 cells through the miR-93-5p carried by the G-exosomes.

Example 6: preparation of G-exosomes overexpressing or blocking miR-93-5p

(1) Centrifuging mics of miR-93-5p, a mics-negative control (mics-NC) and freeze-dried powder (invitobors-negative control, invibitors-NC), dissolving with sterile DEPC water, blowing and beating to obtain a storage solution with a final concentration of 20 mu M, and subpackaging and storing in a refrigerator at-80 ℃;

(2) cells were seeded at 1X 10 per well in 24-well plates⁶G-MDSC, supplemented with 10% calf serum in RPMI1640 medium (Gibico, USA) to450μL；

(3) Preparing a diluent of the mimics, the mimics-NC and the inhibitors-NC: adding mimics and mimics-NC 50nM into a clean EP tube, adding 1.25 μ L of storage solution of the mimics or the mimics-NC, and supplementing to 25 μ L with serum-free RPMI1640 culture solution (Gibico, USA); adding inhibitors and inhibitors-NC100nM into a clean EP tube, adding 2.5 mu L of inhibitors and inhibitors-NC storage liquid, and supplementing to 25 mu L with serum-free RPMI1640 culture solution (Gibico, USA);

(4) preparation of Entrance TM-R transfection reagent dilutions: adding corresponding amount of Entrance TM-R transfection reagent (Beijing Engreen company) into a clean EP tube, controlling the amount of the transfection reagent according to the amount of mimics, mimics-NC, inhibitors and inhibitors-NC, adding 1 mu L of transfection reagent into every 50nM, then supplementing to 25 mu L with a serum-free 1640 culture medium (Gibico company in USA), and standing for 5min at room temperature;

(5) preparation of transfection complexes: adding an Entrance TM-R transfection reagent (Beijing Engreen company) diluent into corresponding mimics, mimics-NC or inhibitors and inhibitors-NC diluents, immediately and fully mixing, and standing for 30min at room temperature;

(6) dripping 50 mu L of transfection compound into the inoculated cells in the step (2), and gently mixing;

(7) observing the cell state after 6 hours of transfection, and if the cell state is good, centrifuging to collect G-MDSC;

(8) g-exosomes overexpressing or blocking miR-93-5p were prepared using G-MDSCs transfected with mimics or inhibitors according to the method in example 2.

Example 7: validation of G-exosomes overexpressing or blocking miR-93-5p

(1) 1X 10 treatment groups in step (6) of example 6 were collected⁶G-MDSC, extracting the total RNA of the G-MDSC of each treatment group according to the method in the step (3) of the example 5, and detecting the concentration of the total RNA;

(2) the content of miR-93-5p of each treatment group G-MDSC is subjected to fluorescence quantitative detection according to the method in example 5, and as a result, the mimics or inhibitors of miR-93-5p are successfully transfected into the G-MDSC as shown in figure 12.

(3) Each treatment group of 3X 10 in step (8) of example 6 was collected⁷G-exosomes secreted from individual G-MDSC were dissolved in 1mL of Trizol (Invitrogen, USA).

(4) Quantitative analysis of miR-93-5P contained in each group of G-exosomes was performed according to the procedure in example 5, and as shown in FIG. 13, the expression amount of miR-93-5P in the G-exosomes secreted by the G-MDSC transfected with miR-93-5P was significantly increased (P <0.01) relative to that of the miR-93-5P in the mimics-NC group, while the expression amount of miR-93-5P in the G-exosomes secreted by the G-MDSC transfected with inhibitor of miR-93-5P was decreased (P <0.05) relative to that of the inhibitor-NC group. These results demonstrate that the G-exosomes over-expressing or blocking miR-93-5p are successfully extracted in the example, and the content of miR-93-5p in the G-exosomes over-expressing miR-93-5p prepared in the example is increased by about 15 times compared with the original G-exosomes.

Example 8: g-exosomes loaded miR-93-5p can inhibit differentiation of Th17 cells in vitro

(1) By adding 60 mu G/mL of G-exosomes over-expressing or blocking miR-93-5P to a Th17 cell induced differentiation system, as shown in FIG. 14, the proportion of Th17 cells in a miR-93-5P over-expressing G-exosomes treatment group (a miR-NC transfected group) is obviously reduced (P <0.01), and the proportion of Th17 cells in an miR-93-5P blocking G-exosomes treatment group (an inhibitor transfected group) is increased (P <0.05) relative to the inhibitor-NC group. These results indicate that miR-93-5p carried by G-exosomes can inhibit the differentiation of Th17 cells in vitro, and the G-exosomes over-expressing miR-93-5p has stronger inhibition capability on the differentiation of Th17 cells. In the embodiment, the G-exosomes over-expressing miR-93-5p have stronger inhibition capacity on the differentiation of Th17 cells compared with the original G-exosomes, and the proportion of Th17 cells is reduced by one time.

(2) The expression level of miR-93-5P in cells in a Th17 induction system after G-exosomes treatment for over-expressing or blocking miR-93-5P is detected according to the operation method in example 5, and as shown in FIG. 15, the G-exosomes treatment group for over-expressing miR-93-5P can obviously improve the miR-93-5P expression level of the induced cells (P <0.01) compared with the mimics-NC group, and the miR-93-5P expression level of the G-exosomes treatment group for blocking miR-93-5P is obviously reduced (P <0.001) compared with the inhibitors-NC group. These results indicate that G-exosomes can deliver miR-93-5p carried by G-exosomes to receptor cells so as to play an inhibiting function, and the miR-93-5p is delivered more efficiently by the G-exosomes over-expressing miR-93-5p prepared by the invention.

Example 9: miR-93-5p loaded on G-exosomes can more effectively inhibit the morbidity of a CIA model mouse and relieve the severity of diseases

(1) Utilizing the CIA model mouse constructed in example 4;

(2) CIA model mice were injected tail vein with G-exosomes overexpressing or blocking miR-93-5p at day 18 and day 24 after the first immunization.

(3) Grouping experiments:

PBS buffer group: each mouse was injected intravenously with 100 μ L of PBS into the tail vein on day 18 and 24 of immunization, respectively;

group of G-exosomes: mice were injected intravenously with 100 μ L of PBS-solubilized G-exosomes to each tail on day 18 and 24 of immunization, respectively;

mimics-NC-exosomes group: g-exosomes secreted by G-MDSC after transfection of mimic-NC dissolved in 100. mu.L PBS were injected intravenously to each mouse tail on day 18 and 24 of immunization, respectively;

mimics-exosomes group: injecting 100 μ L of PBS-solubilized G-exosomes secreted by G-MDSC after transfection with mimic into the tail vein of each mouse at day 18 and 24 of immunization, respectively;

inhibitors-NC-exosomes group: g-exosomes secreted by G-MDSC after tail vein injection of 100. mu.L of PBS-solubilized transfection inhibitors-NC to each mouse at day 18 and 24 of immunization, respectively;

inhibitors-exosomes group: g-exosomes secreted by G-MDSC after tail vein injection of 100. mu.L of PBS-solubilized transfected inhibitors were administered to each mouse on day 18 and 24 of immunization, respectively.

In each of the above groups, 100. mu.g of G-exosomes from different treatment groups were dissolved in 100. mu.L of PBS.

(4) As shown in FIG. 16, the mean arthritis index of the mice in the mimics-exosomes treated group was significantly decreased (P <0.05) compared to the mimics-NC-exosomes treated group, and the mean arthritis index of the mice in the inhibitors-exosomes treated group was significantly increased (P <0.01) compared to the inhibitors-NC-exosomes treated group. As shown in FIG. 17, the toes of the mice in the G-exosomes, mimics-NC-exosomes, mimics-exosomes and inhibitors-NC-exosomes groups had only slight red swelling and normal joint movement; however, most of the toes of the mice in the PBS buffer group and the inhibitors-exosomes group were red and swollen significantly, and the joint movement was limited. As shown in FIG. 18, the bone structures at the toe joints of mice in the groups of G-exosomes, mimics-NC-exosomes, mimics-exosomes and inhibitors-NC-exosomes were relatively intact, the joint spaces were relatively normal, and inflammatory cell infiltration was less; however, the bone structure at the toe joint of the mice in the PBS buffer group and the inhibitors-exosomes group is seriously damaged, the joint space is reduced, and a large amount of inflammatory cells infiltrate. The results show that miR-93-5p carried by G-exosomes can inhibit the morbidity of a CIA model mouse and relieve the severity of diseases, and the G-exosomes over-expressing the miR-93-5p have stronger protective effect on the CIA model mouse.

In the embodiment, compared with the original G-exosomes, the G-exosomes over-expressing miR-93-5p has stronger protective effect on CIA mice, and compared with the original G-exosomes, the average arthritis index of the mice treated by the G-exosomes over-expressing miR-93-5p is reduced by nearly one time, the toe swelling degree is obviously reduced, and the joint structure is more complete.

(5) The popliteal lymph nodes of each group of mice were isolated, single cell suspensions were prepared, and the percentage of Th17 cells in the popliteal lymph node cells of each group of mice was analyzed by flow cytometry as shown in FIG. 19, and the percentage of Th17 cells in the popliteal lymph node cells of the mimics-exosomes-treated group of mice was significantly reduced relative to the mimics-NC-exosomes-treated group (P)<0.01), the proportion of Th17 cells was significantly increased in the inhibitor-exosomes treated group relative to the inhibitor-NC-exosomes treated group (P)<0.001), and no significant difference was observed between the PBS-treated group and either the mimics-NC-exosomes-treated group or the inhibitors-NC-exosomes-treated group (P)>0.05). qRT-PCR detection of draining lymph node CD4 in each group of mice as shown in FIG. 20⁺Expression level of T cell miR-93-5p, mimics-exosomes treatment group mouse draining lymph node CD4⁺Expression level of T cell miR-93-5p relative to mimiThe cs-NC-exosomes treatment group was significantly elevated (P)<0.01), the expression level of miR-93-5P in the inhibition group is obviously reduced compared with that in the inhibition-NC-exosomes treatment group (P)<0.01), no significant difference was observed between the PBS buffer group and the mimics-NC-exosomes-treated group or the inhibitors-NC-exosomes-treated group (P)>0.05). The results show that the G-exosomes can inhibit the proportion of draining lymph node pathogenic Th17 cells of a CIA model mouse and relieve the severity of diseases by transporting miR-93-5p to receptor cells, and the G-exosomes over-expressing miR-93-5p prepared by the invention has higher miR-93-5p transporting efficiency and stronger protection effect on the CIA model mouse.

In the embodiment, the G-exosomes over-expressing miR-93-5p have stronger protective effect on CIA mice compared with the original G-exosomes, the proportion of Th17 cells in popliteal lymph node cells of mice treated by the G-exosomes over-expressing miR-93-5p is reduced by one time compared with the original G-exosomes, and the reduction of the proportion of pathogenic Th17 cells is important for inhibiting the pathogenesis of the CIA mice.

Example 10: g-exosomes loaded miR-93-5p can reduce expression of target protein STAT3

(1) According to the Targetscan website analysis, as shown in FIG. 21, miR-93-5p can be specifically combined with the 248-254 and 495-501 regions of the 3' UTR region of the target gene STAT 3.

(2) Cells in a Th17 induction system after the treatment of G-exosomes for over-expressing or blocking miR-93-5P are collected, and a Wes' trin blot detects the expression level of STAT3 in each treatment group, as shown in FIG. 22, the expression level of STAT3 in the G-exo mes treatment group for over-expressing miR-93-5P is reduced (P <0.05) compared with that in the mimics-NC group, while the expression level of STAT3 in the G-exomes treatment group for blocking miR-93-5P is increased (P <0.05) compared with that in the inhibitors-NC group. The results show that miR-93-5p carried by G-exosomes can inhibit the expression of receptor cell STAT3, so that the differentiation of Th17 cells is inhibited, and the G-exosomes over-expressing miR-93-5p prepared by the invention has a stronger effect of inhibiting the expression of target protein STAT3, so that a stronger immunosuppressive function is exerted. The G-exosomes over-expressing miR-93-5p prepared by the invention has a stronger effect of inhibiting the expression of a target protein STAT3, and compared with the original G-exosomes, the expression level of STAT3 is reduced by one time, so that the function of stronger inhibiting Th17 cell differentiation is exerted.

(3) Collecting the drainage lymph node CD4 of CIA model mouse after the treatment of G-exosomes for over-expressing or blocking miR-93-5p⁺T cells and Western blot detection of the expression level of STAT3 in each treatment group cell, as shown in FIG. 23, the expression level of miR-93-5P over-expressed G-exosomes treatment group is obviously reduced compared with the expression level of miR-NC STAT3 (P is obviously reduced)<0.01) and the G-exosomes treatment group blocking miR-93-5P has an increased expression level of STAT3 (P) relative to the inhibitorss-NC group (P)<0.05). The results show that miR-93-5p carried by G-exosomes can reduce the expression of receptor cell target protein STAT3 in vivo and in vitro of CIA model mice, and the G-exosomes over-expressing miR-93-5p prepared by the invention has stronger capacity of inhibiting the expression of the target protein STAT3, thereby playing a stronger protective role on the CIA model mice. The G-exosomes over-expressing miR-93-5p prepared by the invention has stronger capacity of inhibiting the expression of target protein STAT3, and compared with the original G-exosomes, the expression level of STAT3 is reduced by about 2 times, so that the G-exosomes over-expressing miR-93-5p has stronger protective effect on a CIA model mouse.

Claims

1. Application of G-exosomes over-expressing miR-93-5p in preparation of medicines for treating rheumatoid arthritis.

2. Application of G-exosomes over-expressing miR-93-5p in preparation of drugs for inhibiting rheumatoid arthritis.

3. The use according to any one of claims 1-2, wherein said use is the inhibition of Th17 cell differentiation.

4. The use according to any one of claims 1 to 2, characterized in that it is the inhibition of the expression of the corresponding receptor cell target protein STAT 3.

5. A medicament for treating rheumatoid arthritis, comprising G-exosomes overexpressing miR-93-5 p.