CN107243012B - Application of an exosomes-loaded miR-93-5p in the treatment of rheumatoid arthritis - Google Patents

Abstract

The invention provides an application of miR-93-5p loaded by exosomes in the treatment of rheumatoid arthritis, belonging to the technical field of biomedicine; the invention firstly prepares G-exosomes that overexpress or block miR-93-5p, In vitro experiments proved that G-exosomes overexpressing miR-93-5p more significantly inhibited the differentiation of Th17 cells; and by studying G-exosomes overexpressing and blocking miR-93-5p, G-exosomes overexpressed and blocked miR-93-5p on CIA model mice. It was found that G-exosomes overexpressing miR-93-5p had a stronger protective effect on CIA mice; while G-exosomes blocking miR-93-5p had no such effect; the results showed that overexpressing miR-93 G-exosomes of ‑5p can be used in the treatment of rheumatoid arthritis.

Description

The role of an exosomes loaded miR-93-5p in the treatment of rheumatoid arthritis application

technical field

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

Background technique

Rheumatoid arthritis (RA) is a chronic progressive autoimmune disease of unknown etiology that involves multiple systems of the body. The pathological features are mostly symmetrical chronic inflammation of the facet joints of the hands and feet. The physical harm caused will seriously affect the production and life of human beings. The exact pathogenesis of RA is unknown, and immunological and epidemiological studies believe that it is related to factors such as genetics, hormones, environment, and viral infection. At present, the main purpose of its treatment is to reduce inflammation of the joints, inhibit the development of the disease, protect the joints as much as possible and reduce the activity of the disease. Clinical treatment of RA mainly includes drug therapy and immunological therapy. Drug treatment mainly includes non-steroidal drugs, anti-rheumatic drugs, glucocorticoids, etc., but drugs bring many toxic and side effects, affect the metabolism of liver and kidney, and easily lead to drug dependence. In addition, hormone drugs can cause gastrointestinal Adverse reactions, central obesity, low immunity, osteoporosis, etc. Immunological treatment mainly includes immune purification and biological targeted drug treatment. Among them, immune purification can remove autoantibodies, immune complexes and immunoglobulins in the blood of rheumatoid arthritis patients, but the treatment cost is high, the operation is complicated, and the trauma is relatively large. . Biological targeted drugs mainly include TNF-α antagonists and biological agents targeting RA inflammatory molecules. These drugs have the effect of rapidly inhibiting the inflammatory response and repairing damaged joints, but they are very expensive, and there are cases of tuberculosis recurrence in patients with latent tuberculosis infection. Risk of burning, liver damage, cancer, etc. In addition, biological agents targeting RA inflammatory molecules include IL-1 receptor antagonists and IL-6 receptor antagonists. Traditional drug treatment brings great toxic and side effects. At present, immunological treatment methods are relatively simple and expensive. There is still a lot of research and improvement for the treatment of RA.

Researchers have discovered a new type of CD4 ⁺ helper T cells, and this subset of cells specifically secretes IL-17 and is called helper T cell type 17 (Th17), which can also secrete IL-17. Cytokines such as IL-6, TNF-α, IL-17 and Th17 cells play an important role in the pathogenesis of RA and its animal models.

In recent years, related studies have shown that MDSCs have great potential in the treatment of autoimmune diseases. MDSCs have been used in experiments to treat collagen-induced arthritis (CIA) in mice, and certain curative effects have been achieved; The disadvantages of insufficient source, inconvenient storage and complex composition limit its application. Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of bone marrow-derived cells. In mice, MDSCs are double-positive cells for Gr-1 (composed of Ly6G and Ly6C) and CD11b. Morphology and the expression levels of Ly6G and Ly6C divide MDSCs into two subtypes: CD11b ⁺ Ly6G ⁺ Ly6C ^low granulocyte-like MDSC (G-MDSC) and CD11b ⁺ Ly6G ^- Ly6C ^hi monocyte-like MDSC ( M-MDSCs), in which G-MDSCs mainly inhibit the activation and proliferation of T cells through arginase 1 (Arginine1, Arg-1) and reactive oxygen species (ROS), while M-MDSCs mainly through Arg- 1 and nitric oxide to exert an inhibitory effect.

Studies have shown that G-MDSC-derived exosomes (G-exosomes) have protective effects on inflammatory bowel disease model mice and CIA model mice. Exosomes are biologically active vesicles produced by the endocytic system. It encapsulates various lipids, proteins, and nucleic acids, and can transport the substances carried by them to recipient cells to play biological roles. At present, the applications of exosomes mainly include drug carriers and disease diagnosis. Exosomes are used as drug transporters. Compared with the current liposome carriers and polymer carriers, they have low immunogenicity, high transport efficiency, good stability, and strong targeting. Due to the advantages of crossing the blood-brain barrier, the current drugs using exosomes as carriers mainly include gene drugs (siRNA, mRNA, miRNA), anticancer drugs (doxorubicin, paclitaxel, etc.) and antigens that enhance immunity. Since exosomes secreted by different cells under different diseases contain different nucleic acid and protein components, analysis of the components contained in exosomes isolated from patient body fluids can be used as a basis for disease diagnosis. More and more studies have shown that exosomes play an important role in the inflammatory process of autoimmune diseases, but the components of G-MDSC-derived exosomes are complex, and their specific mechanisms are still unclear.

Many studies have shown that miRNAs carried by exosomes can inhibit the proliferation and differentiation of T cells. For example, Let-7d carried by exosomes derived from Treg cells can inhibit the inflammatory response mediated by Th1 cells and reduce the disease in a mouse model of inflammatory bowel disease in vivo. Severity (Okoye IS, Coomes SM, Pelly VS, Czieso S, Papayannopoulos V, Tolmachova T, et al. MicroRNA-containing T-regulatory-cell-derived exosomes suppress pat hogenic T helper 1cells[J].Immunity.2014,41 (1):89-103.). MicroRNAs (miRNAs, miRNAs) are a series of non-coding small RNAs with a length of 18-22 nucleotides. They mainly inhibit their targets by specifically binding to the 3' untranslated region (3'-UTR) of target genes. The translation of a gene or induce the degradation of its mRNA, thereby inhibiting the expression of the target protein.

According to the structural and functional properties of G-exosomes, the application of the loaded miRNA in the treatment of RA can achieve the purpose of "high safety and strong targeting", and has a good clinical application prospect, which is expected to maintain the body's immune balance. Play an active role and provide a new therapeutic approach for the treatment of RA. The present invention artificially intervenes miRNA in G-exosomes, and can significantly improve the application effect after overexpression, so as to obtain G-exosomes with better therapeutic effect.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the defects in the prior art, and to provide a preparation method of G-exosomes overexpressing miR-93-5p and its application in the preparation of medicines for treating rheumatoid arthritis.

For achieving the above object, the technical means that the present invention takes are as follows:

The present invention first provides an application of miR-93-5p in the preparation of a drug for treating rheumatoid arthritis.

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

The present invention also provides a preparation method of G-exosomes overexpressing miR-93-5p.

The present invention also provides a medicine for treating rheumatoid arthritis, which comprises G-exosomes overexpressing miR-93-5p.

Specifically, the technical solutions adopted are as follows:

The present invention firstly separates and extracts M-MDSC and G-MDSC, and then extracts and identifies the exosomes (G-exosomes and M-exosomes) secreted by these two cells. It is verified through experiments that G-exosomes and M-exosomes are effective for CIA model mice onset The results show that G-exosomes can inhibit the pathogenesis of CIA model mice. Th17 cells are treated with G-exosomes and M-exosomes, which further shows that G-exosomes can inhibit the differentiation of Th17 cells, thereby reducing the incidence of small Mice onset, but M-exosomes had no obvious inhibitory effect.

miRNA extraction and sequencing analysis was performed on G-exosomes and M-exosomes, and the top 20 miRNAs with large absolute differences in the content of G-exosomes and M-exosomes were screened out; further, according to the structural and functional properties of miRNAs Screening was carried out to screen out 9 miRNAs that may have effects on RA, namely: miR-16-5p, miR-29a-3p, miR-93-5p, miR-22-3p, miR-26b-5p, Let- 7f-5p, Let7g-5p, miR-221-3p, miR-92a-3p.

Primers were designed according to the screened 9 miRNA sequences, and the expression changes of 9 miRNAs in Th17 cells treated with G-exosomes were detected by qRT-PCR. The results showed that the expression level of Th17 cells treated with G-exosomes increased the most The three miRNAs were miR-16-5p, miR-29a-3p, and miR-93-5p.

The mimics and mimics-negative controls corresponding to the selected 3 miRNAs (miR-16-5p, miR-29a-3p, miR-93-5p) were transfected into naive T cells and induced to differentiate Th17 cells. PCR detection showed that miR-93-5p could inhibit the differentiation of naive T cells into Th17 cells, and it also indicated that G-exosomes might inhibit the differentiation of Th17 cells through their loading of miR-93-5p.

Further, by transfecting G-MDSCs of mimics and inhibors, G-exosomes overexpressing or blocking miR-93-5p were prepared, and in vitro experiments proved that the G-exosomes overexpressing miR-93-5p were better than the original G-exosomes. -exosomes inhibited the differentiation of Th17 cells more obviously; while G-exosomes blocking miR-93-5p lost the inhibitory effect of the original G-exosomes on the differentiation of Th17 cells. The results show that miR-93-5p carried by G-exosomes inhibits the differentiation of Th17 in vitro, and the G-exosomes overexpressing miR-93-5p prepared by the present invention have stronger inhibitory ability on the differentiation of Th17 cells than the original G-exosomes .

In the present invention, by studying the effect of G-exosomes overexpressing and blocking miR-93-5p on the pathogenesis of CIA model mice, it is found that G-exosomes overexpressing miR-93-5p are more effective than original G-exosomes on CIA mice. The protective effect of miR-93-5p was stronger, while the G-exosomes that blocked miR-93-5p lost the inhibitory effect of the original G-exosomes on the pathogenesis of CIA mice. The results show that the miR-93-5p carried by G-exosomes alleviates the onset of CIA model mice, and the G-exosomes overexpressing miR-93-5p prepared by the present invention can protect CIA model mice compared to the original G-exosomes stronger.

The present invention finds that miR-93-5p loaded by G-exosomes can inhibit the expression of the corresponding receptor cell target protein STAT3 in vitro and in vivo, thereby exerting an inhibitory effect on the differentiation of Th17 cells.

Advantages of the present invention

(1) The present invention finds for the first time that miR-93-5p carried by G-exosomes can alleviate the onset of CIA model mice by inhibiting the differentiation of Th17 cells, providing a new method for the treatment of RA.

(2) The method of the present invention for preparing G-exosomes overexpressing miR-93-5p is short in time, convenient in operation, stable in activity of the components contained, and can be stored at -80°C for more than 1 year; the structure and active components are also stable at room temperature , which is beneficial to clinical application.

(3) Compared with ordinary G-exosomes, the G-exosomes overexpressing miR-93-5p prepared in the present invention have stronger effects of inhibiting the differentiation of Th17 cells and alleviating the severity of the disease in CIA model mice.

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

(5) Using exosomes as a drug transporter has the advantages of low immunogenicity, high transport efficiency, good stability, fast absorption and strong targeting. The G-exosomes overexpressing miR-93-5p prepared by the present invention are relatively The existing drug for the treatment of RA has the advantages of no toxic and side effects, no influence on normal liver and kidney metabolism, simple preparation process, and low cost. In addition, the G-exosomes overexpressing miR-93-5p prepared by the present invention, the drug action mechanism Clear, conducive to the monitoring of drug efficacy.

Description of drawings

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

Figure 2 is a transmission electron microscope image 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 particle size frequency distribution diagram of G-exosomes and M-exosomes prepared by sorting according to the present invention, wherein A is G-exosomes and B is M-exosomes.

Figure 4 shows the expression results of the landmark proteins of G-exosomes and M-exosomes detected by Western blot.

Figure 5 shows the results of the effect of G-exosomes and M-exosomes on the average arthritis index during the onset of CIA model mice.

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

Figure 7 shows the effects of G-exosomes and M-exosomes on the differentiation of Th17 cells in vitro. A is the proportion of Th17 cells in the phosphate buffer control group; B is the proportion of Th17 cells in the N-exosomes treatment group; C is the M-exosomes treatment The proportion of Th17 cells in the group; D is the proportion of Th17 cells in the G-exosomes treatment group.

Figure 8 shows the top 20 miRNAs in the absolute value of the difference in miRNA content loaded by G-exosomes and M-exosomes.

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

Figure 10 shows the changes in the expression levels of miR-93-5p, miR-16-5p and miR-29a-3p in Th17 cells induced to differentiate after transfecting corresponding mimics into naive T cells.

Figure 11 shows the effect of transfecting the corresponding mimics of miR-93-5p, miR-16-5p, and miR-29a-3p into naive T cells, on the induced differentiation of Th17 cells. In the figure, A is the mimics-negative control group Th17 Cell ratio; B is the ratio of Th17 cells in miR-16-5p-mimics treatment group; C is the ratio of Th17 cells in miR-29a-3p-mimics treatment group; D is the ratio of Th17 cells in miR-93-5p-mimics treatment group.

Figure 12 shows the effect of miR-93-5p on the expression of miR-93-5p after transfection of mimics or inhibitors of miR-93-5p into G-MDSC.

Fig. 13 is a graph showing the expression level of miR-93-5p expressed by G-exosomes after transfecting the mimics or inhibitors of miR-93-5p into G-MDSCs.

Figure 14 shows the effect of G-exosomes overexpressing or blocking miR-93-5p on Th17 cell differentiation. In the figure, A is the proportion of Th17 cells in the negative control group, B is the proportion of Th17 cells in the phosphate buffer control group, and C is the proportion of Th17 cells in the negative control group. The proportion of Th17 cells in G-exosomes treatment group, D is the proportion of Th17 cells in the mimics-NC treatment group, E is the proportion of Th17 cells in the mimic treatment group, F is the proportion of Th17 cells in the inhibitor-NC treatment group, and G is the proportion of Th17 cells in the inhibitor-treated group.

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

Figure 16 shows the degree of effect of G-exosomes overexpressing or blocking miR-93-5p on arthritis index in CIA model mice.

Figure 17 is a visual picture of the degree of influence of G-exosomes overexpressing or blocking miR-93-5p on toe swelling in CIA model mice.

Figure 18 shows the results of HE staining of the toes of CIA model mice treated with G-exosomes overexpressing or blocking miR-93-5p, wherein A is the PBS buffer group, B is the G-exosomes group, and C is the mimics- NC-exosomes group, D is mimics-exosomes group, E is inhibors-NC-exosomes group, F is inhibors-exosomes group.

Figure 19 shows the flow cytometry results of the proportion of Th17 cells in the popliteal lymph node cells of CIA model mice treated with G-exosomes overexpressing or blocking miR-93-5p.

Figure 20 shows the results of G-exosomes overexpressing or blocking miR-93-5p on the expression of miR-93-5p in CD4 ⁺ T cells in the draining lymph nodes of CIA model mice.

Figure 21 shows the possible binding sites of miR-93-5p and STAT3.

Figure 22 shows the expression of total STAT3 cells in the Th17 induction system by G-exosomes overexpressing or blocking miR-93-5p, wherein A is the result of Western blot detection, and B is the statistical analysis diagram.

Figure 23 shows the expression of G-exosomes overexpressing or blocking miR-93-5p on the total STAT3 expression of CD4 ⁺ T cells in the draining lymph nodes of CIA model mice, where A is the result of Western blot detection, and B is the statistical analysis graph.

Detailed ways

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

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

(1) Construction of tumor-bearing mouse model: cultured in DMEM medium containing 10% calf serum (purchased from Gibco, USA) and pH 7.3 (purchased from Gibco, USA), cultured at 37°C and 5% CO ₂ Mouse Lewis lung adenocarcinoma cell line (purchased from Shanghai Academy of Life Sciences). During the culture process, closely observe the cell shape, adherence, density, and the color of the culture medium. When the cells grow to a density of about 85% of the bottom of the culture dish, use 0.25% trypsin (purchased from Boster Bioengineering Co., Ltd. company) were digested and passaged. The Lewis cells in logarithmic growth phase were taken, and 3.0×10 ⁶ cells were subcutaneously injected into the right abdomen of each mouse to transplant tumors into 6-8-week-old male C57BL/6 mice (purchased from the Experimental Animal Center of Jiangsu University). During the construction of the model, the living state and tumor size of the mice were closely observed.

(2) Isolation of spleen cells from tumor-bearing mice: On the 30th day after the Lewis cells were injected into mice, blood was collected from the eyeballs and the mice were killed by neck dislocation. After soaking the mice in 75% alcohol for 5 minutes, they were dissected in a clean bench. The spleen was obtained from the mouse; the ground spleen was filtered with a 0.22 μm pore size mesh, washed with PBS (phosphate buffered saline), the suspension was collected and centrifuged at 4°C and 500 g for 5 min, and the supernatant was discarded; in the precipitated cells Add 5 mL of ACK erythrocyte lysate (ACK Lysis Buffer, ACK) (Shanghai Ruigu Biotechnology Co., Ltd.), and after mixing, lyse the erythrocytes for 5 minutes; add RPMI-1640 medium containing 10% calf serum (Gibco, USA) to 10mL, after mixing, centrifuge at 4°C and 500g for 5min, discard the supernatant; when the cells are resuspended to 10mL, a small amount of cells are collected and counted under a microscope.

(3) Magnetic bead sorting G-MDSC: resuspend each 10 ⁸ splenocytes with 350 μL of PBE, add 50 μL of FcR Blocking Reagent (Miltenyi, Germany), mix well and incubate on ice for 10 min; every 10 ⁸ cells Splenocytes were added with 40 μL of anti-Ly-6G Biotin (Miltenyi, Germany), and then incubated on ice for 30 min, and mixed every 10 min; 10 mL of PBE was added, and after mixing, the suspension was placed at 4°C, 500 g Centrifuge for 5 min, discard the supernatant; add 50 μL anti-Ly-6G-Biotin Beads (Miltenyi, Germany) per 10 ⁸ splenocytes, mix well, incubate on ice for 30 min, and mix once every 10 min; add 10 mL PBE , after mixing, centrifuge the suspension at 4°C and 500g for 5 min, discard the supernatant; add 500 μL of PBE, mix well; put the MACS sorting column on the VarioMACS sorter (Miltenyi, Germany), add 3 mL After rinsing once with PBE, add the spleen cell suspension, and after the last drop of cell suspension flows out, wash the sorting column with 3 mL of PBE, repeating 3 times; remove the sorting column from the sorter and place the sorting column on Add 5mL of pre-cooled PBE to a 10mL centrifuge tube, push the piston, and press out the cells in the separation column. Repeat once to obtain 10mL of cell suspension, which is G-MDSC. A small amount is reserved for counting under a microscope.

(4) Magnetic bead sorting M-MDSC: collect the cell suspension flowing out of the sorting column when sorting G-MDSC, centrifuge at ⁴ °C and 500 g for 5 min, discard the supernatant; add 40 μL of anti- Gr-1Biotin (Miltenyi, Germany), incubate on ice for 30 minutes after mixing, and mix once every ¹⁰ minutes; add 10 mL of PBE, centrifuge at 4°C and 500 g for 5 minutes after mixing, and discard the supernatant; Splenocytes were added with 50 μL of anti-Gr-1-Biotin Beads (Miltenyi, Germany), mixed and incubated on ice for 30 min, and mixed every 10 min; 10 mL of PBE was added, and after mixing, the conditions were 4°C and 500 g. Centrifuge for 5 min, discard the supernatant; add 500 μL of PBE, mix well; install the MACS column on a sorter (Miltenyi, Germany), add 3 mL of PBE to rinse once, and then add the above-mentioned to-be-sorted After the last drop of cell suspension is exhausted, add 3 mL of PBE to wash the sorting column, repeat 3 times; withdraw the sorting column and place it on a 10 mL centrifuge tube, add 5 mL of PBE, push the piston, and press out the cells in the column, Repeat once to obtain 10 mL of cell suspension, which is M-MDSC, and leave a small amount to count under a microscope.

(5) Purity identification of G-MDSC and M-MDSC: respectively take 1×10 ⁶ G-MDSC and 1×10 ⁶ M-MDSC obtained in step (4) and step (5) in EP tube, 1 mL Resuspend in PBS, centrifuge at 4°C and 500g for 5 min, discard the supernatant and leave about 100 μL of PBS; after mixing, add 0.5 μL of anti-Ly-6G antibody (eBioscience, USA) to the EP tube of G-MDSC. and 0.5 μL of anti-CD11b antibody (eBioscience, USA), add 0.5 μL of anti-Ly-6C antibody (eBioscience, USA) and 0.5 μL of anti-CD11b antibody (eBioscience, USA) to the EP tube of M-MDSC; respectively at 4°C Incubate for 30 min; resuspend in 1 mL of PBS, centrifuge at 4°C and 500 g for 5 min, discard the supernatant, add 200 μL of PBS to resuspend, and detect by flow cytometry (BD, USA). As a result, 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-MDSCs and M-MDSCs were resuspended in 10% calf serum (Gibco, USA, the serum was centrifuged at 4°C and 100,000g for 16h, the upper 2/3 of the serum was collected, and the serum was removed. exosomes) in RPMI-1640 medium (Gibco, USA), seeded in 24-well plates with 1.5×10 ⁶ cells per well and 1 mL per well; under the conditions of 37°C, 5% CO ₂ After culturing for 24 hours, the culture supernatant was collected.

(2) The collected culture supernatant of G-MDSC and M-MDSC was centrifuged at 4°C and 300g for 20min to collect the supernatant; then the supernatant was centrifuged at 4°C and 1000g for 30min to collect the supernatant; and then the supernatant was collected. Centrifuge at 4°C and 10000g for 30min, and collect the supernatant; add the supernatant to a 100kDa ultrafiltration tube (Milipore, Germany), centrifuge at 4°C and 1000g for 30min, and collect the concentrate in the tube.

(3) Extract exosomes by ExoQuick-TC ^TM Exosome Kit (SBI, USA): mix the concentrate and ExoQuick-TC ^TM Exosome reagent in a volume ratio of 5:1, mix gently, and let stand in a refrigerator at 4°C for more than 16 hours; Centrifuge at 4°C and 1000g for 30min, discard the supernatant, and collect the precipitate, which is G-exosomes or M-exosomes. The prepared exosomes were dissolved in PBS, aliquoted into EP tubes, and stored at -80°C.

(4) After mixing the lysate of exosomes with an equal volume of lysate (RIPA:PMSF=250:1), place it on ice for 1 hour, and shake it on a shaker for 1 minute every 10 minutes; supernatant. The protein concentration in the supernatant of exosomes lysis was detected according to the instructions of the BCA microprotein quantification kit (Beijing Kangwei Century Co., Ltd.) to determine the content of the extracted exosomes. The results show that each G-MDSC or M-MDSC obtained in the present invention can extract about 3×10 ^-6 μg of exosomes after culturing for 24 hours.

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

(1) Observation of the morphology of G-exosomes by transmission electron microscope: Take 20 μL of G-exosomes or M-exosomes suspension and drop it on a 3 mm diameter copper wire mesh, let it stand for 2 min at room temperature; gently blot the liquid with filter paper, add dropwise pH The 2% phosphotungstic acid solution of 6.8 (Shanghai Junrui Technology Co., Ltd.) was placed on the copper mesh, counterstained for 1 min; the filter paper blotted the dye solution, dried under an incandescent lamp, and observed under a transmission electron microscope (Philips, the Netherlands). The results are shown in Figure 2 As shown, G-exosomes and M-exosomes were observed under transmission electron microscope as round or oval microcapsule structures with a complete envelope and low electron density components in the cavity; Figure 3 shows G-exosomes and M-exosomes, respectively The particle size frequency distribution of exosomes, the results show that the particle size of G-exosomes and M-exosomes are mainly distributed between 20-120nm.

(2) Western blot detection of protein molecules CD9 and CD63 contained in G-exosomes and M-exosomes and Calnexin contained in mitochondria: prepare 5% stacking gel and 12% separating gel, use RIPA protein lysis buffer (Shanghai Bi Yuntian Biotechnology Co., Ltd.) cleaved G-exosomes and M-exosomes, loaded the cleaved G-exosomes with a total amount of 250 μg protein, electrophoresed at 100V constant voltage; Block PVDF membrane for 1 h; incubate overnight at 4°C with anti-CD63 monoclonal antibody (eBioscience, USA) and anti-Calnexin monoclonal antibody (eBioscience, USA); wash the membrane with TBS/T (Shanghai Jingke Chemical Technology Co., Ltd.) 10min×3 times, incubate with horseradish peroxidase-labeled secondary antibody (eBioscience, USA) for 30min at 37°C; after taking out, wash the membrane with TBS/T (Shanghai Jingke Chemical Technology Co., Ltd.), 10min×3 times; ImageQuant The LAS 4000 gel imaging system (GE, USA) was exposed to color, and the results were shown in Figure 4. G-exosomes and M-exosomes specifically highly expressed CD9 and CD63 molecules, but G-exosomes and M-exosomes did not contain localization in Calnexin on the endoplasmic reticulum membrane. In view of the morphology, size, and protein expression results of the extracted G-exosomes and M-exosomes, it was proved that G-exosomes and M-exosomes were successfully extracted in this example.

Example 4: Experiments demonstrate the immunosuppressive function of G-exosomes and M-exosomes

(1) Construction of CIA model mice:

The first immunization: mix equal volumes of 2 mg/mL bovine type II collagen (Sigma, USA) and 2 mg/mL complete Freund's adjuvant (Thermo Fisher, USA), and grind on ice to a water-in-oil state; Each DBA/1 mouse (Shanghai Slack Laboratory Animal Co., Ltd.) was subcutaneously injected with 0.1 mL of the above mixture at the base of the tail;

The second immunization: mix equal volumes of 2 mg/mL bovine type II collagen (Sigma, USA) and 2 mg/mL incomplete Freund's adjuvant (Thermo Fisher, USA), grind on ice to a water-in-oil state, On the 21st day after the first immunization, 0.1 mL of the above mixture was subcutaneously injected into the tail base of each DBA/1 mouse (Shanghai Slack Laboratory Animal Co., Ltd.).

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

6-8 weeks old male C57BL/6 mice (Jiangsu University Laboratory Animal Center) were removed from the eyeballs and blood was collected, dropped into anticoagulant blood collection tubes containing EDTA-Na ₂ (Suzhou Lingyan Medical Equipment Co., Ltd.), and centrifuged at 1000 rpm Blood was drawn from the eyeball for 10 min, the plasma was aspirated, and the blood cells were resuspended in 500 μL of PBS. Add 1 mL of mononuclear lymphocyte separation solution (Tianjin Haoyang Biological Products Technology Co., Ltd.) into a 10 mL glass test tube, slowly drop the blood cell suspension into the glass test tube along the tube wall, use a horizontal centrifuge, 4 ℃, 2000rpm centrifugation for 15min. Slowly suck up the other cell layers except the red blood cell layer with a pipette from top to bottom and discard, add 5mL ACK (Shanghai Ruigu Biotechnology Co., Ltd.), mix by pipetting, let stand at room temperature for 5min, add 5mL containing 10% calcium. RPMI 1640 medium of bovine serum (Gibico, USA) was used to terminate the lysis, and after mixing, centrifuge at 500 g for 5 min at 4°C. The supernatant was discarded and washed twice with 10 mL of PBE to obtain neutrophils. Then N-exosomes were extracted according to the method in Example 2.

(3) Experimental grouping:

PBS control group: 100 μL of PBS was injected into the tail vein of each mouse on the 18th and 24th days after the first immunization, respectively;

N-exosomes group: 100 μL of PBS-dissolved N-exosomes were injected into the tail vein of each mouse on the 18th and 24th day after the first immunization, respectively, wherein 100 μL of PBS dissolved 100 μg of N-exosomes;

G-exosomes group: 100 μL PBS-dissolved G-exosomes were injected into the tail vein of each mouse on the 18th and 24th day after the first immunization, respectively, in which 100 μL PBS dissolved 100 μg G-exosomes;

M-exosomes group: 100 μL of PBS-dissolved M-exosomes were injected into each mouse on the 18th and 24th day after the first immunization, respectively, in which 100 μg of M-exosomes was dissolved in 100 μL of PBS.

(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, and AS represented the score of all diseased joints in each mouse The total number of joint lesions was scored using a 5-level scoring method, where no redness and swelling were scored as 0 points, joint redness and swelling were scored as 1 point, mild joint redness and swelling were scored as 2 points, moderate joint redness and swelling were scored as 3 points, and severe joint redness and swelling with associated 4 points for functional impairment. The mean arthritis index (MAI) of mice in each group was calculated, and MAI was divided by the sum of AS of mice in each group by the number of mice in each group. As shown in Figure 5, the average arthritis index of mice in the G-exosomes-treated group was lower than that in the N-exosomes-treated group, while there was no significant difference between the PBS buffer, N-exosomes, and M-exosomes-treated groups; Figure 6 shows The toes of the mice in the G-exosomes-treated group had only mild redness and swelling, and the joint activities were normal; however, the toes of the mice in the PBS buffer, N-exosomes, and M-exosomes-treated groups were mostly red and swollen, and the joint activities were limited. These results It shows that G-exosomes can inhibit the pathogenesis of CIA model mice, while M-exosomes do not significantly inhibit the pathogenesis of CIA model mice.

(5) Establish a Th17 cell differentiation system: coat a 24-well round-bottom culture plate with 200 μL of coating solution containing 2 μg/mL anti-mouse CD3 monoclonal antibody (eBioscience, USA), overnight at 4°C; The coating solution was washed once with PBS, and the initial T cells were sorted by the initial T cell sorting kit from Miltenyi, Germany. The initial T cells were seeded in 24-well plates at a density of 1.5 × 10 ⁶ cells per well. , the total volume of each well is 1mL; each well is added with anti-mouse CD28 monoclonal antibody (eBioscience, USA) with a final concentration of 2μg/mL, recombinant mouse TGF-β (eBioscience, USA) with a final concentration of 5ng/mL, IL-6 with a final concentration of 30 ng/mL (eBioscience, USA), IL-23 with a final concentration of 30 ng/mL (eBioscience, USA), anti-IL-4 with a final concentration of 5 μg/mL (eBioscience, USA), Anti-IFN-γ with a final concentration of 5 μg/mL (eBioscience, USA); RPMI-1640 cell culture medium (U.S. gibico company), cultured at 37°C and 5% CO ₂ for 72h.

(6) Experimental grouping: control group: 100 μL of PBS was added as a control; N-exosomes, G-exosomes and M-exosomes with a final concentration of 60 μg/mL were added to each well of the other groups, as shown in Figure 7, G-exosomes treatment The proportion of Th17 cells in the group was lower than that in the N-exosomes-treated group, but there was no significant difference between the PBS buffer, N-exosomes, and M-exosomes-treated groups. These results indicate that G-exosomes play a role in alleviating the pathogenesis of CIA model mice compared with M-exosomes, inhibiting the differentiation of Th17 cells in vitro, and G-exosomes exhibit stronger immunosuppressive function than M-exosomes .

Example 5: Sequencing results and analysis of miRNAs in G-exosomes and M-exosomes

(1) Entrust Guangzhou Ribo Company to extract and sequence miRNAs carried in G-exosomes and M-exosomes. It is known to those skilled in the art that G-exosomes and M-exosomes contain many specific miRNAs, and there is a certain amount of expression in them. difference. According to the analysis of the sequencing results, combined with the relevant miRNA information on the TargetScan website, the types of miRNAs were determined; as shown in Figure 8, the top 20 absolute differences in the content of related miRNAs contained in G-exosomes and M-exosomes were 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(MIMAT0000677), miR-140-5p(MIMAT0000151), Let7g-5p(MIMAT0000121), miR-26a-5p(MIMAT0000533), miR- 221-3p (MIMAT0000669), miR-93-5p (MIMAT0000540), miR-148b-3p (MIMAT0000580), the numbering in brackets is the corresponding numbering information according to the MIRBASE website analysis, and they are used as the miRNA to be screened.

(2) The miRNA to be screened in the present invention is further screened, because miR-16-5p, miR-29a-3p, miR-93-5p, Let7g-5p are related to the development of osteoarthritis and the 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, miR-221-3p are related to T cell proliferation, differentiation, immune response, etc.; miR-16- The potential targets of 5p, miR-29a-3p, Let-7f-5p, and miR-92a-3p, Smad7, Smad6, FOS, PIK3R1, STAT3, EMOS, PIK3R1, etc., are closely related to the proliferation and differentiation of T cells. Therefore, the present invention further screened out miR-16-5p, miR-29a-3p, miR-93-5p, miR-22-3p, miR-26b-5p, Let-7f-5p, Let7g-5p, miR-221 -3p, miR-92a-3p as candidate miRNAs.

(3) Detect the fold change of the expression level of candidate miRNAs in Th17-induced cells after G-exosomes treatment: collect the control group and Th17-induced cells treated with G-exosomes in step (6) of Example 4, and use 1 mL of Trizol (U.S.A. Invitrogen Company) lysed 1×10 ⁶ Th17 induced cells, fully mixed with a pipette, added 200 μL of pre-cooled chloroform (Shanghai Chemical Reagent Co., Ltd.), immediately shaken for 15-30 s, and stood at room temperature for 10 min; at 4°C Then, centrifuge at 12000g for 15min; the liquid in the EP tube is stratified, suck the upper aqueous layer (containing RNA) into a new clean EP tube, add 500 μL of isopropanol, mix gently with a pipette, and let stand at room temperature 5～10min; centrifuge at 12000g for 15min at 4℃, RNA sinks to the bottom of the tube and becomes translucent; discard the supernatant, add 1mL of 75% ethanol (Shanghai Chemical Reagent Co., Ltd.) to wash; at 4℃ Centrifuge at 7800g for 5 min at room temperature; place it in an ultra-clean bench at room temperature to dry until the RNA becomes translucent; add 5 μL of DEPC (Shanghai Biyuntian Co., Ltd.) to dissolve in water; use a Thermo Fisher detector to detect the RNA concentration.

(4) Perform reverse transcription of the RNA extracted in step (3) according to the requirements of the Japanese Takara reverse transcription kit, design primers according to the miRNA sequence, and then entrust Guangzhou Ribo Company to synthesize miR-16-5p, miR-29a-3p, miR - Forward and backward primers for 93-5p, miR-22-3p, miR-26b-5p, Let-7f-5p, Let7g-5p, miR-221-3p, miR-92a-3p, purchased from Shanghai BIO-RAD Company The qRT-PCR kit was used to detect the expression changes of candidate miRNAs in Th17-induced cells after G-exosomes treatment. are miR-16-5p, miR-29a-3p and miR-93-5p.

(5) The mimics and mimics-negative controls of miR-16-5p, miR-29a-3p and miR-93-5p (Guangzhou Ribo Biotechnology Co., Ltd.) were separately transfected by EntranceTM-R transfection reagent (Beijing Engreen Company). Transfected into the initial T cells sorted in step (5) of Example 4, then induced to differentiate into Th17 cells according to the method in Example 4, and detected by qRT-PCR, the results are shown in Figure 10, the miR in Th17 cells transfected with mimics The expression levels of -16-5p, miR-29a-3p and miR-93-5p increased accordingly; as shown in Figure 11, the proportion of naive T cells transfected with miR-93-5p mimics differentiated to Th17 cells was significantly lower than that of transfection The naive T cells of the mimics-negative control were transfected, while the naive T cells transfected with miR-16-5p corresponding to mimics and miR-29a-3p corresponding to mimics did not change significantly. These results suggest that exogenous miR-93-5p can inhibit the differentiation of naive T cells into Th17 cells, suggesting that G-exosomes may inhibit the differentiation of Th17 cells through the miR-93-5p they carry.

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

(1) The lyophilized powder of miR-93-5p mimics, mimics-negative control (mimics-negitive control, mimics-NC) and inhibitors, inhibitors-negative control (inhibitors-negitive control, inhibitor-NC) (Guangzhou Rui Bo Biotechnology Co., Ltd.) after centrifugation, dissolved in sterile DEPC water, pipetting and mixing to form a storage solution with a final concentration of 20 μM, and stored in a -80°C refrigerator in aliquots;

(2) Inoculate cells, inoculate 1×10 ⁶ G-MDSCs in each well of a 24-well plate, and supplement the system with 10% calf serum RPMI1640 medium (Gibico, USA) to make up the system to 450 μL;

(3) Preparation of mimics, mimics-NC, and inhibitors, inhibitors-NC dilutions: take a clean EP tube, add mimics and mimics-NC 50nM, respectively, add 1.25 μL of mimics or mimics-NC stock solution, then Make up to 25 μL with serum-free RPMI 1640 medium (Gibico, USA); then take a clean EP tube, add inhibitors and inhibitors-NC 100nM respectively, and then add 2.5 μL of inhibitor and inhibitor-NC stock solution respectively, and then use no Serum RPMI 1640 medium (Gibico, USA) was supplemented to 25 μL;

(4) Preparation of EntranceTM-R transfection reagent dilution: take a clean EP tube, add the corresponding amount of EntranceTM-R transfection reagent (Beijing Engreen Company), according to the amount of mimics, mimics-NC, inhibitors and inhibitors-NC Control the amount of transfection reagent, add 1 μL of transfection reagent per 50nM, then supplement to 25 μL with serum-free 1640 medium (Gibico, USA), and let stand for 5 min at room temperature;

(5) Preparation of transfection complex: Add the dilution solution of EntranceTM-R transfection reagent (Beijing Engreen Company) to the corresponding dilution solution of mimics, mimics-NC or inhibitors and inhibitors-NC, then mix well immediately and keep at room temperature. Set 30min;

(6) Drop 50 μL of the transfection complex into the inoculated cells in step (2), and mix gently;

(7) Observe the cell state after 6 hours of transfection. If the cell state is good, collect G-MDSC by centrifugation;

(8) G-exosomes overexpressing or blocking miR-93-5p were prepared by using G-MDSC 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) Collect 1×10 ⁶ G-MDSCs in each treatment group in step (6) of Example 6, extract the total RNA of G-MDSCs in each treatment group according to the method in step (3) of Example 5, and detect its concentration ;

(2) According to the method in Example 5, the content of miR-93-5p in G-MDSCs of each treatment group was quantitatively detected by fluorescence. The results are shown in Figure 12. The mimics or inhibitors of miR-93-5p were successfully transfected into G-MDSCs. - MDSC.

(3) Collect G-exosomes secreted by 3×10 ⁷ G-MDSCs in each treatment group in step (8) of Example 6, and dissolve them with 1 mL of Trizol (Invitrogen, USA).

(4) Quantitative analysis of miR-93-5p contained in G-exosomes in each group was carried out according to the operation method in Example 5. As shown in Figure 13, G-MDSCs secreted by G-MDSCs transfected with miR-93-5p mimics Compared with the mimics-NC group, the expression of miR-93-5p in exosomes was significantly increased (P<0.01), while the G-exosomes secreted by G-MDSCs transfected with inhibitors of miR-93-5p were higher than those in the inhibitor-NC group. The expression of miR-93-5p in the group decreased (P<0.05). These results show that the G-exosomes overexpressing or blocking miR-93-5p were successfully extracted in this example, and the G-exosomes overexpressing miR-93-5p prepared in this example are in phase with the original G-exosomes. The content of miR-93-5p increased approximately 15-fold.

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

(1) Add 60 μg/mL G-exosomes overexpressing or blocking miR-93-5p into the Th17 cell differentiation system. As shown in Figure 14, the G-exosomes overexpressing miR-93-5p treatment group (transformed The proportion of Th17 cells in the mimics group) was significantly lower than that in the mimics-NC group (P<0.01), while the G-exosomes treatment group that blocked miR-93-5p (inhibitors transfection group) was relatively Th17 cells in the inhibitor-NC group. The proportion of cells increased (P<0.05). These results indicate that miR-93-5p carried by G-exosomes can inhibit the differentiation of Th17 cells in vitro, and G-exosomes overexpressing miR-93-5p have a stronger ability to inhibit the differentiation of Th17 cells. Compared with the original G-exosomes, the G-exosomes overexpressing miR-93-5p in this example have a stronger inhibitory ability on the differentiation of Th17 cells, and the proportion of Th17 cells is doubled.

(2) According to the operation method in Example 5, the expression of miR-93-5p in cells in the Th17 induction system was detected after G-exosomes overexpressing or blocking miR-93-5p were treated. As shown in Figure 15, overexpression Compared with the mimics-NC group, the G-exosomes treatment group of miR-93-5p can significantly increase the expression level of miR-93-5p in induced cells (P<0.01), and block the G-exosomes treatment of miR-93-5p The expression level of miR-93-5p in the group was significantly lower than that in the inhibitor-NC group (P<0.001). These results indicate that G-exosomes can transmit the miR-93-5p carried by them to the recipient cells, thereby exerting an inhibitory function, and the G-exosomes overexpressing miR-93-5p prepared by the present invention transmit the miR-93-5p higher efficiency.

Example 9: miR-93-5p loaded by G-exosomes can more effectively inhibit the onset of CIA model mice and alleviate the severity of the disease

(1) using the CIA model mice constructed in Example 4;

(2) G-exosomes overexpressing or blocking miR-93-5p were injected into the tail vein of CIA model mice on the 18th and 24th days after the first immunization.

(3) Experimental grouping:

PBS buffer group: 100 μL of PBS was injected into the tail vein of each mouse on the 18th and 24th day of immunization;

G-exosomes group: 100 μL of PBS-dissolved G-exosomes were injected into the tail vein of each mouse on the 18th and 24th day of immunization, respectively;

mimics-NC-exosomes group: on the 18th and 24th day of immunization, 100 μL of PBS-dissolved G-exosomes secreted by G-MDSCs after transfection of mimic-NC were injected into the tail vein of each mouse, respectively;

mimics-exosomes group: on the 18th and 24th day of immunization, 100 μL of PBS was injected into the tail vein of each mouse to dissolve the G-exosomes secreted by G-MDSCs after mimic transfection;

Inhibitors-NC-exosomes group: G-exosomes secreted by G-MDSCs after transfection with inhibitors-NC were injected into the tail vein of each mouse on the 18th and 24th day of immunization, respectively;

Inhibitors-exosomes group: G-exosomes secreted by G-MDSCs after transfection of G-MDSCs were injected into the tail vein of each mouse on the 18th and 24th days of immunization, respectively.

In each of the above groups, 100 μg of G-exosomes in different treatment groups were dissolved in 100 μL of PBS.

(4) As shown in Figure 16, the average arthritis index of mice in the mimics-exosomes treatment group was significantly lower than that in the mimics-NC-exosomes treatment group (P<0.05). The inhibitor-NC-exosomes treatment group significantly increased (P<0.01). As shown in Figure 17, the toes of mice in the G-exosomes, mimics-NC-exosomes, mimics-exosomes, and inhibitors-NC-exosomes groups had only mild redness and swelling, and the joint activities were normal; however, the PBS buffer group, inhibitors-exosomes The toes of the mice in the group were mostly red and swollen, and the joint movement was limited. As shown in Figure 18, the bone structure at the toe joints of mice in the G-exosomes, mimics-NC-exosomes, mimics-exosomes, and inhibitors-NC-exosomes groups was relatively intact, the joint space was relatively normal, and there was little inflammatory cell infiltration; However, the bone structure at the toe joints of the mice in the PBS buffer group and the inhibitor-exosomes group was severely damaged, the joint space was reduced, and a large number of inflammatory cells were infiltrated. These results suggest that miR-93-5p carried by G-exosomes can inhibit the onset of CIA model mice and alleviate the severity of the disease, and G-exosomes overexpressing miR-93-5p have stronger protection against CIA model mice effect.

Compared with the original G-exosomes, the G-exosomes overexpressing miR-93-5p in this example have a stronger protective effect on CIA mice. The average arthritic index of mice treated with 5p G-exosomes nearly doubled, the degree of toe swelling was significantly reduced, and the joint structure was more complete.

(5) Isolate the popliteal lymph nodes of each group of mice to prepare a single cell suspension. As shown in Figure 19, flow cytometry was used to analyze the proportion of Th17 cells in the popliteal lymph node cells of the mice in each group. Compared with the mimics-NC-exosomes-treated group, the proportion of Th17 cells in the fossa lymph node cells was significantly decreased (P<0.01), and the proportion of Th17 cells in the inhibitor-exosomes-treated group was significantly increased compared with the inhibitor-NC-exosomes-treated group (P<0.001). In addition, there was no significant difference between the PBS treatment group and the mimics-NC-exosomes treatment group or the inhibitors-NC-exosomes treatment group (P>0.05). As shown in Figure 20, qRT-PCR was used to detect the expression of miR-93-5p in the draining lymph node CD4 ⁺ T cells of the mice in each group ^. Compared with the inhibitors-NC-exosomes treatment group, the expression of miR-93-5p in the inhibitor-exosomes treatment group was significantly decreased (P<0.01). Compared with the mimics-NC-exosomes treatment group or the inhibitors-NC-exosomes treatment group, the liquid group had no significant difference (P>0.05). These results indicate that G-exosomes can inhibit the proportion of pathogenic Th17 cells in the draining lymph nodes of CIA model mice by transporting miR-93-5p to recipient cells, and alleviate the severity of the disease, and the miR-93 overexpressed in the present invention -5p G-exosomes have a higher efficiency of transporting miR-93-5p and a stronger protective effect on CIA model mice.

Compared with the original G-exosomes, the G-exosomes overexpressing miR-93-5p in this example have a stronger protective effect on CIA mice. The proportion of Th17 cells in the popliteal lymph node cells of mice treated with 5p G-exosomes doubled, and the reduction in the proportion of pathogenic Th17 cells was critical to suppress the pathogenesis of CIA mice.

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

(1) According to the analysis of the Targetscan website, as shown in Figure 21, miR-93-5p can specifically bind to the 248-254 and 495-501 regions of the 3' UTR region of the target gene STAT3.

(2) Collect the cells in the Th17 induction system treated with G-exosomes that overexpress or block miR-93-5p, and detect the expression level of STAT3 in each treatment group by Western blot. As shown in Figure 22, overexpressing miR- Compared with the mimics-NC group, the expression level of STAT3 in the G-exosomes treatment group with 93-5p decreased (P<0.05), while the G-exosomes treatment group with miR-93-5p blocked the expression level of STAT3 compared with the inhibitor-NC group. The expression level was increased (P<0.05). These results show that miR-93-5p carried by G-exosomes can inhibit the expression of STAT3 in recipient cells, thereby inhibiting the differentiation of Th17 cells, and the G-exosomes overexpressing miR-93-5p prepared in the present invention have stronger inhibitory targets protein STAT3 expression, thereby exerting a stronger immunosuppressive function. The G-exosomes overexpressing miR-93-5p prepared by the present invention have a stronger effect of inhibiting the expression of the target protein STAT3. Compared with the original G-exosomes, the expression level of STAT3 is doubled, thereby exerting a stronger effect. Inhibits the function of Th17 cell differentiation.

(3) Collect CD4 ⁺ T cells in the draining lymph nodes of CIA model mice after G-exosomes overexpressing or blocking miR-93-5p treatment, and Western blot was used to detect the expression level of STAT3 in cells in each treatment group, as shown in Figure 23. The expression level of STAT3 in the G-exosomes treatment group expressing miR-93-5p was significantly lower than that in the mimics-NC group (P<0.01), while the G-exosomes treatment group that blocked miR-93-5p was relative to the inhibitors-NC group. The expression level of STAT3 in the group increased (P<0.05). These results indicate that miR-93-5p carried by G-exosomes can reduce the expression of receptor cell target protein STAT3 in vitro and in vivo in CIA model mice, and the G-exosomes overexpressing miR-93-5p prepared by the present invention have stronger The ability to inhibit the expression of the target protein STAT3, thus exerting a stronger protective effect on CIA model mice. The G-exosomes overexpressing miR-93-5p prepared by the present invention have a stronger ability to inhibit the expression of the target protein STAT3. Compared with the original G-exosomes, the expression level of STAT3 is reduced by nearly 2 times, so that the CIA model can be improved. Mice exert stronger protection.

Claims (5)

1. Application of G-exosomes overexpressing miR-93-5p in the preparation of a drug for the treatment of rheumatoid arthritis. 2. The application of G-exosomes overexpressing miR-93-5p in the preparation of a drug for inhibiting rheumatoid arthritis. 3. The application according to any one of claims 1-2, wherein the application is to inhibit the differentiation of Th17 cells. 4. The application according to any one of claims 1-2, wherein the application is to inhibit the expression of the corresponding receptor cell target protein STAT3. 5. A medicine for the treatment of rheumatoid arthritis, characterized in that the medicine comprises G-exosomes overexpressing miR-93-5p.

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