Application of targeted synovitis macrophage exosome to delivery of miR-155-5p inhibitor in treatment of osteoarthritis

Abstract

The invention relates to application of a miR-155-5p inhibitor delivered by an exosome of a targeted synovitis macrophage to treatment of osteoarthritis, and belongs to the technical field of biological medicines. According to the scheme, the exosome drug delivery system for targeting the synovial inflammatory macrophages in the osteoarthritis, which contains the miR-155-5p inhibitor, is prepared through a genetic engineering technology, so that the exosome derived from stem cells is targeted to inflammatory synovial tissues, and damaged cartilage tissues are repaired and inflammation is resisted based on the stem cell exosome; compared with the prior art, the application has the advantages that the combined action of inhibiting the osteoarthritis injury and inhibiting the miR-155-5p so as to resist synovitis is realized, the osteoarthritis injury repair can be better applicable, a new scheme for treating the osteoarthritis is provided, a support is provided for clinical transformation, and the application value is extremely high.

Description

Technical Field

[0001] This application relates to the application of a miR-155-5p inhibitor delivered via exosomes targeting inflammatory macrophages of the synovium for the treatment of osteoarthritis, and belongs to the field of biomedical technology. Background Technology

[0002] In the pathological progression of osteoarthritis (OA), synovial inflammation precedes the onset of articular cartilage degeneration symptoms. Synovial inflammation significantly affects the degree of cartilage degeneration in OA, especially since macrophages in the synovium are closely related to the pathological development of OA. Further studies have found that an increase in inflammatory macrophages in the synovial surface layer is closely related to synovial inflammation and is an important structural and morphological indicator of synovial inflammation.

[0003] The content of miR-155-5p in cartilage tissue is positively correlated with the progression of osteoarthritis. Although miR-155-5p-based therapies have achieved considerable success, their in vivo delivery remains a challenge. To date, exosomes have been successfully used for the in vivo and in vitro delivery of miRNAs, proteins, and small molecules. Compared to synthetic materials, exosome delivery systems offer several advantages, such as endogenous nature, stability, biocompatibility, nanoscale size, ability to cross the blood-brain barrier, and designability of components. Furthermore, exosomes derived from human adipose-derived stem cells, umbilical cord stem cells, and other sources can influence the regenerative capacity of damaged tissues through multiple pathways, promoting the recovery of damaged tissue structure and function.

[0004] The essential role of microRNA-155 in the pathogenesis of autoimmune arthritis in mice (Arthritis and rheumatism, 63, 1281-1288.10.1002 / art.30281) reports on the relationship between OA and miR-155; the PIEZO1 / miR-155-5p / GDF6 / SMAD2 / 3 signaling axis is involved in inducing the occurrence and progression of osteoarthritis under excessive mechanical stress (Cellular signalling, 118, 111142.10.1016 / j.cellsig.2024.111142) describes the preparation of untargeted drug products; and microRNA-155 as a proinflammatory regulator in clinical and experimental arthritis (Proceedings of the National Academy of Sciences of the United States of America). The article (America, 108, 11193-11198.10.1073 / pnas.1019536108) reports on RA and miR-155, and also mentions macrophages.

[0005] However, a search revealed that current technology lacks experimental results on the delivery of miR-155-5p inhibitors to inflammatory synovial macrophages for the treatment of OA and related cartilage degeneration and synovial inflammation.

[0006] It should be noted that the above background information is used to illustrate the research background of this solution and does not necessarily constitute prior art. Summary of the Invention

[0007] To address the aforementioned issues, this application provides an application for treating osteoarthritis using exosomes delivering miR-155-5p inhibitors targeting synovial inflammatory macrophages. Researchers in this application discovered that during the progression of osteoarthritis, synovial inflammatory macrophages secrete large amounts of exosomes that are absorbed by chondrocytes. Specifically, miR-155-5p is actively loaded onto exosomes derived from synovial inflammatory macrophages. Therefore, an exosome drug delivery system targeting synovial inflammatory macrophages containing miR-155-5p inhibitors was prepared. Based on the combined effects of stem cell exosomes' ability to repair damaged cartilage tissue and reduce inflammation, as well as the inhibition of miR-155-5p to combat synovial inflammation, this system is well-suited for osteoarthritis repair and exhibits significant efficacy advantages compared to existing exosome drug delivery systems containing miR-155-5p inhibitors.

[0008] This application provides an exosome containing a miR-155-5p inhibitor, and the exosome is capable of targeting synovial inflammatory macrophages.

[0009] Optionally, the miR-155-5p inhibitor is selected from one or more of the following:

[0010] a) Knockout of miR-155-5p targeting vector in cells;

[0011] b) miR-155-5p inhibitors;

[0012] c) Viruses expressing the antisense complement sequence of miR-155-5p.

[0013] The synovium plays a vital role in maintaining the normal physiological function of joints. It serves as a bridge connecting the internal structure of the joint with the musculoskeletal tissue. The hyaluronic acid it secretes can keep the articular cartilage surface lubricated, reduce the friction generated during joint movement, and provide nutrition to other soft tissues within the joint, thereby maintaining the integrity of the articular cartilage surface.

[0014] The synovium can be divided into two layers: the inner synovial layer (superficial layer) near the joint cavity and the subsynovial layer. The inner synovial layer has 2 to 3 layers of cells, which can be divided into type A synovial macrophages (SM) and type B synovial fibroblasts (FLS).

[0015] Type A synovial macrophages are the main immune cells in the joint. In the progression of osteoarthritis, they are polarized into a large number of inflammatory macrophages. The surface of inflammatory macrophages has many pseudopodia that penetrate into the synovial space. They can synthesize and release a variety of dissolving enzymes, which have functions such as phagocytosis, degradation, and removal of particulate matter and cell debris in the joint cavity.

[0016] In this application, the inflammatory macrophages of the synovium, namely the aforementioned type A synovial macrophages, are described as exhibiting polarized morphology during the pathological process of osteoarthritis.

[0017] Optionally, the miR-155-5p inhibitor comprises a small molecule containing an antisense complementary sequence of miR-155-5p, or further modified with cholesterol and 2'OMe. The miR-155-5p inhibitor comprises a complementary single chain of mature miR-155-5p; or a complementary single chain of a chemically modified mature miR-155-5p chain (e.g., cholesterol and 2'OMe modification). The miR-155-5p inhibitor includes methylation modification based on miR-155-5p, with optional 2 and 4 base thio modifications at the 5' and 3' ends (e.g., within 10 bases), respectively, and a high-affinity cholesterol modification linked to the 3' end. This can be achieved using conventional techniques in the art. Further information on miR-155-5p inhibitors can be found in the inventor's prior published patent CN117586946A.

[0018] This application provides an exosome containing a miR-155-5p inhibitor and carrying an affinity peptide for synovial inflammatory macrophages.

[0019] Optionally, the miR-155-5p inhibitor is selected from one or more of the following:

[0020] a) Knockout of miR-155-5p targeting vector in cells;

[0021] b) miR-155-5p inhibitors;

[0022] c) Viruses expressing the antisense complement sequence of miR-155-5p;

[0023] Optionally, the affinity peptide may be partially or entirely exposed on the outer side of the exosome;

[0024] Optionally, the affinity peptide comprises the amino acid sequence of LPSGAA;

[0025] Optionally, the affinity peptide is expressed in fusion with an exosome membrane protein;

[0026] Optionally, the exosome membrane protein is a transmembrane protein;

[0027] Optionally, the transmembrane protein is Lamp2b.

[0028] The affinity peptides described in this application are bioactive polypeptides used for affinity interactions with specific tissues or molecules. They can recognize specific tissues or molecules and achieve targeting effects through affinity with those tissues or molecules.

[0029] It should be noted that affinity peptides are only one way to target inflammatory macrophages in the synovium. Those skilled in the art can choose other methods besides affinity peptides to achieve targeting. They can also modify existing affinity peptides, etc. It is understood that as long as they possess affinity for inflammatory macrophages in the synovium, they can achieve the same technical effect as described in this application. For example, the fusion expression of exosome scaffold proteins (CD63 / CD9 / CD81, Lamp2b / PTGFRN) and targeted peptide lipid anchoring technology can also be used to achieve the targeting effect of exosomes, such as using Enzekangtai's ExoCoupl. TM Exosome surface modification bioorthogonal coupling kit for exosome editing and modification.

[0030] Optionally, the affinity peptide may be partially or entirely exposed on the outer side of the exosome;

[0031] Optionally, the affinity peptide comprises the amino acid sequence of LPSGAA.

[0032] Optionally, the affinity peptide is expressed in fusion with an exosome membrane protein;

[0033] Optionally, the exosome membrane protein is a transmembrane protein;

[0034] Optionally, the transmembrane protein is Lamp2b;

[0035] Optionally, the miR-155-5p inhibitor is selected from one or more of the following:

[0036] a) Knockout of miR-155-5p targeting vector in cells;

[0037] b) miR-155-5p inhibitors;

[0038] c) Viruses expressing the antisense complement sequence of miR-155-5p.

[0039] This application provides a method for preparing exosomes, the method comprising the following steps:

[0040] 1) Collect initial exosomes from cultured cells, the initial exosomes carrying affinity peptides for synovial inflammatory macrophages;

[0041] 2) Load the miR-155-5p inhibitor into the initial exosomes to obtain the exosomes.

[0042] Optionally, the affinity peptide may be partially or entirely exposed on the outer side of the exosome;

[0043] Optionally, the affinity peptide comprises the amino acid sequence of LPSGAA.

[0044] Optionally, the affinity peptide is expressed in fusion with an exosome membrane protein;

[0045] Optionally, the exosome membrane protein is a transmembrane protein;

[0046] Optionally, the transmembrane protein is Lamp2b;

[0047] Optionally, the miR-155-5p inhibitor is selected from one or more of the following:

[0048] a) Knockout of miR-155-5p targeting vector in cells;

[0049] b) miR-155-5p inhibitors;

[0050] c) Viruses expressing the antisense complement sequence of miR-155-5p.

[0051] Optionally, the cells are stem cells;

[0052] Alternatively, mesenchymal stem cells may be used;

[0053] Optionally, the mesenchymal stem cells are adipose-derived mesenchymal stem cells.

[0054] Those skilled in the art can choose natural stem cells or genetically engineered stem cells; this application does not make any specific limitation in this regard.

[0055] It should be noted that those skilled in the art can choose a suitable source of exosomes. When the exosomes contain components that have therapeutic or alleviating effects on osteoarthritis, cartilage degeneration, and synovial inflammation, they can theoretically be better used in conjunction with the present application's approach of targeting inflammatory macrophages in the synovium to inhibit miR-155-5p. However, it is understood that other components are not necessary. When the exosomes are blank carriers, such as blank liposomes, it does not hinder the realization of the technical concept and technical effects of this application. In practice, the source of exosomes can be selected and determined by those skilled in the art. In addition to natural exosomes, genetically engineered exosomes can also be selected.

[0056] Optionally, the preparation method includes constructing an expression vector that fuses and expresses a synovial inflammatory macrophage affinity peptide to an exosome membrane protein, and then transfecting the expression vector into the cells in step 1) to produce the initial exosomes.

[0057] It should be noted that the role of the vector here is to enable the expression of the affinity peptide and, through fusion with exosome membrane proteins, to achieve partial or complete exposure of the peptide to the outer side of the exosome. This is due to the effect of the expressed membrane proteins reaching the outer side of the exosome and forming its membrane. The method for achieving partial or complete exposure of the affinity peptide to the outer side of the exosome is not limited.

[0058] Those skilled in the art will understand that there are various ways to achieve the fusion expression of affinity peptides with exosomal membrane proteins, and this application does not impose any specific limitations. Furthermore, there are no specific limitations on the choice of vector; those skilled in the art can choose vectors such as plasmids or viruses. As long as the affinity peptide is partially or completely exposed on the outer side of the exosome, the exosome should have the ability to target synovial inflammatory macrophages, and all such vectors can achieve the technical effects of the solution described in this application.

[0059] For example, to achieve the fusion expression of synovial inflammatory macrophage affinity peptides on exosomal membrane proteins, the MAP-Lamp2b plasmid is constructed using genetic engineering. This plasmid fuses the glycosylated peptide (GNSTM) gene, the affinity peptide (MAP: LPSGAA) gene, and the exosomal transmembrane protein (Lamp2b) gene, thereby achieving the fusion expression of synovial inflammatory macrophage affinity peptides on exosomal membrane proteins and obtaining exosomes targeting synovial inflammatory macrophages. The operation of fusing and expressing multiple genes is a conventional technique for those skilled in the art. The selection of specific gene sequences, the design of corresponding nucleotide sequences based on amino acid sequences, and the construction of expression vectors such as plasmid vectors are all conventional techniques for those skilled in the art.

[0060] This application provides the use of the above-described exosomes or exosomes prepared by the above-described method in the preparation of pharmaceutical products.

[0061] Optionally, the pharmaceutical product is used to relieve or treat one or more of cartilage degeneration, synovitis, and osteoarthritis.

[0062] This application provides a pharmaceutical product comprising the exosomes described above or exosomes prepared by the above-described method for preparing exosomes.

[0063] This application provides a pharmaceutical product that can target synovial inflammatory macrophages, the pharmaceutical product comprising an ingredient that can reduce miR-155-5p activity.

[0064] Optionally, the component capable of reducing miR-155-5p activity is selected from one or more of the following:

[0065] a) Knockout of miR-155-5p targeting vector in cells;

[0066] b) miR-155-5p inhibitors;

[0067] c) Viruses expressing the antisense complement sequence of miR-155-5p.

[0068] Optionally, the component that reduces miR-155-5p activity can reduce the activity of miR-155-5p in synovial inflammatory macrophages.

[0069] For example, targeting vectors that knock out miR-155-5p in cells, miR-155-5p inhibitors, or viruses that express the antisense complementary sequence of miR-155-5p, all known in the art, can reduce the activity of miR-155-5p in synovial inflammatory macrophages. Those skilled in the art can also choose other feasible technical solutions. As long as they can reduce the activity of miR-155-5p in synovial inflammatory macrophages, they can achieve the same technical effect as the solution in this application.

[0070] No specific limitations are made on the form of the drug product. Those skilled in the art can choose an appropriate method as needed. For example, this application uses an exosome form to target inflammatory macrophages of the synovium and carries a component that can reduce miR-155-5p activity, thereby achieving its technical effect. It is understood that other forms that can target inflammatory macrophages of the synovium and carry a component that can reduce miR-155-5p activity can also achieve the same technical effect as this application through routine experiments and adjustments by those skilled in the art.

[0071] It should be noted that the meaning of reducing activity, such as reducing its content, reducing its expression level, knocking out related genes to reduce its content, or reducing its biological activity through modification or inhibitors, or reducing its content by accelerating its degradation, all fall within the scope of reducing activity in this application. It can be understood that the meaning of reducing activity in this application is broad.

[0072] The beneficial effects of this application include, but are not limited to:

[0073] 1. Based on the application of this application for the exosome delivery of miR-155-5p inhibitors targeting synovial inflammatory macrophages to treat osteoarthritis, and based on prior research, this application further clarifies the important role of reducing the activity of miR-155-5p in synovial inflammatory macrophages in the treatment of cartilage degeneration, synovial inflammation, and osteoarthritis. However, no clear research results on this have been found in the current art.

[0074] 2. The application of this application for the treatment of osteoarthritis by targeting synovial inflammatory macrophages with exosome delivery of miR-155-5p inhibitors, which specifically reduces the activity of miR-155-5p in synovial inflammatory macrophages, has significant technological advancements compared to non-targeted approaches. This is of great significance for the treatment of cartilage degeneration, synovial inflammation, and osteoarthritis, and will have a profound impact on future drug development for the treatment of cartilage degeneration, synovial inflammation, and osteoarthritis.

[0075] 3. Based on the application of this application for the exosome delivery of miR-155-5p inhibitors targeting synovial inflammatory macrophages to treat osteoarthritis, and combined with the currently popular exosome technology, an exosome drug that specifically reduces the activity of miR-155-5p in synovial inflammatory macrophages is provided. The technical effect of specifically reducing the activity of miR-155-5p in synovial inflammatory macrophages has been verified, which is of great significance for fully utilizing the application of exosomes in pharmaceutical products. Attached Figure Description

[0076] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments of this application and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0077] Figure 1 This is a diagram illustrating the construction strategy of the synovial inflammatory macrophage mir-155-5p strip knockout mouse involved in Example 1 of this application;

[0078] Figure 2 This is a schematic diagram of an animal experiment involved in Embodiment 1 of this application;

[0079] Figure 3 The images show the morphological display of cartilage tissue before and after knockout of synovial inflammatory macrophages miR-155-5P and the OARSI scoring results in Example 1 of this application (where A is the morphological display of cartilage tissue, and from top to bottom are Safranin O-Rapid Green staining of the same specimen, with scale bars of 200 μm (top) and 50 μm (bottom), respectively; B is the OARSI scoring result).

[0080] Figure 4 The image shows the COL2A1 expression results in cartilage tissue before and after knockout of mir-155-5p inflammatory macrophages involved in Example 1 of this application (where A is an immunohistochemical image of COL2A1 in cartilage tissue, with scale bars of 200μm (top) and 50μm (bottom) in A, and B is a quantitative analysis of COL2A1 positive cells in cartilage tissue).

[0081] Figure 5 The image shows the expression results of MMP13 in cartilage tissue before and after knockout of synovial inflammatory macrophages mir-155-5p in Example 1 of this application (where A is the immunohistochemical image of MMP13 in cartilage tissue, and the scale bars in A are 200μm (top) and 50μm (bottom), respectively; B is the quantitative analysis of MMP13 positive cells in cartilage tissue).

[0082] Figure 6This is a morphological illustration of synovial tissue before and after knockout of mir-155-5p inflammatory macrophages involved in Example 1 of this application (where A is the morphology of synovial tissue, and the scale bars in A are 200μm (top) and 50μm (bottom), respectively; B is the synovial inflammation score result).

[0083] Figure 7 This is a graph showing the effect of knocking out mir-155-5p inflammatory macrophages of the synovium on the proportion of inflammatory macrophages derived from BMDMs before and after the knockout of mir-155-5p in synovial cells involved in Example 1 of this application.

[0084] Figure 8 This is a diagram illustrating the construction strategy of the chondrocyte mir-155-5p strip knockout mouse involved in Example 1 of this application;

[0085] Figure 9 The images show the morphological display of cartilage tissue before and after mir-155-5p knockout of chondrocytes involved in Example 1 of this application and the results of OARSI scoring (where A is the morphological display of cartilage tissue, stained with Safranin O-Rapid Green, scale bar is 200μm, and B is the OARSI scoring result).

[0086] Figure 10 The proceduralized exosomes MAP-EVs involved in Embodiment 2 of this application ADSCs A schematic diagram;

[0087] Figure 11 The engineered exosome MAP-EVs involved in Embodiment 2 of this application ADSCs NTA analysis plot (scale bar at 200 nm);

[0088] Figure 12 The engineered exosome MAP-EVs involved in Embodiment 2 of this application ADSCs Scanning electron microscope image;

[0089] Figure 13 This is a schematic diagram of an animal experiment involved in Embodiment 2 of this application;

[0090] Figure 14 The engineered exosome MAP-EVs involved in Embodiment 2 of this application ADSCs WB electrophoresis image;

[0091] Figure 15 This is an in vivo localization map (scale bar is 100 μm) of the engineered exosomes involved in Example 2 of this application in the synovial inflammatory macrophages of KOA rats;

[0092] Figure 16 This is a schematic diagram of an animal experiment involved in Embodiment 3 of this application;

[0093] Figure 17This is a graph showing the effect of engineered exosomes involved in Example 3 of this application on the content of miR-155-5p in synovial tissue;

[0094] Figure 18 This is a diagram showing the therapeutic effect of engineered exosomes on synovial inflammation according to Example 3 of this application (where A is the morphology of synovial tissue, with HE staining of the same specimen from top to bottom, and scale bars at 200 μm (top) and 50 μm (bottom), respectively; B is the synovial inflammation scoring result, and the legend is the same as above). Figure 17 (The same as B in Chinese);

[0095] Figure 19 This is a figure showing the effect of engineered exosomes on CD206 expression in synovial tissue according to Example 3 of this application (where A is an immunohistochemical image of CD206 in synovial tissue, scale bar is 100 μm, B is quantitative analysis of CD206 positive cells, and the legend is the same as above). Figure 17 (The same as B in Chinese);

[0096] Figure 20 This is a figure showing the effect of engineered exosomes on iNOS in synovial tissue according to Example 3 of this application (where A is an immunohistochemical image of iNOS in synovial tissue, scale bar is 100 μm, B is the quantitative analysis of iNOS positive cells, and the legend is the same as above). Figure 17 (The same as B in Chinese);

[0097] Figure 21 This is a graph showing the effect of engineered exosomes involved in Example 4 of this application on the content of miR-155-5p in cartilage tissue;

[0098] Figure 22 This is a diagram showing the therapeutic effect of engineered exosomes on cartilage degeneration according to Example 4 of this application (where A is the morphological display of cartilage tissue, from top to bottom showing safranin O-rapid green staining of the same specimen, scale bars are 200μm (top) and 50μm (bottom), B is the OARSI score result, and the legend is the same as...). Figure 17 (The same as B in Chinese);

[0099] Figure 23 This is a graph showing the effect of engineered exosomes on the COL2A1 content in cartilage tissue according to Example 4 of this application (where A is the immunofluorescence image of COL2A1 in cartilage tissue, scale bar is 100 μm, B is the quantitative analysis of COL2A1 fluorescence intensity, and the legend is the same as above). Figure 17 (The same as B in Chinese);

[0100] Figure 24 This is a figure showing the effect of engineered exosomes on the proportion of MMP13-positive cells in cartilage tissue according to Example 4 of this application (where A is an immunohistochemical image of MMP13 in cartilage tissue, scale bar is 100 μm, B is the quantitative analysis of MMP13-positive cells, and the legend is the same as above). Figure 17 (The same as B in Chinese);

[0101] Figure 25 This is a figure showing the effect of engineered exosomes on the proportion of GSK-3β-positive cells in cartilage tissue according to Example 4 of this application (where A is an immunohistochemical image of GSK-3β in cartilage tissue, scale bar is 100 μm, B is the quantitative analysis of GSK-3β-positive cells, and the legend is the same as above). Figure 17 (The same as B in Chinese). Detailed Implementation

[0102] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments. Unless otherwise specified, the raw materials and reagents in the embodiments of the present application are all purchased through commercial channels.

[0103] The specific experimental operations involved in this application are as follows. It should be noted that other operations not detailed in this application can be performed with reference to existing technical materials or textbooks, and are all routine operations for those skilled in the art.

[0104] Knee joint sample collection, fixation, and decalcification:

[0105] Mice / rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution. Then, the mice / rats were euthanized by cervical dislocation, and the hind limbs were dissected, with muscle removed as much as possible while maintaining joint continuity; joint detachment was avoided. Ten samples were placed in 4% paraformaldehyde and fixed on a shaker at 4°C for 24 hours (the 4% paraformaldehyde solution was changed once during this period) for morphological staining of the joint tissue.

[0106] Take the above-preserved mouse / rat knee joint samples, place them in deionized water, and rinse them repeatedly 6 times, each time for 15 minutes. Then, transfer them to 0.5M Na2EDTA decalcification solution at 4℃ for 7 days, changing the solution every two days until decalcification is complete (they can be easily pierced by a needle and can be bent without resistance).

[0107] Preparation of paraffin sections of the knee joint:

[0108] The decalcified samples were soaked in 0.01M PBS solution at 4°C for 6-18 hours.

[0109] Gradient alcohol dehydration for 60 min (50%, 70%, 80%, 95%, 95%, 100%, 100% in sequence).

[0110] Xylene I and II, 60 min each; soft wax, 60 min (xylene: paraffin = 1:1, 60℃ 60 min); hard wax, 60 min (paraffin, 60℃ 60 min).

[0111] Paraffin embedding: First, preheat the paraffin embedding machine to melt the paraffin blocks. Remove the dehydrated joint sample and place it in the liquid paraffin pool, embedding it sagittally with the inner surface of the joint facing down. After the liquid paraffin-embedded sample has solidified, place it on a low-temperature ice table to cool and solidify. Store the solidified paraffin block at -20°C. Repeat this embedding process for other samples.

[0112] Paraffin sections of tissue: Sagittal sections are taken from the medial aspect of the joint. The sections are first trimmed to a thickness of 15-20 μm. The sections are then retrieved and observed under a microscope while trimming. When the desired area is reached, the thickness is adjusted to 2-3 μm. The sections are cut, spread, and observed simultaneously. The articular surface in the center of the joint is collected.

[0113] Dewaxing and hydration of the slices: Place the above paraffin slices in a constant temperature oven at 65℃ for 2 hours. After the paraffin melts and drips, dewaxing and hydration are performed.

[0114] Safranin O-Rapid Green staining of paraffin sections of the knee joint:

[0115] a) Baking: Place the paraffin slices in a constant temperature oven and bake at 65°C for 2 hours;

[0116] b) Xylene dewaxing: Xylene I and II, 10 min each;

[0117] c) Hydration: 100% ethanol I and II, 10 min each; 95% and 80% ethanol solutions, 8 min each; 70% and 50% ethanol solutions, 5 min each;

[0118] d) Wash and remove ethanol: Rinse with PBS 3 times, 5 min each time;

[0119] e) Stain the nucleus with hematoxylin for 10 seconds, then rinse several times with deionized water, differentiate with differentiation solution for 3 seconds, and then rinse several times with deionized water.

[0120] f) Stain the sample with 0.2% rapid green staining solution for 20 seconds, without rinsing;

[0121] g) Place the sample in 1% glacial acetic acid for differentiation for 15 seconds, without rinsing;

[0122] h) Stain the sample with 1% safranin O solution for 1-2 minutes without rinsing;

[0123] i) Gradient alcohol dehydration: The specific program is 50%, 70%, 80%, 90%, 100% ethanol I and 100% ethanol II solutions, 3 min each;

[0124] j) Transparent: Xylene I, 3 min; Xylene II, 1 min;

[0125] k) Mounting: Mount the slide with neutral resin, avoiding the formation of air bubbles. Next, observe the slide using an Olympus upright fluorescence microscope (BX53).

[0126] Hematoxylin and eosin staining (HE staining) of paraffin sections of the knee joint:

[0127] a) The four steps of baking, dewaxing, and gradient alcohol hydration for paraffin sections of the knee joint are the same as those for safranin O-rapid green staining.

[0128] b) Use hematoxylin solution to stain for 10 seconds, then rinse with deionized water for 30 seconds;

[0129] c) Use 1% hydrochloric acid alcohol differentiation medium, differentiate for 10 seconds, then rinse with deionized water until the cell nuclei appear blue under an optical microscope;

[0130] d) Stain with eosin solution for 1 min, then rinse with deionized water for 1 min;

[0131] e) Continue until the cell nucleus and cytoplasm appear blue and red respectively when observed under a microscope;

[0132] f) Place the slices in a 37°C oven to air dry, seal, observe and photograph.

[0133] Example 1: Inflammatory macrophages in the synovium, rather than chondrocytes, exacerbate the progression of osteoarthritis.

[0134] 1) Prepare synovial inflammatory macrophage strips (mir-155-5p) for knockout mice ( Figure 1 ): Using transgenic animals LyzM Cre and miR-155 flox / flox Through mating and selective breeding (purchased from Cyagen Biosciences), miR-155 inflammatory macrophage conditional knockout mice were obtained. flox / flox LyzM Cre .

[0135] like Figure 2 The diagram shown is a schematic of an animal experiment.

[0136] like Figure 3 The image shows the morphological results of cartilage tissue before and after knockout of synovial inflammatory macrophages mir-155-5p. According to... Figure 3 The experimental results show that, compared with the control group, the synovial inflammatory macrophage miR-155-5p knockout mice showed significantly slower cartilage degeneration and a significantly lower OARSI score (from 2.95 to 1.95, a decrease of 34%).

[0137] like Figure 4The image shows the COL2A1 expression results in cartilage tissue before and after knockout of mir-155-5p inflammatory macrophages in the synovium. According to... Figure 4 The experimental results show that, compared with the control group, the proportion of COL2A1 positive chondrocytes in the cartilage tissue of synovial inflammatory macrophage mir-155-5p knockout mice was significantly increased (from 43.56% to 57.81%, an increase of 33%).

[0138] like Figure 5 The image shows the expression results of MMP13 in cartilage tissue before and after knockout of mir-155-5p inflammatory macrophages in the synovium. According to... Figure 5 The experimental results show that, compared with the control group, the proportion of MMP13 positive chondrocytes in the cartilage tissue of synovial inflammatory macrophage mir-155-5p knockout mice was significantly reduced (from 72.17% to 50.20%, a decrease of 30%).

[0139] like Figure 6 The image shows the morphological results of synovial tissue before and after knockout of inflammatory macrophages mir-155-5p. According to... Figure 6 The experimental results show that, compared with the control group, the number of synovial inflammatory macrophages mir-155-5p knockout mice was significantly reduced, and the synovial inflammation score was significantly reduced (from 3.81 to 0.54, a reduction of 86%).

[0140] like Figure 7 The image shows the effect of mir-155-5p knockout on the proportion of inflammatory macrophages derived from BMDMs. Based on... Figure 7 The experimental results show that, compared with the control group, the proportion of inflammatory macrophages differentiated from bone marrow-derived macrophages was significantly reduced in synovial inflammatory macrophage knockout mice (from 41.04% to 34.08%, a decrease of 17%).

[0141] 2) Prepare chondrocyte mir-155-5p strips for knockout mice ( Figure 8 ): Using transgenic animals Col2a1 Cre and miR-155 flox / flox Through mating and selective breeding (purchased from Cyagen Biosciences), miR-155-155-5p conditional knockout mice were obtained. flox / flox Col2a1 Cre .

[0142] like Figure 9 The image shows the morphological results of cartilage before and after mir-155-5p knockout of chondrocytes. According to... Figure 9The experimental results show that, compared with the control group, the mir-155-5p chondrocyte knockout mice showed no significant changes in cartilage degeneration and OARSI scores.

[0143] Based on the above results, it can be concluded that miR-155-5p in synovial inflammatory macrophages, rather than in chondrocytes, exacerbates the progression of osteoarthritis.

[0144] Example 2: Preparation of engineered exosomes for targeted delivery of Antagonomi R-155-5p to synovial inflammatory cells

[0145] 1) Isolation and identification of hADSCs

[0146] hADSCs were extracted from healthy human adipose tissue (provided by the 85th Hospital of the People's Liberation Army and approved by the hospital's ethics committee). The adipose tissue was washed three times with PBS, minced, and digested with 0.1% collagenase I for 60 min. After centrifugation at 1200 rpm for 5 min, the cells were cultured in F12 / DMEM medium containing 10% FBS at 37°C and 5% CO2. 2 After culturing in a cell culture incubator for 24 hours, the medium was changed. When the cells reached 80% confluence, they were digested with 0.25% trypsin and passaged at a 1:3 ratio. Seven 500 μL single-cell suspensions were prepared from the digested cells, and 2 μL of anti-human CD44-PE, CD90-FITC, CD73-APC, CD19-FITC, and CD105-PerCP-CyTM 5.5 monoclonal antibodies (BD, 562245) were added to each suspension. The cells were incubated at 37°C for 30 min, washed three times with PBS, and the CD molecules on the surface of hADSCs were detected using flow cytometry. The morphology of hADSCs was observed under a light microscope.

[0147] 2) Enrichment of targeted exosomes derived from ADSCs

[0148] 2.1) Construct a plasmid system MAP-Lamp2b that fuses the affinity peptide (MAP) carrying synovial inflammatory macrophages into an exosome membrane protein (Lamp2b), and compare it with the Lamp2b plasmid, such as... Figure 10 As shown, the construction method is as follows:

[0149] The MAP-Lamp2b plasmid was constructed using genetic engineering. The glycosylation-modified peptide (GNSTM) gene, the affinity peptide (MAP: LPSGAA) gene, and the exosome transmembrane protein (Lamp2b) gene were fused and expressed. This enabled the fusion expression of the affinity peptide for synovial inflammatory macrophages onto the exosome membrane protein, resulting in exosomes targeting synovial inflammatory macrophages. The construction of the MAP-Lamp2b plasmid is a routine technique for those skilled in the art. For example, the content disclosed in the paper "Exosome-mediated delivery of kartogenin for chondrogenesis of synovial fluid-derived mesenchymal stemcells and cartilage regeneration" (Xu X, Biomaterials. 2021 Feb; 269:120539. doi:10.1016 / j.biomaterials.2020.120539.Epub 2020Nov 18.PMID:33243424.) was referenced, and the affinity peptide sequence was replaced with the desired sequence.

[0150] 2.2) After the P3-P7 generation of hADSCs reached 80%, the above plasmid system was transfected into ADSCs cells using Lipo3000, ensuring that the positive rate of each transfection reached more than 90%. After 8 hours of transfection, the culture medium was changed to exosome-free serum (EVs-free FBS) and cultured for another 24 hours. The cell culture supernatant was collected, and the exosomes adhering to the cell surface were washed with PBS and collected as well.

[0151] 2.3) Electroporation was used to transfer AntagonomiR-155-5p into the target exosomes obtained in the above steps, so that the exosomes were coated with miRNA and carried AntagonomiR-155-5p.

[0152] 2.4) The collected supernatant was pre-centrifuged sequentially at 4℃ for 300g x 10min, 2000g x 20min, and 10000g x 30min. After filtration through a 0.22μm filter, the supernatant was carefully transferred to sterilized ultracentrifuge tubes (Beckman Coulter, #lot P22192, USA). After sealing, the tubes were weighed and balanced to two decimal places. The tubes were then centrifuged at 4℃ and 100000g for 70min using an ultracentrifuge (Beckman Coulter, Optima XPN-100, USA). The supernatant was carefully removed with a sterile Pasteur pipette and resuspended in 20-25ml of cold PBS. The tubes were then centrifuged again at 100000g for 70min. Finally, the PBS was removed and each tube was resuspended in 100μL of sterile PBS. The tubes were used immediately or stored at -80℃.

[0153] 2.5) Particle size analysis of exosomes: The concentration and purity of exosomes were determined using a particle size analyzer (NS300). Exosomes were prepared into 1 mL of a solution of appropriate concentration and injected into the sample cell until it was full. The instrument parameters were adjusted to obtain the distribution intensity and scattering intensity of the exosomes.

[0154] 2.6) Transmission electron microscopy (TEM) analysis: Hitachi HT7800 electron microscope (HT-7800, Hitachi, Japan);

[0155] 2.7) WB analysis.

[0156] according to Figure 11 The results show that MAP-EVs ADSCs The particle size is generally distributed between 75 and 180 nm, and the average particle size is 130.8 nm.

[0157] according to Figure 12 The results show that MAP-EVs ADSCs It exhibits a saucer-like or concave hemispherical structure, with a distinct membrane structure, clear three-dimensional structure, and a size of approximately 30–200 nm.

[0158] like Figure 13 As shown, ADSC was transfected into plasmids Lamp2b and MAP-Lamp2b, respectively, and then the corresponding exosomes (EVs) were collected. ADSCs and MAP-EVs ADSCs Next, ADSC cells transfected with the plasmid and the corresponding exosomes were lysed, and Western blot analysis was performed on the exosomal protein markers Alix, Tsg101, CD9, Lamp2b and ER marker Calnexin in the lysate.

[0159] according to Figure 14The results showed that exosome MAP-EVs ADSCs and EVs ADSCs The proteins Alix, Tsg101, CD9, and Lamp2b were highly expressed, but Calnexin was not expressed. The protein expression in the corresponding transfected cells was the opposite, indicating that the expression of the above-mentioned highly expressed proteins differs between exosomes and cells.

[0160] according to Figure 15 The results show that engineered exosomes MAP-EVs ADSCs In animal models for treating osteoarthritis, engineered exosomes MAP-EVs carrying the FAM marker AntagonomiR-155-5p were injected in situ into the joint cavity. ADSCs Immunofluorescence results of knee joint tissue showed: MAP-EVs ADSCs The overlap rate (84%) between FAM-AntagomiR-155-5p (green) and the synovial inflammatory macrophage marker iNOS (red) in the treatment group was significantly higher than that in EVs. ADSCs Treatment group (5%).

[0161] The above results indicate that high-purity stem cell-derived engineered exosomes targeting synovial inflammatory macrophages can be obtained by transfecting ADSCs with the target plasmid, and MAP-EVs ADSCs It specifically targets inflammatory macrophages in the synovium, enabling the targeted absorption of AntagonomiR-155-5p by these macrophages, resulting in better therapeutic effects for osteoarthritis.

[0162] Example 3: The therapeutic effect of engineered exosomes targeting synovial inflammatory cells to deliver AntagonomiR-155-5p on synovial inflammation (animal experiment).

[0163] like Figure 16 The test method is shown below:

[0164] Thirty-five 8-week-old male wild-type (WT) SD rats were randomly divided into 5 groups of 7 rats each:

[0165] Sham group: Seven 8-week-old male wild-type (WT) SD rats underwent sham surgery, incising only the skin and joint capsule at the knee joint, while other tissues remained intact.

[0166] OA group: 28 eight-week-old male wild-type (WT) SD rats underwent single-knee DMM+ACLT surgery and were randomly divided into 4 groups: ①OA+PBS group, ②OA+EVs group. ADSCs Group 3, OA+EVs ADSCs -AntagomiR-155-5p group,④OA+MAP-EVs ADSCs-AntagomiR-155-5p group. 50 μL of EVs were injected orally into the joint cavity. ADSCs or MAP-EVs ADSCs Injections began 4 weeks post-surgery and continued for 6 weeks, once weekly. Knee joint samples were collected 12 weeks post-surgery. Among them, EVs ADSCs It is a wild-type exosome derived from ADSC.

[0167] according to Figure 17 The results showed that, compared with the control group, the exosome group significantly reduced the level of miR-155-5p in the synovial tissue of OA rats. Unmodified EVs ADSCs Exosomes, regardless of whether they carried antagomiR-155-5p, had no significant effect on miR-155-5p levels in the synovial tissue of OA rats (20.52 and 14.61); EVs targeting inflammatory macrophages of the synovium ADSCs Engineered exosomes (MAP-EVs) ADSCs The effect of carrying antagomiR-155-5p was the best (reduced to 7.43% compared to the control group of 36.06%; compared to unmodified EVs). ADSCs Exosomes decreased from 20.52% / 14.61% to 7.43%.

[0168] according to Figure 18 The results showed that, compared with the control group, the exosome group significantly improved synovial inflammation in OA rats (6.40 compared with the control group, EVs alone). ADSCs The exosome count decreased to 4.11 for EVs carrying antagomiR-155-5p. ADSCs Engineered exosomes decreased to 2.81, targeting inflammatory macrophages in the synovium. ADSCs The engineered exosome count decreased to 1.33.

[0169] Among them, EVs carrying antagomiR-155-5p ADSCs Engineered exosomes are superior to individual EVs ADSCs Exosomes (as opposed to individual EVs) ADSCs Exosome group 4.11, EVs carrying antagomiR-155-5p ADSCs Engineered exosomes decreased to 2.81), and EVs targeting synovial inflammatory macrophages ADSCs Engineered exosomes (MAP-EVs) ADSCs Superior to unmodified EVs ADSCs Exosomes (EVs targeting synovial inflammatory macrophages) ADSCs The engineered exosome count was reduced to 1.33, compared to EVs alone. ADSCsThe exosome count decreased to 4.11 for EVs carrying antagomiR-155-5p. ADSCs The engineered exosome count decreased to 2.81.

[0170] EVs targeting inflammatory macrophages in the synovium ADSCs Engineered exosomes compared to EVs carrying antagomiR-155-5p ADSCs The engineered exosome group decreased from 2.81 to 1.33, a reduction of 53%.

[0171] according to Figure 19 The results showed that, compared with the control group, the exosome group significantly increased the proportion of CD206-positive cells in the synovial tissue of OA rats. Among them, EVs carrying antagomiR-155-5p... ADSCs Engineered exosomes are superior to individual EVs ADSCs Exosomes, and EVs targeting inflammatory macrophages of the synovium. ADSCs Engineered exosomes (MAP-EVs) ADSCs Superior to unmodified EVs ADSCs exosomes.

[0172] EVs targeting inflammatory macrophages in the synovium ADSCs Engineered exosomes compared to EVs carrying antagomiR-155-5p ADSCs The engineered exosome group increased from 35.49 to 58.78, an improvement of 66%.

[0173] according to Figure 20 The results showed that, compared with the control group, the exosome group significantly reduced the proportion of iNOS-positive cells in the synovial tissue of OA rats. Among them, EVs carrying antagomiR-155-5p... ADSCs Engineered exosomes are superior to individual EVs ADSCs Exosomes, and EVs targeting inflammatory macrophages of the synovium. ADSCs Engineered exosomes (MAP-EVs) ADSCs Superior to unmodified EVs ADSCs exosomes.

[0174] EVs targeting inflammatory macrophages in the synovium ADSCs Engineered exosomes compared to EVs carrying antagomiR-155-5p ADSCs The engineered exosome group decreased from 46.28 to 27.49, a reduction of 41%.

[0175] Example 4: The therapeutic effect of engineered exosomes targeting synovial inflammatory cells to deliver Antagonomi R-155-5p on cartilage degeneration (animal experiment).

[0176] Thirty-five 8-week-old male wild-type (WT) SD rats were randomly divided into 5 groups of 7 rats each:

[0177] Sham group: Seven 8-week-old male wild-type (WT) SD rats underwent sham surgery, incising only the skin and joint capsule at the knee joint, while other tissues remained intact.

[0178] OA group: 28 eight-week-old male wild-type (WT) SD rats underwent single-knee DMM+ACLT surgery and were randomly divided into 4 groups: ①OA+PBS group, ②OA+EVs group. ADSCs Group 3, OA+EVs ADSCs -AntagomiR-155-5p group,④OA+MAP-EVs ADSCs -AntagomiR-155-5p group. 50 μL of EVs were injected orally into the joint cavity. ADSCs or MAP-EVs ADSCs Injections began 4 weeks post-surgery and continued for 6 weeks, once weekly. Knee joint samples were collected 12 weeks post-surgery. Among them, EVs ADSCs It is a wild-type exosome derived from ADSCs; AntagonomiR-155-5p is obtained by modifying miR-155-5p with cholesterol and 2'OMe, and its function is to inhibit the activity of miR-155-5p. The inhibitor used in this application can also be purchased directly, such as from GemmaGene.

[0179] according to Figure 21 The results showed that, compared with the control group, the exosome groups significantly reduced the level of miR-155-5p in the cartilage tissue of OA rats. EVs carrying antagomiR-155-5p... ADSCs Exosomes (2.00%) compared to wild-type EVs ADSCs (3.95%) significantly reduced the level of miR-155-5p in the cartilage tissue of OA rats. Carriers of antagomiR-155-5p, regardless of whether they underwent targeted modification (targeted synovial inflammatory macrophages: 1.22%; untargeted modification: 2.00%), had no significant effect on the level of miR-155-5p in the cartilage tissue of OA rats.

[0180] according to Figure 22 (In section A, from top to bottom, are safranin O-rapid green stainings of the same specimen. Scale bars are 500 μm (top) and 100 μm (bottom).) The results showed that, compared with the control group, the exosome groups significantly improved cartilage degeneration in OA rats, especially those carrying antagomiR-155-5p. ADSCs Engineered exosomes are superior to individual EVs ADSCsExosomes, and EVs targeting inflammatory macrophages of the synovium. ADSCs Engineered exosomes (MAP-EVs) ADSCs Superior to unmodified EVs ADSCs exosomes.

[0181] EVs targeting inflammatory macrophages in the synovium ADSCs Engineered exosomes compared to EVs carrying antagomiR-155-5p ADSCs The engineered exosome group decreased from 10.40 to 5.07, a reduction of 51%.

[0182] according to Figure 23 The results showed that, compared with the control group, the exosome groups significantly increased the COL2A1 content in the cartilage tissue of OA rats. Among them, EVs carrying antagomiR-155-5p... ADSCs Engineered exosomes are superior to individual EVs ADSCs Exosomes, and EVs targeting inflammatory macrophages of the synovium. ADSCs Engineered exosomes (MAP-EVs) ADSCs Superior to unmodified EVs ADSCs exosomes.

[0183] The EVsADSCs engineered exosomes targeting synovial inflammatory macrophages increased from 62.10% to 75.47%, a 22% improvement, compared to the EVsADSCs engineered exosomes carrying antagomiR-155-5p.

[0184] according to Figure 24 The results showed that, compared with the control group, the exosome group significantly reduced the proportion of MMP13-positive cells in the cartilage tissue of OA rats. Among them, EVs carrying antagomiR-155-5p... ADSCs Engineered exosomes are superior to individual EVs ADSCs Exosomes, and EVs targeting inflammatory macrophages of the synovium. ADSCs Engineered exosomes (MAP-EVs) ADSCs Superior to unmodified EVs ADSCs exosomes.

[0185] EVs targeting inflammatory macrophages in the synovium ADSCs Engineered exosomes compared to EVs carrying antagomiR-155-5p ADSCs The engineered exosome group decreased from 35.90% to 21.27%, a reduction of 41%.

[0186] according to Figure 25The results showed that, compared with the control group, the exosome group significantly increased the proportion of GSK-3β-positive cells in the cartilage tissue of OA rats. Among them, EVs carrying antagomiR-155-5p... ADSCs Engineered exosomes are superior to individual EVs ADSCs Exosomes, and EVs targeting inflammatory macrophages of the synovium. ADSCs Engineered exosomes (MAP-EVs) ADSCs Superior to unmodified EVs ADSCs exosomes.

[0187] EVs targeting inflammatory macrophages in the synovium ADSCs Engineered exosomes compared to EVs carrying antagomiR-155-5p ADSCs The engineered exosome group increased from 66.80% to 82.71%, an increase of 24%.

[0188] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. An exosome, characterized in that, The exosomes contain miR-155-5p inhibitors and are capable of targeting synovial inflammatory macrophages; Optionally, the miR-155-5p inhibitor is selected from one or more of the following: a) Knockout of miR-155-5p targeting vector in cells; b) miR-155-5p inhibitors; c) Viruses expressing the antisense complement sequence of miR-155-5p.

2. An exosome, characterized in that, The exosomes contain miR-155-5p inhibitors and carry affinity peptides for synovial inflammatory macrophages; Optionally, the miR-155-5p inhibitor is selected from one or more of the following: a) Knockout of miR-155-5p targeting vector in cells; b) miR-155-5p inhibitors; c) Viruses expressing the antisense complement sequence of miR-155-5p; Optionally, the affinity peptide may be partially or entirely exposed on the outer side of the exosome; Optionally, the affinity peptide comprises the amino acid sequence of LPSGAA; Optionally, the affinity peptide is expressed in fusion with an exosome membrane protein; Optionally, the exosome membrane protein is a transmembrane protein; Optionally, the transmembrane protein is Lamp2b.

3. The exosomes according to claim 2, characterized in that, The affinity peptide is partially or entirely exposed on the outer side of the exosome; Optionally, the affinity peptide comprises the amino acid sequence of LPSGAA; Optionally, the affinity peptide is expressed in fusion with an exosome membrane protein; Optionally, the exosome membrane protein is a transmembrane protein; Optionally, the transmembrane protein is Lamp2b; Optionally, the miR-155-5p inhibitor is selected from one or more of the following: a) Knockout of miR-155-5p targeting vector in cells; b) miR-155-5p inhibitors; c) Viruses expressing the antisense complement sequence of miR-155-5p.

4. A method for preparing exosomes, characterized in that, The preparation method includes the following steps: 1) Collect initial exosomes from cultured cells, the initial exosomes carrying affinity peptides for synovial inflammatory macrophages; 2) Load the miR-155-5p inhibitor into the initial exosomes to obtain the exosomes.

5. The method for preparing exosomes according to claim 4, characterized in that, The affinity peptide is partially or entirely exposed on the outer side of the exosome; Optionally, the affinity peptide comprises the amino acid sequence of LPSGAA; Optionally, the affinity peptide is expressed in fusion with an exosome membrane protein; Optionally, the exosome membrane protein is a transmembrane protein; Optionally, the transmembrane protein is Lamp2b; Optionally, the miR-155-5p inhibitor is selected from one or more of the following: a) Knockout of miR-155-5p targeting vector in cells; b) miR-155-5p inhibitors; c) Viruses expressing the antisense complement sequence of miR-155-5p.

6. The method for preparing exosomes according to claim 4, characterized in that, The cells in question are stem cells; Alternatively, mesenchymal stem cells may be used; Optionally, the mesenchymal stem cells are adipose-derived mesenchymal stem cells.

7. The method for preparing exosomes according to claim 4, characterized in that, The preparation method includes constructing an expression vector that fuses and expresses a synovial inflammatory macrophage affinity peptide into an exosome membrane protein, and then transfecting the expression vector into the cells in step 1) to produce the initial exosomes.

8. The use of exosomes as described in any one of claims 1 to 3 or exosomes prepared by the method for preparing exosomes as described in any one of claims 4 to 7 in the preparation of pharmaceutical products; Optionally, the pharmaceutical product is used to relieve or treat one or more of cartilage degeneration, synovitis, and osteoarthritis.

9. A pharmaceutical product, characterized in that, The pharmaceutical product comprises exosomes as described in any one of claims 1 to 3 or exosomes prepared by the method for preparing exosomes as described in any one of claims 4 to 7.

10. A pharmaceutical product, characterized in that, The drug product can target inflammatory macrophages of the synovium, and the drug product includes an ingredient that can reduce miR-155-5p activity; Optionally, the component capable of reducing miR-155-5p activity is selected from one or more of the following: a) Knockout of miR-155-5p targeting vector in cells; b) miR-155-5p inhibitors; c) Viruses expressing the antisense complement sequence of miR-155-5p.

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