New subpopulation of human umbilical cord mesenchymal stem cells, exosomes, preparations and applications thereof

Abstract

The application discloses a new subpopulation of human umbilical cord mesenchymal stem cells, exosomes, preparations and applications thereof, and particularly relates to a new subpopulation of human umbilical cord mesenchymal stem cells or exosomes purified from supernatant generated by the new subpopulation of human umbilical cord mesenchymal stem cells, which can be used for preparing a preparation for treating pulmonary fibrosis by atomization. In particular, the application comprises: a new subpopulation of human umbilical cord mesenchymal stem cells or a stem cell preparation; supernatant generated by the new subpopulation of human umbilical cord mesenchymal stem cells or exosomes purified from the supernatant; a preparation of exosomes purified from the supernatant generated by the new subpopulation of human umbilical cord mesenchymal stem cells; and a preparation of exosomes obtained by culturing the new subpopulation of mesenchymal stem cells for treating pulmonary fibrosis by atomization. The application has great value for treating and prognosis of pulmonary fibrosis and related diseases.

Description

Technical Field

[0001] This invention relates to a new subpopulation of human umbilical cord mesenchymal stem cells, their exosomes, and their formulations, and their application in nebulized products for the treatment of pulmonary fibrosis. Specifically, it relates to a new subpopulation of human umbilical cord Wharton interstitial mesenchymal stem cells, their exosomes, and their formulations, and their application in products for the treatment of pulmonary fibrosis. Background Technology

[0002] Stem cell-derived exosomes are extracellular vesicles secreted by stem cells under stimuli such as resting or hypoxic stress, radiation, and oxidative damage. [1] It can selectively transport proteins, mRNA, and microRNA to act as signaling molecules between stem cells and differentiated cells. [2] From 2007 to 2017, research in the field of mesenchymal stem cell exosomes primarily focused on basic research into the mechanisms of exosomes. From 2017 to 2018, research mainly focused on exosome expression, differentiation, growth, and regeneration, and initial exploration and trials of exosomes in disease treatment began. From 2018 to the present, with increased understanding and research into mesenchymal stem cell exosomes, exploration into their use in treating more diseases has begun, with gradual trials in the treatment of heart disease, kidney disease, orthopedic diseases, inflammation, and related conditions. Existing research has demonstrated that mesenchymal stem cell exosomes can reduce the area of ​​myocardial infarction. [3] Reduce limb ischemia [4] Promotes wound healing [5,6] Improve graft-versus-host disease (GVHD) [6,7] Reduce kidney damage [8] Promotes liver regeneration [9] Reduce retinal damage

[10] and recent improvements in cartilage

[11] and bone regeneration

[12] It plays an important role in aspects such as...

[0003] Currently, the field of mesenchymal stem cell exosomes still faces several bottlenecks. For example, standardized methods for exosome isolation and purification suitable for large-scale drug formulation applications have not yet been established, affecting the reproducibility of research results. The fate, biodistribution, pharmacokinetics, and organ-specificity of exosomes after entering recipient cells, as well as their therapeutic mechanisms, are not fully elucidated. Compared to monoclonal antibody products, their large-scale production, component heterogeneity, and the need for cold chain storage are all issues that need to be addressed for industrialization and standardization in clinical applications.

[0004] CN112826833A does not mention exosomes targeting GP130-positive stem cells for pulmonary fibrosis.

[0005] CN114269358A discloses the treatment of pulmonary fibrosis with exosomes derived from mesenchymal stem cells (MSCs), but makes no disclosure regarding GP130-positive stem cells. Summary of the Invention

[0006] The purpose of this invention is to provide a new subpopulation of human umbilical cord mesenchymal stem cells, their exosomes, and their formulations for use in nebulized products for the treatment of pulmonary fibrosis.

[0007] In one aspect of the invention, an exosome of a mesenchymal stem cell is provided, wherein the GP130 positivity rate of the mesenchymal stem cell is above 95%.

[0008] In one embodiment of the present invention, the exosomes of the mesenchymal stem cells are characterized in that: the mesenchymal stem cells are umbilical cord mesenchymal stem cells prepared from umbilical cord Wharton's zone tissue blocks.

[0009] The method for preparing umbilical cord mesenchymal stem cells includes the following steps: using umbilical cord Wharton's zone tissue blocks as raw materials, umbilical cord mesenchymal stem cells are prepared, and a low serum culture medium is used in the preparation process.

[0010] In one embodiment of the present invention, the exosomes of the mesenchymal stem cells are characterized in that the umbilical cord mesenchymal stem cells are primary umbilical cord mesenchymal stem cells or passaged umbilical cord mesenchymal stem cells.

[0011] In one embodiment of the present invention, the method for preparing the umbilical cord mesenchymal stem cells includes the following steps:

[0012] (1) Culture isolated umbilical cord Wharton's zone tissue blocks in low serum medium for 7-10 days;

[0013] (2) After completing step (1), discard the tissue block and culture the cells in a low serum medium until 80% confluence.

[0014] (3) After completing step (2), collect the cells, digest them with pancreatic enzymes, and collect the cells, which are P0 generation umbilical cord mesenchymal cells;

[0015] (4) After completing step (3), the P0 generation umbilical cord mesenchymal cells were passaged 1:2 and cultured in low serum medium until 80% confluence.

[0016] (5) After completing step (4), collect the cells, digest them with pancreatic enzymes, and collect the cells, which are P1 generation umbilical cord mesenchymal cells.

[0017] (6) After completing step (5), the P1 generation umbilical cord mesenchymal cells were passaged 1:2 and cultured in low serum medium until 80% confluence.

[0018] (7) After completing step (6), collect the cells, digest them with pancreatic enzymes, and collect the cells, which are P2 generation umbilical cord mesenchymal cells;

[0019] (8) After completing step (7), the P2 generation umbilical cord mesenchymal cells were passaged 1:2 and cultured in low serum medium until 80% confluence.

[0020] (9) After completing step (8), collect the cells, digest them with pancreatic enzymes, and collect the cells to obtain the cell preparation.

[0021] In one embodiment of the present invention, the exosomes of the mesenchymal stem cells are characterized in that the positive rates of CD29, CD44, CD90, and CD105 of the mesenchymal stem cells are all above 90%, and the positive rates of CD31 and CD34 are below 5%.

[0022] In one embodiment of the present invention, the method for preparing the exosomes of the mesenchymal stem cells includes the following steps:

[0023] (1) Collect the culture supernatant of the umbilical cord mesenchymal stem cells within 10 generations;

[0024] (2) Centrifuge 800g for 5 minutes at room temperature for the first time;

[0025] (3) Collect the supernatant and centrifuge it a second time at 2000g for 10 minutes at room temperature;

[0026] (4) Then filter with a 0.22 μm filter membrane;

[0027] (5) Load into an ultracentrifuge tube and centrifuge at 11000g for 1 hour and 30 minutes at 4℃;

[0028] (6) Discard the supernatant, resuspend in 1 ml PBS and measure the concentration.

[0029] In one aspect of the invention, there is a medicament for treating pulmonary fibrosis, comprising exosomes of mesenchymal stem cells as defined above.

[0030] In one embodiment of the present invention, the drug is an atomized dosage form.

[0031] In one aspect of the invention, there is a connection to the use of the exosomes of the aforementioned mesenchymal stem cells in the preparation of a medicament for treating pulmonary fibrosis.

[0032] In one embodiment of the invention, the drug is administered to the lungs via nebulization.

[0033] The exosomes of this invention directly reach the alveoli, influencing the immune microenvironment of the patient's lungs, regulating the patient's immune balance, and dissolving the extracellular matrix, thereby achieving the therapeutic effect of pulmonary fibrosis. The exosome nebulization formulation provided by this invention is suitable for cell therapy of pulmonary fibrosis and related diseases. These related diseases include complications of pulmonary fibrosis and diseases with similar pathogenesis, such as lung injury and pneumonia. This invention has significant application value for the treatment of pulmonary fibrosis and related diseases. Attached Figure Description

[0034] Figure 1 The results are for markers of mesenchymal stem cells (CD29, CD44, CD90, CD105).

[0035] Figure 2 This represents the typical gene expression levels of the GP130 subset of mesenchymal stem cells.

[0036] Figure 3 The results are from the observation of phenotypes in mice with pulmonary fibrosis.

[0037] Figure 4 The results are from pathological examination of mouse lungs.

[0038] Figure 5 The results are from a MicroCT scan of mouse lungs. Detailed Implementation

[0039] This invention first protects this new subpopulation of mesenchymal stem cells, as well as the supernatant and the purified exosomes.

[0040] In the above applications, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells. These umbilical cord mesenchymal stem cells are prepared from Wharton's zone tissue blocks of the umbilical cord. Serum-free culture medium is used in the preparation of umbilical cord mesenchymal stem cells from Wharton's zone tissue blocks. The umbilical cord is an ex vivo umbilical cord. The umbilical cord mesenchymal stem cells can be primary umbilical cord mesenchymal stem cells or passaged umbilical cord mesenchymal stem cells. The new subpopulation of mesenchymal stem cells is obtained by flow cytometry sorting using the cell surface marker GP130. The passaged umbilical cord mesenchymal stem cells can be GP130-positive umbilical cord mesenchymal stem cells passaged up to 20 times, specifically GP130-positive umbilical cord mesenchymal stem cells passaged up to 10 times, and more specifically GP130-positive umbilical cord mesenchymal stem cells passaged up to 5 times. The supernatant is a cell culture medium used to passage mesenchymal stem cells to a confluence of 90-95%. The novel subpopulation of mesenchymal stem cell exosomes is the supernatant obtained after culturing GP130-positive mesenchymal stem cells, which is then purified into exosomes. The exosome preparation is prepared by diluting exosomes to a specific particle number using physiological saline. The exosome preparation for treating pulmonary fibrosis refers to a concentration where the number of exosome particles is 2 x 10^6 particles per milliliter of physiological saline. 9 The solution.

[0041] This invention also protects the application of nebulized treatment of new subpopulation cells or supernatant and exosome preparations for pulmonary fibrosis.

[0042] In the above applications, the preparation method of the cell preparation includes the following steps: using umbilical cord Wharton's zone tissue block as raw material, umbilical cord mesenchymal stem cells are prepared, and serum-free culture medium is used in the preparation process.

[0043] The umbilical cord mesenchymal stem cells can be primary umbilical cord mesenchymal stem cells or passaged umbilical cord mesenchymal stem cells.

[0044] The method for preparing primary umbilical cord mesenchymal stem cells includes the following steps in sequence:

[0045] (1) Culture the umbilical cord Wharton's zone tissue block in a low serum medium until cells crawl out;

[0046] (2) Cells were cultured using a low-serum culture medium;

[0047] (3) Collect cells, digest with pancreatic enzymes, and collect the cells, which are the primary umbilical cord mesenchymal cells.

[0048] The passaged umbilical cord mesenchymal stem cells can be umbilical cord mesenchymal stem cells passaged up to 20 times, specifically umbilical cord mesenchymal stem cells passaged up to 10 times, and more specifically umbilical cord mesenchymal stem cells passaged up to 5 times. The passage method can specifically be as follows: passage umbilical cord mesenchymal cells at a ratio of 1:3, culture the cells in serum-free medium, collect the cells, digest them with enzymes, and collect the cells again.

[0049] Furthermore, the method for preparing the mesenchymal stem cells includes the following steps:

[0050] (1) Culture isolated umbilical cord Wharton's zone tissue blocks in serum-free medium for 7-10 days;

[0051] (2) After completing step (1), discard the tissue block and culture the cells in serum-free medium until 80% confluence.

[0052] (3) After completing step (2), collect the cells, digest them with pancreatic enzymes, and collect the cells, which are P0 generation umbilical cord mesenchymal cells;

[0053] (4) After completing step (3), the P0 generation umbilical cord mesenchymal cells were passaged 1:2 and cultured in serum-free medium until 80% confluence.

[0054] (5) After completing step (4), collect the cells, digest them with pancreatic enzymes, and collect the cells, which are P1 generation umbilical cord mesenchymal cells.

[0055] (6) After completing step (5), the P1 generation umbilical cord mesenchymal cells were passaged at a ratio of 1:3 and cultured in serum-free medium until 80% confluence.

[0056] (7) After completing step (6), collect the cells, digest them with pancreatic enzymes, and collect the cells, which are P2 generation umbilical cord mesenchymal cells;

[0057] (8) After completing step (7), the P2 generation umbilical cord mesenchymal cells were passaged at a ratio of 1:3 and cultured in serum-free medium until 80% confluence.

[0058] (9) After completing step (8), collect the cells, digest them with pancreatic enzymes, and collect the cells to obtain the cell preparation.

[0059] The cell preparations described above are GP130-positive, with a GP130 positivity rate of 95% or higher, preferably 99% or higher. The cell preparations described above are also cell preparations with positivity rates of CD29, CD44, CD90, and CD105 all above 90%, preferably 95% or higher, more preferably 99% or higher. The positivity rates of CD31 and CD34 in the cell preparations described above are below 5%, preferably below 3%. CD29, CD44, CD90, and CD105 are all commonly used surface markers for identifying mesenchymal stem cells; high positivity rates of CD29, CD44, CD90, and CD105 indicate that the cell preparation is a mesenchymal stem cell and has high purity. CD31 is a marker for endothelial progenitor cells, CD45 is a marker for leukocytes, and CD34 is a marker for hematopoietic stem cells; low positivity rates of these three markers (CD31, CD45, and CD34) indicate high purity of the cell preparation. GP130 is a surface marker used in this invention to identify the quality of mesenchymal stem cells and the effectiveness of treatment. Figure 2 This represents the typical gene expression levels of the GP130 subset of mesenchymal stem cells.

[0060] In the above applications, the preparation method of the supernatant includes the following steps:

[0061] (1) The umbilical cord mesenchymal stem cells within 10 generations were cultured in serum-free medium until they reached 90-95% confluence.

[0062] (2) Collect the culture supernatant of the cells from step (1), which is the culture supernatant.

[0063] In the above applications, the exosome preparation method includes the following steps:

[0064] (7) Collect the culture supernatant of umbilical cord mesenchymal stem cells within 10 generations;

[0065] (8) Centrifuge 800g for 5 minutes at room temperature for the first time;

[0066] (9) Collect the supernatant and centrifuge for a second time at 2000g for 10 minutes at room temperature;

[0067] (10) Then filter with a 0.22 μm filter membrane;

[0068] (11) Load into an ultracentrifuge tube and centrifuge at 11000g for 1 hour and 30 minutes at 4℃;

[0069] (12) Discard the supernatant, resuspend in 1 ml PBS and measure the concentration.

[0070] This invention also protects an nebulized drug for treating exosomes in pulmonary fibrosis.

[0071] The active ingredient of the drug for nebulized treatment of pulmonary fibrosis protected by this invention is the supernatant or exosome preparation obtained from GP130-positive mesenchymal stem cell culture.

[0072] In the aforementioned drug, the mesenchymal stem cells are umbilical cord mesenchymal stem cells. These umbilical cord mesenchymal stem cells are prepared from Wharton's zone tissue blocks of the umbilical cord. A low-serum culture medium is used during the preparation of umbilical cord mesenchymal stem cells from Wharton's zone tissue blocks. The umbilical cord is an isolated umbilical cord. The umbilical cord mesenchymal stem cells can be primary umbilical cord mesenchymal stem cells or passaged umbilical cord mesenchymal stem cells. Passaged umbilical cord mesenchymal stem cells can be umbilical cord mesenchymal stem cells passaged up to 20 times, specifically umbilical cord mesenchymal stem cells passaged up to 10 times, and more specifically umbilical cord mesenchymal stem cells passaged up to 5 times.

[0073] The serum-free culture medium mentioned above is a cell culture medium that does not contain serum.

[0074] The composition of any of the serum-free culture media mentioned above is as follows: human epidermal growth factor 10 ng / mL, human basic fibroblast growth factor 10 ng / mL, recombinant human insulin-like growth factor 5 μg / mL, platelet-derived factor 10 ng / mL, heparin 5 μg / mL, hydrocortisone 1 μg / mL, ascorbic acid 10 μg / mL, non-essential amino acid solution 1% (volume percentage), L-glutamine 2 mmol / L, and the balance being DMEM high glucose medium.

[0075] The non-essential amino acid solution contains 10mM Glycine, 10mM L-Alanine, 10mM L-Asparagine, 10mM L-Aspartic acid, 10mM L-Glutamic Acid, 10mM L-Proline, and 10mM L-Serine.

[0076] This invention employs a serum-free culture medium for cell culture. In mesenchymal stem cell culture, 10% (volume percentage) fetal bovine serum is typically used, with some serum concentrations reaching as high as 20% (volume percentage). Serum contains various plasma proteins, polypeptides, carbohydrates, growth factors, hormones, etc. Serum composition is complex, and variations exist between batches, making consistency uncertain. Furthermore, while serum contains many components beneficial to cell growth, it inevitably contains some harmful components such as complement, antibodies, and endotoxins. Therefore, cells cultured from high-concentration serum are unsuitable for clinical applications, increasing the risk of clinical allergies. The inventors of this invention have discovered that a serum-free culture medium has no adverse effects on cell growth, proliferation, morphology, or function.

[0077] Commonly used mesenchymal stem cells (MSCs) for immunotherapy include bone marrow MSCs, umbilical cord MSCs, adipose-derived MSCs, and umbilical cord blood MSCs. Compared with commonly used bone marrow MSCs, umbilical cord Wharton's zone MSCs have many advantages, including abundant source, no impact on the donor, ease of collection and transportation, low potential for carcinogenicity, low probability of viral contamination, and no social, ethical, or legal controversies. More importantly, MSCs isolated from the Wharton's zone of the umbilical cord have a high content, and the resulting exosomes have low immunogenicity, high stability and homogeneity, and are easy to store.

[0078] Exosomes directly reach the alveoli, influencing the immune microenvironment of the patient's lungs, regulating the patient's immune balance, and dissolving the extracellular matrix, thereby achieving the goal of treating pulmonary fibrosis. The exosome nebulization formulation provided by this invention is suitable for cell therapy of pulmonary fibrosis and related diseases. The related diseases of pulmonary fibrosis include complications of pulmonary fibrosis and diseases with similar pathogenesis, such as lung injury and pneumonia.

[0079] This invention has significant application value in the treatment of pulmonary fibrosis and related diseases.

[0080] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0081] The formulations or sources of the reagents used in the following examples:

[0082] Serum-free culture medium: human epidermal growth factor 10 ng / mL, human basic fibroblast growth factor 10 ng / mL, recombinant human insulin-like growth factor 5 μg / mL, platelet-derived factor 10 ng / mL, heparin 5 μg / mL, hydrocortisone 1 μg / mL, ascorbic acid 10 μg / mL, non-essential amino acid solution 1% (volume percentage), L-glutamine 2 mmol / L, with the remainder being DMEM high glucose medium.

[0083] Human epidermal growth factor (hEGF): GenScript, product name: EGF Recombinant Human Protein, catalog number: Z00333.

[0084] Human basic fibroblast growth factor (b-FGF): GenScript, catalog number Z03116.

[0085] Recombinant human insulin-like growth factor (IGF): GenScript, catalog number Z03017.

[0086] Platelet-derived factor (PDGF): GenScript, catalog number Z02529.

[0087] Freund's complete adjuvant: Chondrex, catalog number 7001.

[0088] Freund's incomplete adjuvant: Chondrex, catalog number 7002.

[0089] Heparin: Maichen Company.

[0090] Hydrocortisone: Maichen Company.

[0091] Ascorbic acid: Maichen Company.

[0092] The non-essential amino acid solution is Gibco. TM MEM Non-Essential Amino Acids Solution, (100X), catalog number 11140050. Website: https: / / www.thermofisher.com / cn / zh / home / technical-resources / media-formulation.165.html. This product contains seven amino acids, with the following composition: Glycine 10.0mM, L-Alanine 10.0mM, L-Asparagine 10.0mM, L-Aspartic acid 10.0mM, L-Glutamic Acid 10.0mM, L-Proline 10.0mM, and L-Serine 10.0mM.

[0093] C57 mice: Beijing Vital River Co., Ltd.

[0094] Example 1: Preparation of umbilical cord Wharton's interval mesenchymal stem cell preparation

[0095] I. Obtaining the umbilical cord from the fetus

[0096] The umbilical cord of a full-term newborn without congenital diseases was collected; the mother had no infectious diseases such as hepatitis, syphilis, or AIDS, and both the mother and her family gave informed consent to the use of the umbilical cord for experimental research.

[0097] II. Obtaining mesenchymal cells from the Wharton area of ​​the umbilical cord

[0098] 1. Pretreatment of the umbilical cord

[0099] Inside a sterile laboratory table, the umbilical cord is repeatedly rinsed with physiological saline to remove residual blood. Using sterile surgical instruments, the umbilical cord is cut into 2-3 cm segments. The cord is then longitudinally incised, and the umbilical artery, umbilical vein, and amnion are removed. The Wharton's area is harvested and cut into 0.5-1 mm segments. 3 Small pieces on the left and right.

[0100] 2. Obtaining primary umbilical cord mesenchymal cells

[0101] Primary umbilical cord mesenchymal cells were obtained using the tissue block culture method. The specific steps are as follows:

[0102] (1) Spread the Wharton zone tissue block obtained in step 1 evenly in a sterile culture dish with a diameter of 10cm, covering 60-70% of the bottom area of ​​the dish, and invert it and place it in a 37℃ incubator for 15min; turn the culture dish over, gently add 10mL of low serum culture medium, and incubate in a 37℃, 5% CO2 incubator for 7-10 days. At this time, cells will crawl out evenly from under the tissue block.

[0103] (2) After completing step (1), take the culture dish, wash it twice with PBS buffer, discard the tissue block (at this time the cells have adhered and grown), add 10 mL of fresh low serum culture medium to each dish (change the medium once every 3-4 days), and culture until the cells cover about 80% of the bottom of the culture dish.

[0104] (3) After completing step (2), collect the cells, digest them with 0.25% trypsin for 1-2 minutes, centrifuge at 1000 rpm for 3 minutes, and collect the cells. These are the primary umbilical cord mesenchymal cells, also known as P0 generation umbilical cord mesenchymal cells.

[0105] 3. Obtaining P3 generation umbilical cord mesenchymal cells

[0106] (1) Divide the P0 generation umbilical cord mesenchymal cells obtained in step 2 into two sterile culture dishes with a diameter of 10 cm (passage at a ratio of 1:2). Add 10 mL of fresh low serum culture medium to each culture dish (change the medium every 3-4 days) and culture until the cells cover about 80% of the bottom of the culture dish.

[0107] (2) After completing step (1), collect the cells, digest them with 0.25% trypsin for 1-2 minutes, centrifuge at 1000 rpm for 3 minutes, and collect the cells, which are P1 generation umbilical cord mesenchymal cells.

[0108] (3) After completing step (2), divide the P1 generation umbilical cord mesenchymal cells into sterile culture dishes with a diameter of 10 cm (passage at 1:2). Add 10 mL of fresh low serum culture medium to each culture dish (change the medium every 3-4 days) and culture until the cells cover about 80% of the bottom of the culture dish.

[0109] (4) After completing step (3), collect the cells, digest the cells with 0.25% trypsin for 1-2 minutes, centrifuge at 1000 rpm for 3 minutes, and collect the cells, which are P2 generation umbilical cord mesenchymal cells.

[0110] (5) After completing step (4), the P2 generation umbilical cord mesenchymal cells are evenly divided into sterile culture dishes with a diameter of 10 cm (passed at a ratio of 1:2). 10 mL of fresh low serum culture medium is added to each culture dish (the medium is changed every 3-4 days). The cells are cultured until they cover about 80% of the bottom of the culture dish.

[0111] (6) After completing step (5), collect the cells, digest the cells with 0.25% trypsin for 1-2 minutes, centrifuge at 1000 rpm for 3 minutes, and collect the cells, which are P3 generation umbilical cord mesenchymal cells (also known as P3 cell preparation).

[0112] Example 2: Preparation of P3 cell exosomes by atomization

[0113] The test sample was the P3 cell preparation prepared in Example 1.

[0114] The expression of CD29, CD44, CD90, CD105, CD126, CD31, CD45, CD34 and HLA-DR in the test sample was detected by flow cytometry. The specific steps are as follows: After incubating the cells (test sample) with the corresponding antibodies, excess antibodies were washed away, and the cells were resuspended in PBS buffer. The positive rate of each indicator was detected using a BD LSR Fortessa instrument.

[0115] The results of the detection of mesenchymal stem cell markers (CD29, CD44, CD90, CD105, and CD126) are shown in [the table below]. Figure 1 The results showed that the test samples highly expressed biomarkers for mesenchymal stem cells (with positive rates all above 97%).

[0116] GP130-positive cells from the test sample were sorted using flow cytometry. The specific steps are as follows: After co-incubating the cells (test sample) with the corresponding antibody, excess antibody was washed away, the cells were resuspended in PBS buffer, and GP130-positive cells were sorted using a BD LSR Tortessa instrument.

[0117] Serum-free medium was used to culture GP130-positive umbilical cord mesenchymal stem cells obtained from sorting until 90-95% confluence was achieved. The culture supernatant was collected. The cells were centrifuged at 800g for 5 minutes at room temperature. The supernatant was then collected and centrifuged a second time at 2000g for 10 minutes at room temperature. The mixture was then filtered through a 0.22μm filter, placed in an ultracentrifuge tube, and centrifuged at 11000g for 1 hour and 30 minutes at 4°C. The supernatant was discarded, and the cells were resuspended in 1ml of PBS and their concentration was measured.

[0118] Example 3: The therapeutic effect of GP130 subset cell exosomes on pulmonary fibrosis

[0119] I. Model Preparation and Drug Administration

[0120] Test mice: 6-8 weeks old, C57BL / 6 mice, acclimatized for one week (in a clean-grade animal housing with free access to food and water). A pneumonia model was induced in the mice using bleomycin. Based on whether and how much bleomycin was administered, the mice were divided into the following groups:

[0121] Normal control group (n=5): Each mouse was administered 0.05 ml of physiological saline via tracheal infusion. Starting from the second day of tracheal infusion, physiological saline was nebulized for 30 minutes daily for 14 consecutive days.

[0122] Exosome treatment group (n=8): Each mouse was administered 0.05 ml of bleomycin (2 mg / ml) via tracheal infusion. Starting from the second day after tracheal infusion, GP130 subset exosomes or umbilical cord mesenchymal stem cell exosomes were nebulized for 30 min daily for 14 consecutive days.

[0123] Model group (n=8): Each mouse was administered 0.05 ml of bleomycin (2 mg / ml) via tracheal infusion. Starting from the second day of tracheal infusion, the mice were nebulized with physiological saline for 30 minutes daily for 14 consecutive days.

[0124] Mice were euthanized on the day of intratracheal instillation, and samples were collected on the 18th day.

[0125] II. Phenotypic Observation

[0126] Figure 3 The results are from the observation of phenotypes in mice with pulmonary fibrosis.

[0127] On day 17 of the experiment, MicroCT images were taken of mice in each group to assess the pulmonary fibrosis.

[0128] See results Figure 5 (Ctrl represents the normal control group, saline represents the model group, GP130-EXO represents the GP130 subset exosome nebulization treatment group, and UCMSC-EXO represents the umbilical cord mesenchymal stem cell exosome nebulization treatment group.) Compared with the model group, the lung shadow area of ​​mice in the exosome nebulization treatment group was significantly reduced, indicating that exosome nebulization has a significant therapeutic effect on pulmonary fibrosis.

[0129] III. Pathological examination of mouse lungs

[0130] On day 18 of the experiment, lungs of mice were taken and paraffin sections were prepared (section thickness was 4 μm), and then H&E and Masson staining were performed.

[0131] See results Figure 4 (Ctrl represents the normal control group, saline represents the model group, GP130-EXO represents the GP130 subset cell exosome nebulization treatment group, and UCMSC-EXO represents the umbilical cord mesenchymal stem cell exosome nebulization treatment group.) The lungs of normal control group mice are typically pink or light red. Under H&E staining, the alveolar walls of normal mice are thin, the epithelial cells are neatly arranged, and there are some light-colored fine linear structures in the interstitium, which are tissue septa such as blood vessels and bronchi. Masson staining can more clearly show components such as elastic fibers and collagen fibers in the lung tissue, appearing as a light blue color. The lungs of model group mice are dark red and have a harder texture. Under H&E staining, fibrotic areas of mouse lung tissue sections usually show a reduction in alveoli, and the stained areas appear pink or rose-red, forming a clear contrast with the surrounding normal lung tissue. In addition, pathological changes such as inflammatory cell infiltration and angiogenesis may also be observed in the fibrotic areas. Masson staining more clearly shows the degree and extent of fibrosis, with fibrotic areas appearing as dark blue or purple. In cases of severe pulmonary fibrosis, there may also be a large amount of collagen deposition and alveolar structure destruction.

[0132] The above results indicate that GP130-positive mesenchymal stem cell exosomes have a better therapeutic effect on pulmonary fibrosis than umbilical cord mesenchymal stem cell exosomes.

[0133] List of cited references

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Claims

1. An exosome of mesenchymal stem cells, wherein the positive rate of GP130 in the mesenchymal stem cells is above 95%, and the mesenchymal stem cells are umbilical cord mesenchymal stem cells prepared from umbilical cord Wharton's zone tissue blocks.

2. The exosomes of mesenchymal stem cells as described in claim 1, characterized in that: The method for preparing umbilical cord mesenchymal stem cells includes the following steps: using umbilical cord Wharton's zone tissue blocks as raw materials, umbilical cord mesenchymal stem cells are prepared, and a low serum culture medium is used in the preparation process.

3. The exosomes of mesenchymal stem cells as described in claim 2, characterized in that: The umbilical cord mesenchymal stem cells are primary umbilical cord mesenchymal stem cells or passaged umbilical cord mesenchymal stem cells.

4. The exosomes of mesenchymal stem cells as described in claim 2, characterized in that: The method for preparing the umbilical cord mesenchymal stem cells includes the following steps: (1) Culture isolated umbilical cord Wharton's zone tissue blocks in low serum medium for 7-10 days; (2) After completing step (1), discard the tissue block and culture the cells in a low serum medium until 80% confluence. (3) After completing step (2), collect the cells, digest them with pancreatic enzymes, and collect the cells, which are P0 generation umbilical cord mesenchymal cells; (4) After completing step (3), passage the P0 generation umbilical cord mesenchymal cells at a ratio of 1:2 and culture them in a low serum medium until they reach 80% confluence. (5) After completing step (4), collect the cells, digest them with pancreatic enzymes, and collect the cells, which are P1 generation umbilical cord mesenchymal cells. (6) After completing step (5), the P1 generation umbilical cord mesenchymal cells were passaged 1:2 and cultured in low serum medium until 80% confluence. (7) After completing step (6), collect the cells, digest them with pancreatic enzymes, and collect the cells, which are P2 generation umbilical cord mesenchymal cells; (8) After completing step (7), the P2 generation umbilical cord mesenchymal cells were passaged 1:2 and cultured in low serum medium until 80% confluence. (9) After completing step (8), collect the cells, digest them with pancreatic enzymes, and collect the cells, which are the umbilical cord mesenchymal stem cells.

5. The exosomes of mesenchymal stem cells as described in claim 1, wherein the positive rates of CD29, CD44, CD90 and CD105 of the mesenchymal stem cells are all above 90%, and the positive rates of CD31 and CD34 are below 5%.

6. The exosomes of mesenchymal stem cells as described in claim 1, wherein the method for preparing the exosomes comprises the following steps: (1) Collect the culture supernatant of the mesenchymal stem cells within 10 generations; (2) Centrifuge 800g for 5 minutes at room temperature for the first time; (3) Collect the supernatant and centrifuge 2000g for 10 minutes at room temperature; (4) Then filter with a 0.22 μm filter membrane; (5) Load into an ultracentrifuge tube and centrifuge at 11000g for 1 hour and 30 minutes at 4℃; (6) Discard the supernatant, resuspend in 1 ml PBS and measure the concentration.

7. A medicament for treating pulmonary fibrosis, comprising exosomes of mesenchymal stem cells according to any one of claims 1-6.

8. The drug as described in claim 7, wherein the drug is an atomized dosage form.

9. Use of the exosomes of mesenchymal stem cells according to any one of claims 1-6 in the preparation of a medicament for treating pulmonary fibrosis.

10. The application as described in claim 9, wherein the drug is administered to the lungs via nebulization.

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