Preparation method and application of high-expression IL-10 human umbilical cord mesenchymal stem cell exosome for treating acute lung injury

Abstract

The invention discloses a preparation method and application of a high-expression IL-10 human umbilical cord mesenchymal stem cell exosome for treating acute lung injury. The method comprises the following steps: constructing a recombinant lentivirus for highly expressing IL-10, transfecting human umbilical cord mesenchymal stem cells with the recombinant lentivirus, and inducing the human umbilical cord mesenchymal stem cells to secrete exosomes for highly expressing IL-10 by using recombinant human IFN-gamma, PDGF-BB and EPO. Through aerosol inhalation of the exosome prepared in the invention, the content of TNF-alpha, IL-6, IL-1beta and other proinflammatory factors in serum of rats with acute lung injury can be effectively reduced, and the content of an anti-inflammatory factor IL-10 in the serum can be increased. The human umbilical cord mesenchymal stem cell exosome with high expression of IL-10 prepared by the invention can be used for preparing medicines for treating or preventing acute lung injury, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of exosome preparation, and in particular to a preparation method and application of human umbilical cord mesenchymal stem cell exosomes highly expressing IL-10 for treating acute lung injury. Background Art

[0002] Human umbilical cord mesenchymal stem cells are a type of multipotent stem cells derived from the early developmental mesoderm. They are one of the most studied and important adult stem cells. They can differentiate into osteoblasts, chondrocytes, and adipocytes under specific conditions, and still have multidirectional differentiation potential after continuous subculture and cryopreservation. Mesenchymal stem cells derived from the umbilical cord have the characteristics of easy collection, no ethical controversy, low immunogenicity, fast self-renewal, stable doubling rate, and strong proliferation ability. Therefore, umbilical cord mesenchymal stem cells are suitable for clinical research and application, and are the first choice for cell therapy and regenerative medicine. Human umbilical cord mesenchymal stem cells also have immunoregulatory functions and can exert therapeutic effects by secreting a variety of cytokines.

[0003] Human umbilical cord mesenchymal stem cell exosomes (Exos) are a type of tiny vesicles with a diameter of 50 to 150 nm. Exosomes are powerful and can transfer biological molecules such as lipids, carbohydrates, proteins, mRNA, miRNA and DNA from one cell to another. They are important media for intercellular genetic information exchange and communication, and are also excellent delivery vehicles for drugs and natural compounds.

[0004] IL-10 is the main regulator of inflammatory response. It has multiple effects on immune regulation and inflammation. It can not only downregulate Th1 cytokines, class II MHC antigens and co-stimulatory molecules of macrophages, but also enhance B cell survival, proliferation and antibody secretion. IL-10 also inhibits the migration of leukocytes to the inflammatory site to a certain extent by inhibiting the synthesis of multiple chemokines. At the same time, in the process of tissue repair, it inhibits the high expression of pro-inflammatory cytokines such as IL-6, TNF-α and IL-1β, thus playing a role in limiting and terminating the inflammatory response. Human umbilical cord mesenchymal stem cells can secrete IL-10 through immunoregulatory function, but the content of IL-10 protein obtained in human umbilical cord mesenchymal stem cell exosomes in the conventional 2D planar culture system is scarce, and the effect on inhibiting inflammatory factors is not obvious enough.

[0005] Acute lung injury (ALI) refers to acute and progressive respiratory failure caused directly or indirectly by various factors other than cardiogenic lung injury, which eventually develops into acute respiratory distress syndrome (ARDS). It is currently believed that ALI is mainly manifested by the aggregation of multiple types of inflammatory cells (such as macrophages, neutrophils, and vascular endothelial cells). The released inflammatory mediators directly lead to lung inflammation, and the imbalance of inflammatory response aggravates the damage of epithelial cells or endothelial cells. Alveolar macrophages release a large number of TNF-α, IL-1β, and IL-6 cytokines to induce inflammatory response. On the one hand, these proinflammatory factors can cause endothelial cells to produce NO and endothelin. On the other hand, they can also stimulate surrounding cells and generate alveolar cavity chemokines such as mononuclear lymphocytes and neutrophils, promote the production of inflammatory mediators, and then trigger a junction reaction, resulting in a large number of capillary membranes in alveolar cells being damaged. Therefore, it is of great significance to develop an exosome that can highly express anti-inflammatory cytokines, inhibit the levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6, thereby regulating the cellular microenvironment and alleviating the inflammatory response. Summary of the invention

[0006] In order to overcome the above-mentioned defects and shortcomings in the prior art, the present invention provides a method for preparing and using human umbilical cord mesenchymal stem cell exosomes with high expression of IL-10 for treating acute lung injury.

[0007] The first object of the present invention is to provide a method for preparing human umbilical cord mesenchymal stem cell exosomes.

[0008] The second object of the present invention is to provide the application of the above preparation method in increasing the IL-10 content in human umbilical cord mesenchymal stem cell exosomes.

[0009] The third object of the present invention is to provide an application of the above-mentioned preparation method in increasing the yield of exosomes containing IL-10 from human umbilical cord mesenchymal stem cells.

[0010] The fourth object of the present invention is to provide human umbilical cord mesenchymal stem cell exosomes containing IL-10 prepared by the above preparation method.

[0011] The fifth object of the present invention is to provide a use of human umbilical cord mesenchymal stem cell exosomes containing IL-10 in the preparation of a drug for treating acute lung injury.

[0012] Therefore, the present invention claims the following: A method for preparing human umbilical cord mesenchymal stem cell exosomes comprises the following steps: culturing IL-10 stably transfected human umbilical cord mesenchymal stem cells with a complete culture medium containing PDGF-BB, EPO and IFN-γ, collecting the cell culture fluid, and isolating the exosomes from the cell culture fluid.

[0013] Preferably, the final concentration of PDGF-BB in complete culture medium is 5-100 ng / mL.

[0014] More preferably, the final concentration of PDGF-BB in complete culture medium is 50 ng / mL.

[0015] Preferably, the final concentration of IFN-γ in complete culture medium is 5 to 100 ng / mL.

[0016] More preferably, the final concentration of IFN-γ in complete culture medium is 50 ng / mL.

[0017] More preferably, the IFN-γ is recombinant human IFN-γ.

[0018] Preferably, the final concentration of EPO in the complete culture medium is 1-20 ng / mL.

[0019] More preferably, the final concentration of EPO in complete culture medium is 10 ng / mL.

[0020] Preferably, the IL-10 stably transfected human umbilical cord mesenchymal stem cells are obtained by transfecting human umbilical cord mesenchymal stem cells with a recombinant lentivirus that overexpresses IL-10.

[0021] More preferably, the human umbilical cord mesenchymal stem cells are of passages P3 to P5.

[0022] More preferably, the method for preparing the recombinant lentivirus overexpressing IL-10 is: constructing a lentiviral vector containing IL-10, and packaging the lentiviral vector containing IL-10 into an IL-10 recombinant lentivirus in cells with the assistance of packaging plasmid sPAX2 and envelope plasmid PMG2G.

[0023] More preferably, the method for constructing the lentiviral vector containing IL-10 is: inserting the IL-10 target gene fragment into the 3541-4077 sites of the lentiviral vector pLVX-EF1alpha-eGFP-2×Strep-IRES-Puro.

[0024] More preferably, the nucleotide sequence of the IL-10 target gene fragment is as shown in SEQ ID NO:1.

[0025] Preferably, the method for isolating exosomes is: subjecting the cell culture fluid to differential centrifugation, collecting the supernatant, and then subjecting the supernatant to ultracentrifugation to obtain exosomes.

[0026] Application of any of the above preparation methods to increasing the IL-10 content in exosomes of human umbilical cord mesenchymal stem cells.

[0027] Application of any of the above preparation methods in increasing the yield of exosomes containing IL-10 from human umbilical cord mesenchymal stem cells.

[0028] The human umbilical cord mesenchymal stem cell exosomes containing IL-10 prepared by any of the above preparation methods, wherein IL-10 is overexpressed in the human umbilical cord mesenchymal stem cell exosomes.

[0029] Use of human umbilical cord mesenchymal stem cell exosomes containing IL-10 in the preparation of a drug for treating acute lung injury, wherein the human umbilical cord mesenchymal stem cell exosomes are prepared by any of the above-mentioned preparation methods.

[0030] Preferably, the drug is an aerosolized drug.

[0031] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses a preparation method and application of human umbilical cord mesenchymal stem cell exosomes that highly express IL-10 for treating acute lung injury. The present invention constructs a recombinant lentivirus that highly expresses IL-10, transfects it into human umbilical cord mesenchymal stem cells, and uses recombinant human IFN-γ, PDGF-BB and EPO to induce human umbilical cord mesenchymal stem cells to secrete exosomes that highly express IL-10. The exosomes prepared by the present invention can effectively reduce the content of pro-inflammatory factors such as TNF-α, IL-6, IL-1β in the serum of rats with acute lung injury by atomization inhalation, and increase the content of anti-inflammatory factor IL-10 in the serum. The human umbilical cord mesenchymal stem cell exosomes that highly express IL-10 prepared by the present invention can be used to prepare drugs for treating or preventing acute lung injury, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Morphological diagram of human umbilical cord mesenchymal stem cells; a: primary cells of human umbilical cord mesenchymal stem cells crawling out of the tissue block (100×), b: P1 cells of human umbilical cord mesenchymal stem cells (100×), c: P2 cells of human umbilical cord mesenchymal stem cells (100×), d: P3 cells of human umbilical cord mesenchymal stem cells (100×).

[0033] Figure 2 Identification results of cell surface markers of human umbilical cord mesenchymal stem cells P3.

[0034] Figure 3Results of the three-lineage differentiation experiment of human umbilical cord mesenchymal stem cells P3; a: adipogenic differentiation (200×), b: osteogenic differentiation (200×), c: chondrogenic differentiation.

[0035] Figure 4 Results of the blood adsorption experiment of human umbilical cord mesenchymal stem cells P3 (100×).

[0036] Figure 5 This is the skeleton diagram of the lentiviral vector pLVX-EF1alpha-eGFP-2×Strep-IRES-Puro.

[0037] Figure 6 Transmission electron microscopy imaging results of human umbilical cord mesenchymal stem cell exosomes; a: Exos, b: IL10-Exos, c: TFS-IL10-Exos.

[0038] Figure 7 Particle size results of human umbilical cord mesenchymal stem cell exosomes; a: Exos, b: IL10-Exos, c: TFS-IL10-Exos.

[0039] Figure 8 is the TNF-α content in the serum of rats in each group after 7 days of administration.

[0040] Fig. 9 is the IL-6 content in the serum of rats in each group after 7 days of administration.

[0041] Fig.10 is the IL-1β content in the serum of rats in each group after 7 days of administration.

[0042] Fig.11 is the IL-10 content in the serum of rats in each group 7 days after administration.

[0043] Fig.12 The effect of different high-expression IL-10 exosome complete culture media in Example 4 on the IL-10 content in human umbilical cord mesenchymal stem cell exosomes. DETAILED DESCRIPTION

[0044] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0045] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0046] Example 1 Preparation and identification of human umbilical cord mesenchymal stem cells 1. Experimental Methods 1. Isolation of Human Umbilical Cord Tissue Under the sterile conditions of a Class 100 biosafety cabinet, a healthy and normal full-term caesarean section umbilical cord tissue sample was obtained. The umbilical cord Wharton's jelly was separated using medical sterile scissors and sterile tweezers. The umbilical cord was fully rinsed 3 to 5 times with 0.9% sodium chloride injection (w / v). The removed umbilical cord tissue was cut into 0.5 to 2.0 cm pieces in a beaker. 3 For large and small tissue blocks, inoculate them in a 150 mm culture dish in a chessboard pattern and place them in a 37°C, 5% CO2 incubator for 30 to 60 min to allow the tissue blocks to adhere firmly to the wall. The culture medium is DMEM complete medium, and then 15 mL / dish of DMEM complete medium is added to the inoculated 150 mm culture dish, and placed in a 37°C, 5% CO2 incubator for static culture, waiting for the human umbilical cord mesenchymal stem cell primary cells to crawl out of the tissue block ( Figure 1 a) in the figure.

[0047] 2. Cultivation, purification and expansion of human umbilical cord mesenchymal stem cells (1) After 7 days of static culture, change half of the medium. Depending on the cell growth, change half of the medium every 3 days. (2) When the cells reach a confluence of 80% to 90%, digest them with 0.25% pancreatic enzyme solution (w / v). During the digestion process, observe the cell digestion with an inverted microscope. When most of the cell bodies shrink and become round, terminate the digestion. The cells are subcultured and recorded as P1 ( Figure 1 b); (3) During the subculture process, the medium was completely replaced every 3 days. When the cells reached a confluence of 80% to 90%, step (2) was repeated and recorded as P2. Figure 1 c); (4) Repeat step (3) and record it as P3 generation ( Figure 1 (d) The cells were collected and frozen in liquid nitrogen at -196°C for recovery.

[0048] 3. Identification of human umbilical cord mesenchymal stem cells (1) Identification of surface markers Flow cytometry was used to detect P3 human umbilical cord mesenchymal stem cell markers CD73 (antibody brand: BioLegend, catalog number: 344004), CD90 (antibody brand: BioLegend, catalog number: 328108) and CD105 (antibody brand: BioLegend, catalog number: 800504), confirming that the obtained cells were human umbilical cord mesenchymal stem cells.

[0049] (2) Trilineage differentiation ① Cell recovery: DMEM complete medium was used to recover P3 human umbilical cord mesenchymal stem cells at a rate of 1×10 6The cells were inoculated at a density of 1.5 cells / mL and cultured at 37°C in 5% CO2.

[0050] ② Osteogenesis induction: The revived human umbilical cord mesenchymal stem cells were inoculated in a 6-well plate, with 5×10 5 When the cell confluence reaches 80% to 90%, replace the osteogenic induction medium (containing dexamethasone, sodium β-glycerophosphate, and L-ascorbic acid). Replace the medium every 3 days for 2 to 3 weeks. Use Alizarin red staining or alkaline phosphatase (ALP) staining to identify osteoblasts.

[0051] ③ Adipogenic induction: The revived human umbilical cord mesenchymal stem cells were inoculated in a 6-well plate, with 5×10 5 When the cell confluence reaches 80% to 90%, replace the adipogenic induction medium (containing dexamethasone, insulin, isoproterenol and thiazolidinedione). Replace the medium every 3 days for 2 to 3 weeks. Use Oil Red O staining to identify adipogenic cells.

[0052] ④ Chondrogenic induction: The revived human umbilical cord mesenchymal stem cells were inoculated in a 6-well plate, with 5×10 5 When the cell confluence reaches 80% to 90%, replace it with chondrogenic induction medium (containing transforming growth factor-β (TGF-β) and insulin transfer factor). Change the medium every 3 to 4 days for 2 to 3 weeks. Use Alecin blue staining to identify chondrogenic cells.

[0053] (3) Blood adsorption test P3 human umbilical cord mesenchymal stem cells were revived with DMEM complete medium and 5 × 10 5 Cells were cultured at 37°C and 5% CO2 for one week. The cell culture medium was discarded and the monolayer cells were gently rinsed three times with calcium-free and magnesium-free PBS. 5 mL of mixed suspension of guinea pig red blood cells and chicken red blood cells was added (mixed before adding) to evenly cover the monolayer cells. After adding the red blood cell suspension, the monolayer cells were incubated at 2-8°C for 30 min, and then incubated at 20-25°C for 30 min. Gently rinsed with PBS three times and examined under a microscope to observe and record the red blood cell adsorption of the monolayer cells.

[0054] 2. Experimental Results Flow cytometry results showed that CD73 of P3 human umbilical cord mesenchymal stem cells + 、CD90 + 、CD105 +The expression results were greater than 95%, and the expression results of negative markers were less than 2%, indicating that P3 generation human umbilical cord mesenchymal stem cells highly expressed CD105, CD73, and CD90, and negative markers were not expressed or expressed very little, proving that P3 generation human umbilical cord mesenchymal stem cells still have high stem cell characteristics.

[0055] The results of adipogenic induction showed that lipid droplets appeared in umbilical cord mesenchymal stem cells, and Oil Red O staining was positive ( Figure 3 a in (a); The results of osteogenic induction showed that umbilical cord mesenchymal stem cells were able to form calcified nodules and ALP activity increased ( Figure 3 b); The results of chondrogenic induction showed that umbilical cord mesenchymal stem cells can form cartilage-like tissue ( Figure 3 c); The results of tri-lineage differentiation showed that P3 human umbilical cord mesenchymal stem cells still had high differentiation properties.

[0056] The blood adsorption results showed that ( Figure 4 ), there was no red blood cell adsorption on the surface of P3 human umbilical cord mesenchymal stem cells, indicating that the prepared human umbilical cord mesenchymal stem cells were not infected by viruses.

[0057] Example 2 Preparation and identification of human umbilical cord mesenchymal stem cell exosomes that highly express IL-10 1. Experimental Methods 1. Construction of recombinant lentivirus with high expression of IL-10 (1) Obtain the IL-10 gene DNA sequence from NCBI: (SEQ ID NO: 1).

[0058] (2) Design homologous recombination primers based on the DNA sequence of the IL-10 gene. Use cDNA as a template, use primers and high-fidelity enzymes to perform PCR amplification reaction, perform agarose gel electrophoresis on the PCR products and recover the gel to obtain the target gene fragment.

[0059] (3) Insert the target gene fragment into the lentiviral vector pLVX-EF1alpha-eGFP-2×Strep-IRES-Puro (Brand: Addgene, Catalog No.: 141395, Figure 5 ) at sites 3541 to 4077, driving the target gene and Puromycin The resistance gene is expressed, thereby preparing a lentiviral vector that highly expresses IL-10. The lentiviral vector that highly expresses IL-10 is transformed into Escherichia coli competent cells, and the lentiviral vector plasmid that highly expresses IL-10 is extracted using a kit.

[0060] (4) Resuscitation of 293T cells: Use DMEM complete medium at a cell seeding density of 1×10 6cells / mL were inoculated into 150 mm cell culture dishes and cultured in a 37°C, 5% CO2 cell culture incubator. When the confluence of the cultured cells reached 70% to 80%, transfection was prepared. 2 h before transfection, serum-free culture medium was replaced.

[0061] (5) Lentivirus transfection: Add the lentiviral vector plasmid with high expression of IL-10, packaging plasmid sPAX2, and envelope plasmid PMG2G in a mass ratio of 4:3:1, shake gently, and let it stand for 5 minutes. Then slowly drip Lipofectamine2000 into the tube containing the plasmid, shake gently, and incubate at room temperature for 15 minutes. Then evenly add it to the 293T cell culture dish and place it in a 37℃, 5% CO2 cell culture incubator for culture. After 8 hours of transfection, replace the complete culture medium. After 48 hours, collect the supernatant, which is the recombinant lentiviral solution with high expression of IL-10, and store it at 4℃. Add complete culture medium, collect the supernatant again after 72 hours, and store it at 4℃.

[0062] (6) Concentration of high-expression IL-10 recombinant lentivirus: The collected high-expression IL-10 recombinant lentivirus solution was filtered using a 0.45 μm filter and centrifuged at 72,000 g / min for 2 h. After centrifugation, the solution was resuspended in DMEM basal medium and stored at -80°C for later use. The high-expression IL-10 recombinant lentivirus was obtained.

[0063] 2. Preparation of human umbilical cord mesenchymal stem cell exosomes (1) Preparation of human umbilical cord mesenchymal stem cell exosomes (Exos) in the control group ① Resuscitate P3 human umbilical cord mesenchymal stem cells with DMEM complete medium at a rate of 1×10 6 cells / mL and incubated at 37°C, 5% CO2 for 102 h; ② Collect the culture supernatant of human umbilical cord mesenchymal stem cells, centrifuge at 300 g for 10 min, retain the supernatant, discard the precipitate, and remove the cells in the culture supernatant; ③ Centrifuge the supernatant at 2000 g for 20 min, retain the supernatant, discard the precipitate, and remove cell debris; ④ Centrifuge the supernatant at 10,000 g for 70 min, retain the supernatant, discard the precipitate, and remove cell debris again; ⑤ Centrifuge the supernatant at 100,000 g for 70 min, discard the supernatant, and retain the precipitate; ⑥ After adding PBS to the precipitate to resuspend, centrifuge at 100,000 g for 70 min, discard the supernatant, and retain the precipitate. The precipitate is the purified human umbilical cord mesenchymal stem cell exosomes (Exos). Add PBS to resuspend and store at -80°C.

[0064] (2) Preparation of human umbilical cord mesenchymal stem cell exosomes (IL10-Exos) that highly express IL-10 ① Use DMEM complete medium to revive P3 human umbilical cord mesenchymal stem cells at a rate of 1×10 6 cells / mL and incubated at 37°C, 5% CO2 for 48 h. ② Add 1 mL of high-expression IL-10 recombinant lentivirus and 100 μg / mL Polybrene, infect at 37°C, 5% CO2 for 8 h, replace with fresh complete medium, and then incubate at 37°C, 5% CO2 for 72 h; ③ Collect the culture supernatant of human umbilical cord mesenchymal stem cells, centrifuge at 300 g for 10 min, retain the supernatant, discard the precipitate, and remove the cells in the culture supernatant; ④ Centrifuge the supernatant at 2000 g for 20 min, retain the supernatant, discard the precipitate, and remove cell debris; ⑤ Centrifuge the supernatant at 10,000 g for 70 min, retain the supernatant, discard the precipitate, and remove cell debris again; ⑥ Centrifuge the supernatant at 100,000 g for 70 min, discard the supernatant, and retain the precipitate; ⑦ After adding PBS to the precipitate for resuspending, centrifuge at 100,000 g for 70 min, discard the supernatant, and retain the precipitate. The precipitate is the purified human umbilical cord mesenchymal stem cell exosomes (IL10-Exos) with high expression of IL-10. Add PBS for resuspending and store at -80°C.

[0065] (3) Preparation of human umbilical cord mesenchymal stem cell exosomes (TFS-IL10-Exos) induced by the three-factor system with high expression of IL-10 ① Prepare the three-factor system (TFS) high-expression IL-10 exosome complete medium: add recombinant human IFN-γ, PDGF-BB and EPO to DMEM complete medium to make the final concentrations of 50 ng / mL, 50 ng / mL and 10 ng / mL respectively; ② Use DMEM complete medium to revive P3 human umbilical cord mesenchymal stem cells at a rate of 1×10 6 The cells / mL density was inoculated and cultured at 37°C, 5% CO2 for 24 h. ③ Add 1 mL of high-expression IL-10 recombinant lentivirus and 100 μg / ml Polybrene, and infect at 37°C, 5% CO2 for 8 h; ④ Discard the supernatant, replace with the three-factor system (TFS) high-expression IL-10 exosome complete culture medium, and culture at 37°C, 5% CO2 for 72 h; ⑤ Collect the culture supernatant of human umbilical cord mesenchymal stem cells, centrifuge at 300 g for 10 min, retain the supernatant, discard the precipitate, and remove the cells in the culture supernatant; ⑥ Centrifuge the supernatant at 2000 g for 20 min, retain the supernatant, discard the precipitate, and remove cell debris; ⑦ Centrifuge the supernatant at 10,000 g for 70 min, retain the supernatant, discard the precipitate, and remove cell debris again; ⑧ Centrifuge the supernatant at 100,000 g for 70 min, discard the supernatant, and retain the precipitate; ⑨ After adding PBS to the precipitate for re-suspending, centrifuge at 100,000 g for 70 min, discard the supernatant, and retain the precipitate. The precipitate is the purified human umbilical cord mesenchymal stem cell exosomes (TFS-IL10-Exos) induced by the three-factor system with high expression of IL-10. Add PBS for re-suspending and store at -80°C.

[0066] 3. Identification of exosomes from human umbilical cord mesenchymal stem cells (1) Transmission electron microscopy observation 10 μL of exosomes Exos, IL10-Exos and TFS-IL10-Exos prepared in step 2 were respectively taken and dropped on the copper grid for precipitation for 1 min, and the floating liquid was removed by filter paper. 10 μL of uranyl acetate was dropped on the copper grid and precipitated for 1 min, and the floating liquid was removed by filter paper. Dry at room temperature for 10 min, and perform electron microscopy imaging under 80 kV.

[0067] (2) Particle size analysis 10 μL of the exosomes Exos, IL10-Exos and TFS-IL10-Exos prepared in step 2 were respectively taken and diluted to 30 μL. The particle size and concentration of the exosomes were detected using a transmission electron microscope (brand: Hitachi, model: HT-7700).

[0068] (3) IL-10 content detection The ELISA kit (Cat. No.: E-EL-M0046, Brand: Elabscience) was used to detect the content of IL-10 in the prepared exosomes Exos, IL10-Exos and TFS-IL10-Exos.

[0069] 2. Experimental Results Electron microscopy results Figure 6As shown in a to c in the figure, under transmission electron microscopy, exosomes Exos, IL10-Exos and TFS-IL10-Exos are all cup-shaped round membrane vesicles, and the double membrane structure of the vesicles can be seen.

[0070] The particle size results are as follows Figure 7 As shown in a~c in the figure, the peak value is distributed in the range of 60~80 nm, which is consistent with the peak particle size range of umbilical cord mesenchymal stem cell exosomes (50~150 nm).

[0071] The results of IL-10 content are shown in Table 1. The IL-10 content of exosomes in the TFS-IL10-Exos group was 2.34 times that of the Exos group; the IL-10 content of exosomes in the TFS-IL10-Exos group was 1.45 times that of the IL10-Exos group.

[0072] Table 1 Exosomal IL-10 content

[0073] Example 3 Application of human umbilical cord mesenchymal stem cell exosome aerosol liquid highly expressing IL-10 in the treatment of acute lung injury 1. Experimental Methods (1) Animal grouping Adult male SPF rats were selected and randomly divided into 5 groups, namely blank control group, TFS-IL10-Exos group, IL10-Exos group, Exos group and model group. There were 10 rats in each group, which were weighed and marked respectively. The rats were adaptively raised in a laboratory with a constant temperature of 20℃-22℃ and a constant humidity of 60%-70%.

[0074] (2) Animal models The blank control group received no treatment. The rats in the TFS-IL10-Exos group, IL10-Exos group, Exos group and model group were injected with 2.5 mg / kg lipopolysaccharide into the trachea on the 1st and 14th days. On the non-injection day, they were smoked with cigarettes once a day for half an hour for 2 weeks to establish the animal model of lung qi deficiency, thus establishing the acute lung injury inflammatory model.

[0075] (3) Grouped medication: a. Blank control group: Normal feeding, normal records, no model treatment, and 2 mL of normal saline nebulization inhalation.

[0076] b. Model group: An acute lung injury and inflammation model was established and 2 mL of normal saline was nebulized and inhaled.

[0077] c. TFS-IL10-Exos group: An acute lung injury inflammatory model was constructed, and the animal was intubated and connected to a ventilator and an ultrasonic nebulizer to establish a respiratory pathway. 2 mL of the exosome TFS-IL10-Exos prepared in Example 2 after measuring the IL-10 content was administered for aerosol inhalation.

[0078] d. IL10-Exos group: An acute lung injury inflammatory model was constructed, and the animals were intubated and connected to an animal ventilator and an ultrasonic nebulizer to establish a respiratory pathway. 2 mL of the exosome IL10-Exos prepared in Example 2 after measuring the IL-10 content was administered for aerosol inhalation.

[0079] e. Exos group: An acute lung injury inflammatory model was constructed, and the animal was intubated and connected to a ventilator and an ultrasonic nebulizer to establish a respiratory pathway. 2 mL of the exosomes Exos prepared in Example 2 after measuring the IL-10 content was administered for aerosol inhalation.

[0080] (4) Collection of serum samples Rats were killed on day 7 for sampling, and intraperitoneal injection anesthesia was performed. After anesthesia, blood samples were collected from the abdominal aorta. After collecting 5 mL of blood, it was placed in an anticoagulant tube for about 1 hour, centrifuged at 3500 rpm for 5 minutes, and the upper clear liquid was taken out and packaged, and stored in a -80℃ refrigerator for use, and serum was obtained.

[0081] (5) Index detection ELISA kits were used to detect the levels of TNF-α, IL-6, IL-1β and IL-10 in serum.

[0082] 2. Experimental Results 1. TNF-α content in serum The results are shown in Table 2 and Figure 8 As shown. There was no significant difference between the results of the TFS-IL10-Exos group and the blank control group (p<0.05), and there were significant differences between the results of the TFS-IL10-Exos group and the IL10-Exos group, the Exos group and the model group (p>0.05), indicating that the therapeutic effect of the TFS-IL10-Exos group was significant. After 7 days of continuous administration, the level of inflammatory factor TNF-α could be reduced, and there was no difference in the level of inflammatory factor TNF-α compared with the blank control group, and the level returned to normal, indicating that the exosomes TFS-IL10-Exos prepared in Example 2 have obvious anti-inflammatory effect in inhibiting acute lung injury inflammation.

[0083] Table 2 TNF-α content in serum

[0084] 2. IL-6 content in serum The results are shown in Table 3 and Fig. 9 As shown. The results of the TFS-IL10-Exos group were not significantly different from those of the blank control group and the IL10-Exos group (p<0.05), and there were significant differences among the TFS-IL10-Exos group, the Exos group, and the model group (p>0.05), indicating that the therapeutic effects of the TFS-IL10-Exos group and the IL10-Exos group were significant. After 7 days of continuous administration, the level of inflammatory factor IL-6 was reduced, which was no different from that of the blank control group and returned to normal levels, indicating that the TFS-IL10-Exos prepared in Example 2 has a significant inhibitory effect on inflammation in inhibiting acute lung injury inflammation.

[0085] Table 3 IL-6 levels in serum

[0086] 3. IL-1β content in serum The results are shown in Table 4 and Fig.10 As shown. The results of TFS-IL10-Exos group were significantly different from those of blank control group, IL10-Exos group, Exos group and model group (p>0.05), indicating that the therapeutic effect of TFS-IL10-Exos group was significant. After 7 days of continuous administration, the level of inflammatory factor IL-1β was reduced, which was different from the level of inflammatory factor IL-1β in the blank control group and restored to normal extremely low level, indicating that TFS-IL10-Exos prepared in Example 2 had obvious anti-inflammatory effect in inhibiting acute lung injury inflammation.

[0087] Table 4 IL-1β content in serum

[0088] 4. IL-10 content in serum The results are shown in Table 5 and Fig.11 As shown. The results of TFS-IL10-Exos group were significantly different from those of blank control group, IL10-Exos group, Exos group and model group (p>0.05), indicating that the IL-10 expression level of TFS-IL10-Exos group was much higher than that of IL10-Exos group and Exos group after 7 days of continuous administration, indicating that TFS-IL10-Exos prepared in Example 2 has obvious anti-inflammatory effect in inhibiting inflammation of acute lung injury.

[0089] Table 5 IL-10 content in serum

[0090] Example 4 Effects of three factors on IL-10 content in exosomes of human umbilical cord mesenchymal stem cells 1. Experimental Methods Complete culture medium for highly expressed IL-10 exosomes was prepared according to the following groups: ① Control group: DMEM complete medium; ② P+E group: PDGF-BB and EPO were added to DMEM complete medium to a final concentration of 50 ng / mL and 10 ng / mL, respectively; ③ Group I: recombinant human IFN-γ was added to DMEM complete medium to a final concentration of 50 ng / mL; ④ P+E+I group: recombinant human IFN-γ, PDGF-BB and EPO were added to DMEM complete medium to make the final concentrations of 50 ng / mL, 50 ng / mL and 10 ng / mL, respectively.

[0091] The above-mentioned complete medium for exosomes highly expressing IL-10 was used to prepare human umbilical cord mesenchymal stem cell exosomes highly expressing IL-10 according to step (3) in Example 2, and the ELISA kit (Cat. No.: E-EL-M0046, Brand: Elabscience) was used to detect the content of IL-10 in the prepared exosomes.

[0092] 2. Experimental Results The results are shown in Table 6 and Fig.12 Compared with the culture medium of group ② and group ③, the IL-10 content in the exosomes prepared using the culture medium of group ④ was further increased and the difference was significant (P<0.05), indicating that the combination of recombinant human IFN-γ, PDGF-BB and EPO has a synergistic effect on the expression of IL-10 in exosomes.

[0093] Table 6 IL-10 content in exosomes

[0094] Example 5 Effect of IFN-γ on exosomes of human umbilical cord mesenchymal stem cells 1. Experimental Methods Complete culture medium for highly expressed IL-10 exosomes was prepared according to the following groups: ① Control group: DMEM complete medium; ② TNF-α group: recombinant human TNF-α, PDGF-BB and EPO were added to DMEM complete medium to make the final concentrations of 50 ng / mL, 50 ng / mL and 10 ng / mL respectively; ③ IL-1β group: recombinant human IL-1β, PDGF-BB and EPO were added to DMEM complete medium to make the final concentrations of 50 ng / mL, 50 ng / mL and 10 ng / mL, respectively; ④ IFN-γ group: recombinant human IFN-γ, PDGF-BB and EPO were added to DMEM complete medium to make the final concentrations of 50 ng / mL, 50 ng / mL and 10 ng / mL respectively; ⑤ IL-17 group: recombinant human IL-17, PDGF-BB and EPO were added to DMEM complete medium to make the final concentrations of 50 ng / mL, 50 ng / mL and 10 ng / mL, respectively.

[0095] The above-mentioned complete medium for highly expressing IL-10 exosomes was used respectively to prepare human umbilical cord mesenchymal stem cell exosomes highly expressing IL-10 according to the method of “Preparation of human umbilical cord mesenchymal stem cell exosomes highly expressing IL-10 induced by three-factor system (TFS-IL10-Exos)” in Example 2.

[0096] The prepared exosomes were characterized using nanoparticle tracking analysis (NTA).

[0097] Equal masses of exosomes from the Control group, TNF-α group, IL-1β group, IFN-γ group, and IL-17 group were co-cultured with regulatory T cells (Treg), and the cells were labeled with FITC anti-human CD63 antibody (Brand: BioLegend, Catalog No.: 353005) and CFSE dye. The proliferation of Treg cells was detected by flow cytometry, the proportion of Treg cells was analyzed, and the effects of exosomes from different groups on the generation of Treg cells were detected.

[0098] CFSE dye-labeled PBMCs were co-cultured with equal amounts of exosomes from the control group, TNF-α group, IL-1β group, IFN-γ group, and IL-17 group for 5 days. After co-culture, the proliferation of PBMCs was analyzed by flow cytometry.

[0099] 2. Experimental Results The results are shown in Table 7. In terms of the number of exosomes, the number of exosomes prepared using the culture medium of group ④ was the largest, indicating that IFN-γ plays a significant role in promoting the production and secretion of exosomes.

[0100] In terms of exosome diameter, the exosomes prepared using group ④ culture medium had the smallest diameter, indicating that IFN-γ is related to the maturity and secretion efficiency of exosomes.

[0101] In terms of CD63 positivity rate, the CD63 positivity rate of the culture medium-treated group ④ was significantly higher than that of other groups, indicating that IFN-γ can enhance the expression of marker proteins of exosomes and improve the recognition and function of exosomes.

[0102] In terms of the proportion of Treg cells, the proportion of Treg cells in the IFN-γ-treated group was significantly higher than that in other groups, indicating that IFN-γ helps exosomes play an important role in immune regulation.

[0103] In terms of inhibiting the proliferation rate of PBMCs, the exosomes in the IFN-γ-treated group showed the strongest ability to inhibit the proliferation of PBMCs, indicating that IFN-γ is related to the content and activity of immunomodulatory molecules in exosomes.

[0104] Table 7 Effects of IFN-γ on exosomes of human umbilical cord mesenchymal stem cells

[0105] Example 6 Effects of the dosage of IFN-γ, PDGF-BB and EPO on exosomes of human umbilical cord mesenchymal stem cells 1. Experimental Methods Complete culture medium for highly expressed IL-10 exosomes was prepared according to the following groups: ① Control group: DMEM complete medium; ② IFN-γ group: only recombinant human IFN-γ was added to DMEM complete medium, and its final concentration was set to 5 ng / mL, 50 ng / mL, and 100 ng / mL respectively; ③ PDGF-BB group: only PDGF-BB was added to DMEM complete medium, and its final concentration was set to 5 ng / mL, 50 ng / mL, and 100 ng / mL respectively; ④ EPO group: only EPO was added to DMEM complete medium, and its final concentration was set to 1 ng / mL, 10 ng / mL, and 20 ng / mL respectively; ⑤ Three-factor group: recombinant human IFN-γ, PDGF-BB and EPO were added to DMEM complete medium, and low, medium and high concentrations were set, that is, their final concentrations were: Low concentration: recombinant human IFN-γ 5 ng / mL, PDGF-BB 5 ng / mL, EPO 1 ng / mL; Medium concentration: recombinant human IFN-γ 50 ng / mL, PDGF-BB 50 ng / mL, EPO 10 ng / mL; High concentration: recombinant human IFN-γ 100 ng / mL, PDGF-BB 100 ng / mL, EPO 20 ng / mL.

[0106] The above-mentioned complete medium for highly expressing IL-10 exosomes was used respectively to prepare human umbilical cord mesenchymal stem cell exosomes highly expressing IL-10 according to the method of “Preparation of human umbilical cord mesenchymal stem cell exosomes highly expressing IL-10 induced by three-factor system (TFS-IL10-Exos)” in Example 2.

[0107] The prepared exosomes were characterized using nanoparticle tracking analysis (NTA).

[0108] The activity of human umbilical cord mesenchymal stem cells was detected using MTT kit.

[0109] 2. Experimental Results The results are shown in Table 8. The experimental results show that PDGF-BB has the strongest promoting effect on the secretion of exosomes by umbilical cord mesenchymal stem cells at a concentration of 50 ng / mL. IFN-γ also shows a good promoting effect at a concentration of 50 ng / mL, while EPO has a better effect at a concentration of 10 ng / mL.

[0110] In terms of cell growth, the increases of PDGF-BB, EPO, and IFN-γ were most obvious at concentrations of 50 ng / mL and 100 ng / mL, and the differences were not large. PDGF-BB and IFN-γ can be selected at 50 ng / mL, and EPO can be selected at 10 ng / mL, which can save costs without affecting the results.

[0111] In terms of exosome production, the three-factor group induced the largest number of cell exosomes. The exosome production of IFN-γ and PDGF-BB at 50 ng / mL and 100 ng / mL was high and the difference was not large. Then, the exosome production of EPO at 10 ng / mL and 20 ng / mL was high and the difference was not large. Therefore, the concentrations of PDGF-BB and IFN-γ can be 50 ng / mL, and the concentration of EPO can be 10 ng / mL, which can save costs without affecting the production.

[0112] In terms of exosome size, the exosome size distribution of all experimental groups varied, but was within the normal range (50-150 nm).

[0113] In terms of cell viability, there was no decrease in cell viability in all experimental groups, indicating that these factors were not toxic to cells at the tested concentrations.

[0114] Table 8 Effects of IFN-γ, PDGF-BB and EPO dosage on exosomes of human umbilical cord mesenchymal stem cells

[0115] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing human umbilical cord mesenchymal stem cell exosomes, characterized in that: The IL-10 stably transfected human umbilical cord mesenchymal stem cells are cultured in a complete culture medium containing PDGF-BB, EPO and IFN-γ, the cell culture fluid is collected, and exosomes are separated from the cell culture fluid to obtain the exosomes.

2. The preparation method according to claim 1, characterized in that The final concentration of the PDGF-BB in the complete culture medium is 5 to 100 ng / mL.

3. The preparation method according to claim 1, characterized in that The final concentration of the IFN-γ in the complete culture medium is 5 to 100 ng / mL.

4. The preparation method according to claim 1, characterized in that The final concentration of the EPO in the complete culture medium is 1-20 ng / mL.

5. The preparation method according to claim 1, characterized in that: The IL-10 stably transfected human umbilical cord mesenchymal stem cells are obtained by transfecting human umbilical cord mesenchymal stem cells with a recombinant lentivirus that overexpresses IL-10.

6. The preparation method according to claim 5, characterized in that: The human umbilical cord mesenchymal stem cells are of passages P3 to P5.

7. Use of the preparation method according to any one of claims 1 to 6 in increasing the IL-10 content in exosomes of human umbilical cord mesenchymal stem cells.

8. Use of the preparation method according to any one of claims 1 to 6 in increasing the yield of exosomes containing IL-10 from human umbilical cord mesenchymal stem cells.

9. Use of human umbilical cord mesenchymal stem cell exosomes containing IL-10 in the preparation of a drug for treating acute lung injury, characterized in that: The human umbilical cord mesenchymal stem cell exosomes are prepared by the preparation method according to any one of claims 1 to 6.

10. The use according to claim 9, characterized in that: The medicine is an aerosolized medicine.

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