Application of extracellular vesicles derived from decidual mesenchymal stem cells in pulmonary fibrosis

Abstract

The present invention belongs to the application field of extracellular vesicles, and specifically relates to the application of extracellular vesicles derived from decidual mesenchymal stem cells in pulmonary fibrosis. The present invention uses pEGFP-N2-GFE2-Lamp2b plasmid to transfect decidual mesenchymal stem cells I, obtains decidual mesenchymal stem cells II with targeted localization effect, and after culturing with dispersed polymers and supplement culture medium, adopts centrifugation combined with sucrose density gradient purification to obtain extracellular vesicles derived from decidual mesenchymal stem cells with high purity, stable performance, and also having targeted localization effect. It is used in pulmonary fibrosis, which can achieve the application effect of small doses, precise onset, and alleviation of the process of pulmonary fibrosis.

Description

Technical Field

[0001] The present invention belongs to the application field of extracellular vesicles, and specifically relates to the application of extracellular vesicles derived from decidual mesenchymal stem cells in pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis is a progressive, usually fatal interstitial lung disease of unknown etiology, characterized by fibroblast proliferation and accumulation of large amounts of extracellular matrix, accompanied by inflammatory damage and destruction of tissue architecture. The most common type of disease is idiopathic pulmonary fibrosis, a severe inflammatory interstitial lung disease with a poor prognosis, high mortality, and increasing morbidity and prevalence. Several drugs have been tested for the treatment of idiopathic pulmonary fibrosis; however, their efficacy remains unsatisfactory. Lung transplantation is the best treatment option to improve the survival of patients with idiopathic pulmonary fibrosis. However, this approach is limited by a lack of donors. Therefore, there is an urgent need to find new ways to treat pulmonary fibrosis. With the rapid development of cell therapy, stem cell therapy has become a strategy for the treatment of a variety of diseases.

[0003] The low survival rate and high immune rejection of stem cell transplantation have resulted in unsatisfactory clinical treatment effects. Stem cell extracellular vesicles (EVs) are a good alternative. Over the past decade, extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies, have been considered a new paracrine mediator for the transfer of biological components. The contents of EVs mainly consist of microRNA (miRNA), proteins, and lipids. EVs are emerging as new intercellular communication mediators and possible biomarkers for various types of lung diseases. In fact, EVs detected in various biological fluids, including sputum, mucus, blood, urine, and bronchoalveolar lavage fluid, provide useful tools for studying the pathogenesis of lung diseases and discovering biomarkers. EV-derived compositions are an emerging treatment option for lung diseases.

[0004] Some studies have shown that EVs derived from mesenchymal stem cells (MSCs) can reverse inflammation associated with chronic lung diseases. In addition, EVs derived from specific cells or tissues have become a new cell-free model for the treatment of IPF. The applicant has previously confirmed that decidual mesenchymal stem cells (DMSCs) from early pregnancy decidual tissue have been found to have extremely strong proliferation and anti-inflammatory abilities. There are a large number of DMSCs in the decidual tissue stroma, and the clinical material collection is relatively simple. It comes from clinical waste tissue and has a rich tissue source, which makes it possible to extract EVs in large quantities by engineering. We focus on the role and mechanism of DMSCs-derived EVs in improving pulmonary fibrosis. Summary of the invention

[0005] Based on the above problems, the present invention chooses to obtain decidual mesenchymal stem cells I, which have lower immunogenicity and do not cause serious bioethical issues. After plasmid transfection, the obtained decidual mesenchymal stem cells II have better targeting effect. Combined with the application of dispersed polymers, supplement culture medium, sucrose density gradient purification and other technologies, the extracellular vesicles derived from decidual mesenchymal stem cells obtained are not only high in purity and stable in performance, but also have targeting effect, thereby achieving better application effect in pulmonary fibrosis.

[0006] The specific technical scheme for the application of the decidual mesenchymal stem cell-derived extracellular vesicles in pulmonary fibrosis is as follows:

[0007] S1. Obtain decidual tissue from patients undergoing curettage and curettage for early pregnancy abortion in the outpatient clinic, wash, digest with enzymes, and culture under hypoxic conditions to obtain decidual mesenchymal stem cells I;

[0008] S2. Transfecting the plasmid into the decidual mesenchymal stem cells I obtained in step S1 to obtain decidual mesenchymal stem cells II;

[0009] S3. Using a dispersed polymer and a supplement medium to culture the decidual mesenchymal stem cells II obtained in step S2, and obtaining extracellular vesicles derived from decidual mesenchymal stem cells after centrifugation and sucrose density gradient purification;

[0010] S4. The decidual mesenchymal stem cell-derived extracellular vesicles obtained in step S3 are used for treating pulmonary fibrosis.

[0011] Preferably, the anoxic condition in step S1 is preferably 5% carbon dioxide culture, the PBS buffer containing antibiotics is preferably used for washing, and the culture is carried out using complete culture medium;

[0012] Preferably, the plasmid in step S2 is pEGFP-N2-GFE2-Lamp2b, the amino acid sequence of GFE2 is shown in SEQ ID NO.1, and the corresponding mRNA sequence is shown in SEQ ID NO.2. When constructing the plasmid, restriction sites of XhoⅠ and BamHI are preferred.

[0013] Preferably, the dispersed polymer in step S3 is prepared from calcium chloride, fructose, and 4-hydroxyethylpiperazineethanesulfonic acid, and the specific preparation steps are as follows:

[0014] M1-1. Weigh calcium chloride, fructose and 4-hydroxyethylpiperazineethanesulfonic acid, mix and stir to obtain a mixture 1, and add water to sodium alginate to prepare a sodium alginate solution;

[0015] M1-2. The sodium alginate solution is added to the mixed solution 1 by a peristaltic pump at a speed of 60-90 rpm. After the addition, a dispersed polymer can be seen in the mixed solution 1. The dispersed polymer can be obtained by filtration. The dispersed polymer is washed with PBS buffer and stored in PBS buffer at 4°C for later use.

[0016] In particular, in step M1-1, the mass ratio of calcium chloride: fructose: 4-hydroxyethylpiperazineethanesulfonic acid is 13-15: 1-1.2: 2.5-3.0, and the mass concentration of the sodium alginate solution is 5-8%;

[0017] In particular, before the sodium alginate solution is mixed with the mixed solution 1 in step M1-2, the mixed solution 1 is precooled, and the precooling is carried out by adding ice cubes to a beaker or storing at 4°C, and the volume ratio of the sodium alginate solution to the mixed solution 1 is 1:5-8;

[0018] In particular, the dispersed polymer obtained in step M1-2 needs to be placed in a room temperature environment and the PBS buffer solution is warmed up before use;

[0019] Preferably, the supplement culture medium in step S3 is prepared by adding human blood lysate and sodium glycerophosphate to DMEM culture medium, wherein the amount of human blood lysate added is 5-8% by mass, and the amount of sodium glycerophosphate added is 0.8-1.3% by mass.

[0020] Preferably, the medium used for sucrose density gradient purification in step S3 is obtained by mixing ultrapure water, sucrose and tris(hydroxymethyl)aminomethane in a mass ratio of 10-14:2.5-2.8:0.2-0.3 and stirring them uniformly.

[0021] Preferably, the extracellular vesicles derived from decidual mesenchymal stem cells in step S4 can be directly used for pulmonary fibrosis, or can be used as a carrier in combination with other substances to act on pulmonary fibrosis.

[0022] The beneficial effects of the present invention are as follows:

[0023] The present invention obtains decidual tissue of patients who undergo early pregnancy abortion and curettage in an outpatient clinic, obtains decidual mesenchymal stem cells I after enzymatic digestion, designs a pEGFP-N2-GFE2-Lamp2b plasmid and performs transfection to obtain decidual mesenchymal stem cells II, and decidual mesenchymal stem cells II can express GFE2 protein that is not present in decidual mesenchymal stem cells I. The GFE2 protein has a good homing effect and can target the membrane dipeptidase MDP of lung epithelial cells and endothelial cells, so that the extracellular vesicles derived from the decidual mesenchymal stem cells finally obtained also contain GFE2 protein outside the membrane, thereby helping the extracellular vesicles to aggregate in the lung tissue, avoiding the problems of nonspecific uptake and systemic clearance when the extracellular vesicles are administered by intravenous injection, and achieving the effect of a small dose being able to exert a precise effect.

[0024] The present invention uses dispersed polymers and supplement culture medium to culture decidual mesenchymal stem cells I. The dispersed polymer can bring a better three-dimensional culture environment, reduce differentiation, and maintain cell stemness. The supplement culture medium replaces the commonly used fetal bovine serum, which improves safety. The present invention uses centrifugation combined with sucrose density gradient purification to obtain extracellular vesicles. Compared with differential ultracentrifugation, the gold standard for obtaining extracellular vesicles, the extracellular vesicles obtained by the present invention have higher purity, better and more stable performance in batch preparation, and combined with the aforementioned precise targeting design, the extracellular vesicles derived from decidual mesenchymal stem cells prepared by the present invention can have a better application effect in pulmonary fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0026] Figure 1 This is the cell morphology of decidual mesenchymal stem cells I;

[0027] Figure 2 This is the specific phenotype diagram of decidual mesenchymal stem cells I;

[0028] Figure 3 The figure shows the morphology of extracellular vesicles derived from decidual mesenchymal stem cells;

[0029] Figure 4 The particle size diagram of extracellular vesicles derived from decidual mesenchymal stem cells;

[0030] Figure 5 This is a potential map of extracellular vesicles derived from decidual mesenchymal stem cells;

[0031] Figure 6 This is a characterization diagram of the marker proteins of extracellular vesicles derived from decidual mesenchymal stem cells;

[0032] Figure 7 It is the expression diagram of GFE2 protein;

[0033] Figure 8 This is a purity chart of extracellular vesicles derived from decidual mesenchymal stem cells;

[0034] Fig. 9 This is an image of a mouse after extracellular vesicles derived from decidual mesenchymal stem cells labeled with the fluorescent dye Dil were instilled into the lungs via the trachea;

[0035] Fig.10 HE and Masson effect diagram of lung tissues in mice with pulmonary fibrosis treated with extracellular vesicles derived from decidual mesenchymal stem cells;

[0036] Fig.11 This is the effect of extracellular vesicles derived from decidual mesenchymal stem cells on collagen I, fibronectin and α-SMA in lung tissue after treating pulmonary fibrosis mice;

[0037] The EVs in the figure are extracellular vesicles derived from decidual mesenchymal stem cells, and DMSC are decidual mesenchymal stem cells. DETAILED DESCRIPTION

[0038] Example 1: This example provides a method for obtaining decidual mesenchymal stem cells I, and the specific steps are as follows:

[0039] The decidual tissue was obtained from patients who underwent early pregnancy abortion and curettage in the outpatient clinic. The decidual tissue was repeatedly rinsed three times with PBS buffer containing 1% penicillin-streptomycin, and the tissue was cut into small pieces as much as possible with tissue scissors. 0.2% collagenase IV and 0.25% pancreatin were added until the liquid surface covered the tissue, and the decidual tissue was fully digested in a 37°C water bath shaker with shaking at 150 rpm. The digestion fluid was collected into a 50 ml centrifuge tube and neutralized with complete culture medium. A sterile filter was placed on a large cell culture dish for filtration, and the tube was centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the tube was placed in a culture dish and cultured in an environment of 5% carbon dioxide and 37°C. At this time, the adherent cells were decidual mesenchymal stem cells, and the decidual mesenchymal stem cells I of this embodiment can be obtained by passage to the third generation.

[0040] Example 2: This example provides a method for obtaining decidual mesenchymal stem cells II, and the specific steps are as follows:

[0041] V1. According to the mRNA sequence of GFE2 protein, the corresponding DNA sequence was obtained and delivered to Yunzhou Biotechnology (Guangzhou) Co., Ltd. to design and obtain the plasmid: pEGFP-N2-GFE2-Lamp2b. During the process, the restriction sites of XhoⅠ and BamHI were selected;

[0042] V2. The decidual mesenchymal stem cells I obtained in Example 1 were placed in a 6-well plate using lipofectamine 2000 transfection reagent, with 3×10 6 Decidual mesenchymal stem cells I, 2.5 mL complete culture medium, 100 μL of the plasmid in step V1, 100 μL of lipofectamine 2000 transfection reagent, and culture them in an environment of 5% carbon dioxide and 37°C. After checking that the transfection is successful, decidual mesenchymal stem cells II are obtained.

[0043] Example 3: This example provides a method for obtaining extracellular vesicles derived from decidual mesenchymal stem cells, and the specific steps are as follows:

[0044] M1. Calcium chloride, fructose and 4-hydroxyethylpiperazineethanesulfonic acid were weighed in a mass ratio of 13:1:2.5, mixed and stirred to obtain a mixed solution 1, sodium alginate was prepared into a sodium alginate solution with a mass concentration of 5%, sodium alginate solution and mixed solution 1 were weighed in a volume ratio of 1:5, mixed solution 1 was placed in a beaker, ice was added to the beaker to exceed the liquid surface by 2 cm, and after maintaining for 30 minutes, sodium alginate solution was added to mixed solution 1 by a peristaltic pump, and the speed of the peristaltic pump was 60 rpm. After the end, dispersed polymers were visible in mixed solution 1, and the dispersed polymers were obtained after filtration and washed with PBS buffer for 3 times, and stored in PBS buffer for use at 4°C;

[0045] M2. The dispersed polymer and PBS buffer obtained in step M1 are placed at room temperature for 15 minutes. After the dispersion polymer is removed from the PBS buffer, the dispersed polymer and the supplement medium are obtained at a mass ratio of 1:10. The supplement medium is prepared by adding 5% human platelet lysate and 0.8% sodium glycerophosphate to DMEM medium. After the dispersed polymer and the supplement medium are mixed, the decidual mesenchymal stem cells II are transferred thereto, cultured at 5% carbon dioxide and 37°C for 48 hours, centrifuged at 4°C and 400g for 8 minutes, and supernatant 1 is collected. Supernatant 1 is centrifuged at 4°C and 1000g for 60 minutes, and supernatant 2 is collected. Supernatant 2 is ultrafiltered through a 100kDa membrane to obtain a concentrate.

[0046] M3. Ultrapure water, sucrose and tris(hydroxymethyl)aminomethane are weighed in a mass ratio of 10:2.5:0.2, mixed and stirred to obtain a medium, the concentrated solution obtained in step M3 is purified by the medium, centrifuged at 4°C and 80,000 g for 120 minutes, the sucrose layer is collected with a needle syringe, washed twice with 4°C pre-cooled PBS buffer, centrifuged at 4°C and 100,000 g for 100 minutes, precipitated and resuspended in PBS buffer to obtain extracellular vesicles derived from decidual mesenchymal stem cells.

[0047] Example 4: This example provides a method for obtaining extracellular vesicles derived from decidual mesenchymal stem cells, and the specific steps are as follows:

[0048] M1. Calcium chloride, fructose and 4-hydroxyethylpiperazineethanesulfonic acid were weighed in a mass ratio of 14:1.1:2.8, mixed and stirred to obtain a mixed solution 1, sodium alginate was prepared into a sodium alginate solution with a mass concentration of 6%, sodium alginate solution and mixed solution 1 were weighed in a volume ratio of 1:6, mixed solution 1 was placed in a beaker, ice was added to the beaker to exceed the liquid surface by 2 cm, and after maintaining for 30 minutes, sodium alginate solution was added to mixed solution 1 by a peristaltic pump, and the speed of the peristaltic pump was 80 rpm. After the end, dispersed polymers were visible in mixed solution 1, and the dispersed polymers were obtained after filtration and washed with PBS buffer for 3 times, and stored in PBS buffer for use at 4°C;

[0049] M2. The dispersed polymer and PBS buffer obtained in step M1 are placed at room temperature for 15 minutes. After the dispersion polymer is removed from the PBS buffer, the dispersed polymer and the supplement medium are obtained at a mass ratio of 1:12. The supplement medium is prepared by adding 6% human platelet lysate and 1% sodium glycerophosphate to DMEM medium. After the dispersed polymer and the supplement medium are mixed, the decidual mesenchymal stem cells II are transferred thereto, cultured at 5% carbon dioxide and 37°C for 48 hours, centrifuged at 4°C and 400g for 8 minutes, and supernatant 1 is collected. The supernatant 1 is centrifuged at 4°C and 1000g for 60 minutes, and supernatant 2 is collected. The supernatant 2 is ultrafiltered through a 100kDa membrane to obtain a concentrate.

[0050] M3. Ultrapure water, sucrose and tris(hydroxymethyl)aminomethane are weighed in proportions of 12:2.6:0.25 by mass, mixed and stirred to obtain a medium, the concentrated solution obtained in step M3 is purified by the medium, centrifuged at 4°C and 80,000 g for 120 min, the sucrose layer is collected with a needle syringe, washed twice with 4°C precooled PBS buffer, centrifuged at 4°C and 100,000 g for 100 min, precipitated and resuspended in PBS buffer to obtain extracellular vesicles derived from decidual mesenchymal stem cells.

[0051] Example 5: This example provides a method for obtaining extracellular vesicles derived from decidual mesenchymal stem cells, and the specific steps are as follows:

[0052] M1. Calcium chloride, fructose and 4-hydroxyethylpiperazineethanesulfonic acid were weighed in a mass ratio of 15:1.2:3.0, mixed and stirred to obtain a mixed solution 1, sodium alginate was prepared into a sodium alginate solution with a mass concentration of 8%, sodium alginate solution and mixed solution 1 were weighed in a volume ratio of 1:8, mixed solution 1 was placed in a beaker, ice was added to the beaker until it exceeded the liquid surface by 2 cm, and after maintaining for 30 minutes, sodium alginate solution was added to mixed solution 1 by a peristaltic pump, and the speed of the peristaltic pump was 90 rpm. After the end, dispersed polymers were visible in mixed solution 1, and the dispersed polymers were obtained after filtration and washed with PBS buffer for 3 times, and stored in PBS buffer for use at 4°C;

[0053] M2. The dispersed polymer and PBS buffer obtained in step M1 are placed at room temperature for 15 minutes. After the dispersion polymer is removed from the PBS buffer, the dispersed polymer and the supplement medium are obtained at a mass ratio of 1:15. The supplement medium is prepared by adding 8% human platelet lysate and 1.3% sodium glycerophosphate to DMEM medium. After the dispersed polymer and the supplement medium are mixed, the decidual mesenchymal stem cells II are transferred thereto, cultured at 5% carbon dioxide and 37°C for 48 hours, centrifuged at 4°C and 400g for 8 minutes, and supernatant 1 is collected. The supernatant 1 is centrifuged at 4°C and 1000g for 60 minutes, and supernatant 2 is collected. The supernatant 2 is ultrafiltered through a 100kDa membrane to obtain a concentrate.

[0054] M3. Ultrapure water, sucrose and tris(hydroxymethyl)aminomethane are weighed in a mass ratio of 14:2.8:0.3, mixed and stirred to obtain a medium, the concentrated solution obtained in step M3 is purified by the medium, centrifuged at 4°C and 80,000 g for 120 minutes, the sucrose layer is collected with a needle syringe, washed twice with 4°C pre-cooled PBS buffer, centrifuged at 4°C and 100,000 g for 100 minutes, precipitated and resuspended in PBS buffer to obtain extracellular vesicles derived from decidual mesenchymal stem cells.

[0055] The decidual mesenchymal stem cells II used in Examples 3-5 were all prepared in Example 2.

[0056] Comparative Example 1: This comparative example provides a method for obtaining extracellular vesicles derived from decidual mesenchymal stem cells, and the specific steps are as follows:

[0057] This comparative example uses the decidual mesenchymal stem cells II obtained in Example 2 to culture and obtain extracellular vesicles. The difference from Example 4 is that this comparative example does not use dispersed polymers and supplement culture medium for culture, and also does not use sucrose density gradient purification.

[0058] The decidual mesenchymal stem cells II obtained in Example 2 were cultured in DMEM medium, cultured in an environment of 5% carbon dioxide and 37°C for 48 hours, and centrifuged at 500g for 10 min, 2500g for 30 min, and 12000g for 60 min. The centrifugation was performed at 4°C. The precipitate was discarded after each centrifugation to obtain the supernatant. The supernatant was filtered with a 0.22 μm filter. The supernatant after filtration was centrifuged at 110000g for 70 min. The precipitate was collected and resuspended in PBS buffer to obtain the extracellular vesicles derived from the decidual mesenchymal stem cells of this comparative example.

[0059] Comparative Example 2: This comparative example provides a method for obtaining extracellular vesicles derived from decidual mesenchymal stem cells, and the specific steps are as follows:

[0060] In this comparative example, the decidual mesenchymal stem cells I obtained in Example 1 were cultured and extracellular vesicles were obtained, and the remaining steps were consistent with Example 4 to obtain the extracellular vesicles derived from the decidual mesenchymal stem cells of this comparative example.

[0061] Experimental testing:

[0062] 1. Phenotypic identification of decidual mesenchymal stem cells I:

[0063] After digestion of the decidual mesenchymal stem cells I obtained in Example 1, the cells were counted and the number of decidual mesenchymal stem cells added to each EP tube was maintained at 1×10 6 Decidual mesenchymal stem cells were incubated with stem cell phenotype CD105-APC, CD73-PerCP, CD90-FITC, CD34-FITC and CD45-PerCP antibodies for 30 minutes in the dark, and then filtered through a 200-mesh filter to wash away excess antibodies, and then detected by flow cytometry.

[0064] The cell morphology of the decidual mesenchymal stem cells I obtained in Example 1 is as follows Figure 1 As shown by Figure 1 It can be seen that the decidual mesenchymal stem cells I obtained in Example 1 are in the shape of a long fusiform spindle.

[0065] The specific phenotype of the decidual mesenchymal stem cells I obtained in Example 1 is as follows Figure 2 As shown by Figure 2 It can be seen that the decidual mesenchymal stem cells I obtained in Example 1 significantly expressed the stem cell characteristic markers CD73, CD90 and CD105, but did not express CD34 and CD45.

[0066] 2. Characterization of Decidual Mesenchymal Stem Cell-derived Extracellular Vesicles:

[0067] The following experiment is to characterize the extracellular vesicles derived from decidual mesenchymal stem cells obtained in Example 3:

[0068] 1. Transmission electron microscopy observation: The extracellular vesicles derived from decidual mesenchymal stem cells were fixed with glutaraldehyde and dropped onto a copper grid. The grid was adsorbed at room temperature for about 10 min. A phosphotungstic acid solution was added to the copper grid. The extracellular vesicles derived from decidual mesenchymal stem cells were stained at room temperature. The copper grid was dried at room temperature and the morphology of the extracellular vesicles derived from decidual mesenchymal stem cells was observed by a machine.

[0069] 2. Dynamic light scattering detection of particle size: Freshly extracted extracellular vesicles derived from decidual mesenchymal stem cells were diluted, filtered with a 0.22 μm filter, and then the particle size and potential of the extracellular vesicles derived from decidual mesenchymal stem cells were detected using a Malvern particle size potential detector.

[0070] 3. Protein immunoblotting to identify extracellular vesicle marker proteins: Use RIPA lysis buffer and protease inhibitor mixed solution to extract total tissue or cell protein. After calculating the sample weight, take the sample and mix it with PBS. After adding 6× loading buffer, heat it in a 100℃ metal bath for 7 minutes. Prepare SDS-PAGE gel, 70V constant voltage electrophoresis, 300mA constant flow transfer. Block with 5% skim milk at room temperature for 2h. Incubate the primary antibody at 4℃ overnight, and incubate the secondary antibody the next day. Detect the bands with an infrared fluorescence scanning system.

[0071] The results are as follows Figure 3 , Figure 4 , Figure 5 , Figure 6 As shown, Figure 3 The morphology of extracellular vesicles derived from decidual mesenchymal stem cells is shown, which are basically spherical. Figure 4 The morphology and particle size of extracellular vesicles derived from decidual mesenchymal stem cells are shown, with an average particle size of 240 nm. Figure 5 The morphology and potential of extracellular vesicles derived from decidual mesenchymal stem cells are shown. The zeta potential is 11.2 mV. Figure 6 The marker proteins of extracellular vesicles derived from decidual mesenchymal stem cells are displayed, demonstrating the positive marker proteins (TSG-101, Alix) and negative marker proteins (GM130) of extracellular vesicles derived from decidual mesenchymal stem cells.

[0072] 3. Characterization of GFE2 protein expression:

[0073] The expression of GFE2 protein in the decidual mesenchymal stem cells I obtained in Example 1, the decidual mesenchymal stem cells II obtained in Example 2, the extracellular vesicles derived from the decidual mesenchymal stem cells obtained in Example 3, the extracellular vesicles derived from the decidual mesenchymal stem cells obtained in Example 4, and the extracellular vesicles derived from the decidual mesenchymal stem cells obtained in Example 5 were examined. The primary antibody selected the receptor membrane dipeptidase MDP of the GFE2 protein, and β-Actin was used as an internal reference. The protein immunoblotting method was used for inspection, and the other experimental steps referred to the method for identifying extracellular vesicle marker proteins by protein immunoblotting. Figure 7 The expression of GFE2 in decidual mesenchymal stem cells I, decidual mesenchymal stem cells II, and extracellular vesicles derived from decidual mesenchymal stem cells is shown. Figure 7 It can be seen that compared with the decidual mesenchymal stem cells I obtained in Example 1, the decidual mesenchymal stem cells II obtained in Example 2 and the extracellular vesicles derived from the decidual mesenchymal stem cells obtained in Examples 3-5 all showed a higher level of GFE2 protein, proving that the plasmid transfection was effective and that the obtained extracellular vesicles also contained GFE2 protein. The relative expression of GFE2 protein in the extracellular vesicles derived from the decidual mesenchymal stem cells obtained in Examples 3-5 was relatively stable.

[0074] 4. Comparison of the purity of extracellular vesicles derived from decidual mesenchymal stem cells:

[0075] 1. Protein determination:

[0076] A micro BCA protein quantification kit was purchased from Beijing Biolab Technology Co., Ltd., with the item number WE0273 and the English name: MicroBCA Protein Assay Kit. It was used to detect the protein concentration Pr of the extracellular vesicles derived from decidual mesenchymal stem cells obtained in Example 3, Example 4, Example 5, Comparative Example 1, and Comparative Example 2, in μg / mL. The specific operation steps were carried out according to the instructions of the kit.

[0077] 2. Nanoparticle tracking analysis:

[0078] Nanoparticle tracking analysis was performed using a ZetaView_ParticleMetrix instrument from the German brand ParticleMetrix. The sample pool was cleaned three times with pure water. After cleaning, the instrument was calibrated using a standard (100 nm PS beads, polystyrene microspheres, manufactured by ThermoFisher, model 3100A). After calibration, subsequent testing was performed. The sample pool was cleaned with 1×PBS, and the sample was diluted with 1×PBS and added to the sample pool. The real-time dynamic images of the extracellular vesicles derived from decidual mesenchymal stem cells obtained in Example 3, Example 4, Example 5, Comparative Example 1, and Comparative Example 2 were observed on a computer display screen, and corresponding information was collected to obtain the particle concentration Pa, in units of Particles / mL.

[0079] The purity of extracellular vesicles derived from decidual mesenchymal stem cells is the ratio of Pa / Pr, with the unit of particles / 1 μg of protein.

[0080] The results are as follows Figure 8 As shown by Figure 8 It can be seen that the purity of the extracellular vesicles derived from decidual mesenchymal stem cells obtained in Comparative Example 1 is relatively low. In comparison, the purity of the extracellular vesicles derived from decidual mesenchymal stem cells obtained in Example 3, Example 4, Example 5 and Comparative Example 2 is obviously higher in quality.

[0081] 5. Application of extracellular vesicles derived from decidual mesenchymal stem cells:

[0082] The decidual mesenchymal stem cell-derived extracellular vesicles used in this experimental step were prepared in Example 5.

[0083] 1. In vivo tracing of extracellular vesicles derived from decidual mesenchymal stem cells in mice:

[0084] C57BL / 6J mice were anesthetized by intraperitoneal injection of tribromoethanol (500 mg / kg). Then, BLM (3 mg / kg) was intratracheally administered once on day 0, and extracellular vesicles (100 μg / mouse) derived from decidual mesenchymal stem cells labeled with Dil cell membrane fluorescent probe were instilled through the trachea on day 7 after BLM treatment. The dynamic changes of extracellular vesicles entering the pulmonary fibrosis mouse model were tracked by small animal in vivo imaging; BLM-induced mice that were not treated with extracellular vesicles derived from decidual mesenchymal stem cells were the BLM group; mice that did not receive any treatment were the Control group.

[0085] The results are as follows Fig. 9 As shown by Fig. 9 It can be seen that the extracellular vesicles derived from decidual mesenchymal stem cells obtained in Example 5 accumulated in the lungs of mice.

[0086] 2. Effects of extracellular vesicles derived from decidual mesenchymal stem cells on pulmonary fibrosis:

[0087] (1) Obtaining, fixing and embedding mouse lung tissue: Place the euthanized mouse in a supine position on a clean dissecting board, expose the trachea, heart and lungs, use scissors and forceps to remove the skin and muscles covering the ventral chest and neck area, use scissors to cut the ribs on both sides of the chest cavity, fully expose the chest cavity of the heart and lungs, cut the trachea near the jaw, use forceps to gently pull up the trachea, use scissors to cut the lung tissue connection until the entire lung tissue is removed from the body, fill the lungs with 4% paraformaldehyde fixative, tie them with thread, and place them in paraformaldehyde for further fixation for 48 hours. Dehydrate, paraffin embed, and slice.

[0088] (2) HE staining of lung tissue: Dewax the paraffin sections, then rehydrate them in a gradient alcohol solution, stain them with hematoxylin for 2 minutes, and differentiate them in an acidic ethanol solution for 15 seconds. Stain them with eosin stain for 2 minutes, then rinse them under tap water. Dehydrate them quickly with an ethanol solution, make them transparent in xylene, add neutral gum, and seal the sections, then bake them in a 56°C oven for 24 hours.

[0089] (3) Masson trichrome staining: Dewax the paraffin sections, then rehydrate them in a gradient alcohol solution, stain them with Weiger iron hematoxylin for 7 minutes, differentiate them in an acidic ethanol differentiation solution, and reverse blue them with a bluing solution. Stain them in a Ponceau red staining solution for 7 minutes, differentiate them in 1% phosphomolybdic acid, stain them in aniline blue solution for 2 minutes, and wash them in a 1% glacial acetic acid solution. After rapid dehydration with an ethanol solution, make them transparent in xylene, add a drop of neutral gum, and seal the sections. Bake them in a 56°C oven for 24 hours.

[0090] (4) Immunohistochemical staining: Dewax the paraffin sections and then hydrate them in a gradient alcohol solution. Place the sections in a 3% H2O2 solution to remove endogenous peroxidase, then prepare a citric acid-sodium citrate buffer solution, place the slides in it and boil them in a microwave oven to repair the antigen. After cooling naturally, block with 50% goat serum, add the primary antibody (CollagenI, FN1, α-SMA) and incubate at 4°C overnight. The next day, recover the primary antibody and add horseradish peroxidase, and use DAB reagent for color development. Take pictures under an upright microscope.

[0091] The results are as follows Fig.10 , Fig.11 As shown, Fig.10 HE and Masson effect diagram of lung tissue in mice with pulmonary fibrosis treated with extracellular vesicles derived from decidual mesenchymal stem cells. Fig.10 It can be seen that extracellular vesicles derived from decidual mesenchymal stem cells alleviate the destruction of lung tissue structure and collagen deposition in mice with pulmonary fibrosis. Fig.11This is the immunohistochemistry of collagenI, fibronectin and α-SMA in lung tissue of mice with pulmonary fibrosis treated with extracellular vesicles derived from decidual mesenchymal stem cells. Fig.11 It can be seen that extracellular vesicles derived from decidual mesenchymal stem cells reduce the degree of fibrosis in lung tissue of mice with pulmonary fibrosis.

[0092] The above-described embodiments are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of extracellular vesicles derived from decidual mesenchymal stem cells in the preparation of a drug for treating pulmonary fibrosis, characterized in that: The decidual mesenchymal stem cell-derived extracellular vesicles are prepared by the following steps: S1. Obtain decidual tissue from patients undergoing curettage and curettage for early pregnancy abortion in the outpatient clinic, wash, digest with enzymes, and culture under hypoxic conditions to obtain decidual mesenchymal stem cells I; S2. Transfecting the plasmid into the decidual mesenchymal stem cells I obtained in step S1 to obtain decidual mesenchymal stem cells II; S3. Using a dispersed polymer and a supplement medium to culture the decidual mesenchymal stem cells II obtained in step S2, and obtaining extracellular vesicles derived from decidual mesenchymal stem cells after centrifugation and sucrose density gradient purification; S4. using the decidual mesenchymal stem cell-derived extracellular vesicles obtained in step S3 to prepare a pulmonary fibrosis drug; The dispersed polymer is prepared from calcium chloride, fructose, and 4-hydroxyethylpiperazineethanesulfonic acid, and the specific preparation steps are as follows: M1-1. Weigh calcium chloride, fructose and 4-hydroxyethylpiperazineethanesulfonic acid, mix and stir to obtain a mixture 1, and add water to sodium alginate to prepare a sodium alginate solution; M1-2. The sodium alginate solution is added to the mixed solution 1 by a peristaltic pump, the rotation speed of the peristaltic pump is 60-90rpm, and after the end, a dispersed polymer is visible in the mixed solution 1. The dispersed polymer can be obtained by filtration, and the dispersed polymer is washed with PBS buffer, stored in PBS buffer, and stored at 4°C for use; In step M1-1, the mass ratio of calcium chloride: fructose: 4-hydroxyethylpiperazineethanesulfonic acid is 13-15: 1-1.2: 2.5-3.0, and the mass concentration of the sodium alginate solution is 5-8%; Before the sodium alginate solution is mixed with the mixed solution 1 in step M1-2, the mixed solution 1 is precooled by adding ice cubes to a beaker or storing at 4°C. The volume ratio of the sodium alginate solution to the mixed solution 1 is 1:5-8. The supplement culture medium is prepared by adding human blood lysate and sodium glycerophosphate to DMEM culture medium, wherein the amount of human blood lysate added is 5-8% by mass, and the amount of sodium glycerophosphate added is 0.8-1.3% by mass; The mass ratio of the dispersed polymer to the supplement culture medium is 1:10, or 1:12, or 1:15; The plasmid in step S2 is pEGFP-N2-GFE2-Lamp2b, the amino acid sequence of GFE2 is shown in SEQ ID NO.1, and the corresponding mRNA sequence is shown in SEQ ID NO.

2.

2. The use of the extracellular vesicles derived from decidual mesenchymal stem cells according to claim 1 in the preparation of a drug for treating pulmonary fibrosis, characterized in that: The medium used for sucrose density gradient purification in step S3 is obtained by mixing ultrapure water, sucrose and tris(hydroxymethyl)aminomethane in a mass ratio of 10-14:2.5-2.8:0.2-0.3 and stirring them evenly.

3. The use of the extracellular vesicles derived from decidual mesenchymal stem cells according to claim 2 in the preparation of a drug for treating pulmonary fibrosis, characterized in that: The extracellular vesicles derived from decidual mesenchymal stem cells in step S4 can be directly used to prepare drugs for treating pulmonary fibrosis, or can be used as a carrier in combination with other substances to prepare drugs for treating pulmonary fibrosis.

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