Preparation method and application of human umbilical cord mesenchymal stem cells and exosomes

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a preparation method and application of human umbilical cord mesenchymal stem cells and exosomes. A systemic sclerosis model mouse is induced by bleomycin, the systemic sclerosis model mouse is subjected to intervention treatment by the prepared human umbilical cord mesenchymal stem cell and the exosome thereof, MSC and MSC-exo can significantly inhibit infiltration of inflammatory cells in SSc mouse lungs and skin tissues, and inhibit expression of fibrosis indexes HYP and alpha-SMA, so that the human umbilical cord mesenchymal stem cell and the exosome thereof can be used for treating the systemic sclerosis. The content of CD4 + CD25 + FoxP3 + T regulatory T lymphocytes (Treg) infiltrated by the spleen of the SSc mouse is obviously increased, and the differentiation of CD11b + F4 / 80 + total macrophages infiltrated by the spleen of the SSc model mouse and the proportion of the CD11c + F4 / 80 + M1 type macrophages to the CD163 + F4 / 80 + M2 type macrophages are regulated.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a preparation method and application of human umbilical cord mesenchymal stem cells and exosomes. Background Art

[0002] Systemic sclerosis (SSc) is a refractory autoimmune disease with unclear etiology and pathogenesis. Progressive skin and multi-organ fibrosis is its most prominent feature.

[0003] Epidemiological studies have shown that the incidence of SSc is only 0.6 / 1 million to 122 / 1 million, which is a rare disease. The disease is distributed worldwide and is more common in people aged 30 to 50 years old. The incidence rate in women is higher than that in men, with a male-to-female ratio of about 1: (3 to 14). The mortality and disability rates are high, and the prognosis is poor. There is currently no effective cure. Therefore, the development of effective treatment strategies is a difficult problem that needs to be solved urgently in this research field. Summary of the invention

[0004] The purpose of the present invention is to provide a preparation method and application of human umbilical cord mesenchymal stem cells and exosomes to solve the problems raised in the above background technology.

[0005] In order to achieve the above technical objectives, the technical solution of the present invention is: A method for preparing human umbilical cord mesenchymal stem cells and exosomes thereof for preparing a drug for treating systemic sclerosis, wherein the method comprises the following steps: S11. Obtain the umbilical cord under sterile conditions, clean the arterial and venous blood in the umbilical cord and soak it in sterile saline, use sterile scissors to cut the umbilical cord into 3-4 cm small segments, use a scalpel to peel off the outer membrane and scrape out Wharton's jelly; S12, cut Wharton's Jelly into 1~3mm tissue fragments, resuspend in 15mL F12 / DMEM medium, and add T-75cm 2 In the culture bottle, ensure that the cells are evenly distributed and occupy 40% to 50% of the bottom area of ​​the culture bottle. When the cell confluence reaches 80% to 90% of the bottom area of ​​the culture bottle, the cells can be subcultured. The F12 / DMEM medium contains 10% FBS and 1% double antibody; S13, discard the cell supernatant in the culture flask, wash with 1×PBS, then add 1 mL of trypsin to digest for 3 minutes, then add 1 mL of F12 / DMEM complete medium to terminate the digestion, collect the cells and place them in a 15 mL centrifuge tube, centrifuge at 12 rpm for 5 minutes, and after the centrifugation, collect the cell precipitate to obtain human umbilical cord mesenchymal stem cells; The human umbilical cord mesenchymal stem cell exosomes are prepared by ultra-high-speed centrifugation of human umbilical cord mesenchymal stem cells.

[0006] As an improvement, the mesenchymal stem cells and exosomes thereof are used in the preparation of a drug for treating collagen deposition in skin tissue.

[0007] As a further improvement, the mesenchymal stem cells and exosomes thereof are used in the preparation of drugs for regulating the expression of chitinase 3-like protein 1 and α-smooth muscle actin in skin tissue.

[0008] As a further improvement, the use of the mesenchymal stem cells and their exosomes in the preparation of a drug for regulating the proportion of spleen-infiltrating regulatory T cells, wherein the regulatory T cells are CD4 + CD25 + FoxP3 + Treg cells.

[0009] As a further improvement, the mesenchymal stem cells and their exosomes are used to prepare CD11b + F4 / 80 + Application of macrophage differentiation drugs.

[0010] As a further improvement, the mesenchymal stem cells and their exosomes are used in the preparation of drugs for regulating hydroxyproline in skin tissue.

[0011] As a further improvement, the method for preparing human umbilical cord mesenchymal stem cell exosomes comprises the following specific steps: S21, taking out the cell cryopreservation tube of human umbilical cord mesenchymal stem cells from the liquid nitrogen tank, bathing in a constant temperature water bath at 37°C for 3 minutes, transferring to a 15 mL centrifuge tube, washing with 1×PBS, centrifuging at 1200 rpm for 5 minutes, and discarding the supernatant to obtain the mesenchymal stem cells; S22. The mesenchymal stem cells were resuspended in 1 mL of Baso serum-free cell culture medium and added to a T-75 culture flask. After three days of culture, the supernatant was collected and exosomes were obtained by ultrahigh-speed centrifugation.

[0012] As a further improvement, the specific steps of the ultracentrifugation method are as follows: at 4°C, the supernatant is ultracentrifuged at 10,000g for 30 minutes, then ultracentrifuged at 120,000g for 70 minutes, the supernatant is discarded, and then resuspended with 1×PBS and ultracentrifuged at 120,000g for 70 minutes to obtain human umbilical cord mesenchymal stem cell exosomes.

[0013] As a further improvement, the Baso serum-free cell culture medium contains 5% UltraGRO TM -Advanced and 1% double resistance.

[0014] Due to the adoption of the above technical solution, the beneficial effects of the present invention are: The present invention provides an application of human umbilical cord mesenchymal stem cells and exosomes thereof in the preparation of a drug for treating systemic sclerosis. Systemic sclerosis model mice are induced by bleomycin, and the prepared human umbilical cord mesenchymal stem cells and exosomes thereof are used for intervention treatment of the systemic sclerosis model mice. MSC and MSC-exo can significantly inhibit the infiltration of inflammatory cells in the lung and skin tissues of SSc mice, and inhibit the expression of fibrosis indicators HYP and α-SMA.

[0015] Compared with SSc mice, the CD4 + CD25 + FoxP3 + Tregs were significantly increased and regulated the CD11b infiltration in the spleen of SSc model mice + F4 / 80 + Differentiation of total macrophages and CD11c + F4 / 80 + M1 macrophages and CD163 + F4 / 80 + The proportion of M2 macrophages significantly improved inflammation and fibrosis.

[0016] Intervention with MSC and MSC-exo can significantly reduce the expression of CHI3L1 in the skin and lungs and alleviate the degree of fibrosis. MSC and MSC-exo have good therapeutic effects on systemic sclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : is a diagram of the identification results of human umbilical cord mesenchymal stem cells in Example 1, wherein A is a diagram of umbilical cord tissue samples, B is a diagram of two-step enzyme digestion method culture samples, C is a diagram of tissue block climbing method in vitro culture samples, D is a diagram of flow cytometry detection results, and E is a diagram of in vitro induction results of human umbilical cord mesenchymal stem cells; Figure 2 : are the results of the identification experiment of human umbilical cord mesenchymal stem cell exosomes in Example 2, wherein A is a diagram of the morphological structure of exosomes observed by transmission electron microscopy, B is a diagram of the expression of exosome surface marker proteins CD9, TSG101, and GAPDH detected by Western blot, and C is a diagram of the particle size analysis of exosomes by DLS; Figure 33 is a graph showing the experimental results of inflammation and fibrosis in the skin and lungs of SSc mice in Example 3, wherein A is a flowchart showing the construction of an SSc mouse model and the intervention with MSC and MSC-exo, B is a graph showing the results of the HYP content in mouse skin tissue, C is a graph showing the results of the thickness of the dermis skin of mice, D is a graph showing the results of HE staining of mouse skin and lung tissue, E is a graph showing the results of Masson staining of mouse skin and lung tissue, F is a graph showing the expression of CHI3L1 in mouse skin and lung tissue detected by IHC, and G is a graph showing the expression of α-SMA in mouse skin and lung tissue; Figure 4 The CD4 infiltrates in the spleen of mice in Example 3 + CD25 + FoxP3 + Treg cell ratio result diagram; Figure 5 The mouse spleen infiltrating CD11b in Example 3 + F4 / 80 + Total macrophage results graph; Figure 6 The mouse spleen infiltrating CD11c in Example 3 + F4 / 80 + M1 macrophage ratio results; Figure 7 The mouse spleen infiltrating CD163 in Example 3 + F4 / 80 + Results of the proportion of M2 macrophages. DETAILED DESCRIPTION

[0018] The technical scheme of the present invention will be clearly and completely described below in conjunction with specific embodiments, but it will be appreciated by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work, all belong to the scope of protection of the present invention. If specific conditions are not indicated in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be purchased commercially. Example 1

[0019] This embodiment provides a method for preparing human umbilical cord mesenchymal stem cells and identifying the obtained human umbilical cord mesenchymal stem cells. The specific steps are as follows: S1. Obtain the umbilical cord under sterile conditions, clean the arterial and venous blood in the umbilical cord and soak it in sterile saline. Use sterile scissors to cut the umbilical cord into small sections of about 3 to 4 cm, peel off the outer membrane with a scalpel and scrape out Wharton's jelly; S2. Cut Wharton's gel into 1-3 mm tissue fragments, resuspend in 15 mL F12 / DMEM medium (10% FBS, 1% double antibody), and add T-75 cm 2 In the culture flask, ensure that they are evenly distributed and occupy about 40% to 50% of the bottom area. When the cell confluence reaches 80% to 90% of the bottom area of ​​the culture flask, the cells can be subcultured; S3, discard the cell supernatant in the culture flask, wash with 1×PBS, then add 1mL trypsin to digest for 3min, then add 1mL F12 / DMEM complete medium to terminate the digestion, collect the cells and place them in a 15mL centrifuge tube, centrifuge at 12rpm for 5min, after the centrifugation, collect the cell precipitate to obtain human umbilical cord mesenchymal stem cells, and freeze them in liquid nitrogen; Resuspend the obtained human umbilical cord mesenchymal stem cells in 1×PBS, take 200 μL into a flow tube, and detect the expression of CD44, CD73, CD80, CD105, CD45, and CD34 by flow cytometry. According to the instructions of the Pronocell (PD-017 / 018 / 019) human umbilical cord mesenchymal stem cell osteogenic, chondrogenic, and adipogenic induction medium, the induction of osteogenic, chondrogenic, and adipogenic was carried out respectively.

[0020] The results of this example are as follows Figure 1 As shown, Figure A is a picture of the umbilical cord tissue sample, Figure B is a picture of the two-step enzyme digestion method culture sample, Figure C is a picture of the tissue block climbing method in vitro culture sample, Figure D is a picture of the flow cytometry test results, and Figure E is a picture of the in vitro induction results of human umbilical cord mesenchymal stem cells. Example 2

[0021] This embodiment provides a method for preparing human umbilical cord mesenchymal stem cell exosomes, and identifies the obtained human umbilical cord mesenchymal stem cell exosomes, and the specific steps are as follows: S1. Take out the cell cryopreservation tube of human umbilical cord mesenchymal stem cells from the liquid nitrogen tank, place it in a constant temperature water bath at 37°C for 3 minutes, transfer it to a 15 mL centrifuge tube, fill it up with 1×PBS for washing, centrifuge it at 1200 rpm for 5 minutes, and discard the supernatant to obtain the mesenchymal stem cells; S2, mesenchymal stem cells were cultured with 1 mL of Baso (BC-T4) serum-free cell culture medium (5% UltraGRO TM-Advanced, 1% double antibody) was resuspended and added to a T-75 culture flask. After three days of culture, the supernatant was collected and exosomes were obtained by ultracentrifugation. The specific steps of the ultracentrifugation method were as follows: at 4°C, the supernatant was ultracentrifuged at 10000g for 30min, then at 120000g for 70min, the supernatant was discarded, and then resuspended with 1×PBS and ultracentrifuged at 120000g for 70min to obtain human umbilical cord mesenchymal stem cell exosomes.

[0022] The morphological structure of exosomes was observed by transmission electron microscopy (scale = 200 nm), the expression of exosome surface marker proteins CD9 and TSG101 was detected by Western blot, and the particle size of exosomes was analyzed by DLS.

[0023] The experimental results of this embodiment are as follows Figure 2 As shown, Figure A is a diagram of the morphological structure of exosomes observed by transmission electron microscopy, Figure B is a diagram of the expression of exosome surface marker proteins CD9, TSG101, and GAPDH detected by Western blot, and Figure C is a diagram of the particle size analysis of exosomes by DLS. Example 3

[0024] This example provides the use of human umbilical cord mesenchymal stem cells and exosomes derived therefrom in the preparation of a drug for treating scleroderma. The specific experimental method is as follows.

[0025] Sixteen wild-type B6-WT (B6 wild type) female mice aged 6 to 8 weeks were selected to induce scleroderma mouse model through bleomycin. Before modeling, the hair of the same area of ​​about 1.5 cm × 1.5 cm on the back of each group of mice was removed and they were randomly divided into 4 groups, 4 mice in each group, namely blank control group, BLM modeling untreated SSC group, BLM modeling MSC treatment group, BLM modeling MSC-exo treatment group. The four groups of mice were housed in an SPF environment.

[0026] Then, the mice were injected with bleomycin (BLM, 500ug / mL) every day, with a daily injection volume of 100μL for 4 consecutive weeks to construct the SSc mouse model. After the model was successfully constructed, the MSC treatment group and the MSC-exo treatment group were treated with MSC and MSC-exo, respectively. The mice in the MSC treatment group were injected with the mesenchymal stem cells prepared in Example 1 through the tail vein at an injection volume of 5×10 5 / mouse, and the mice in the MSC-exo treatment group were injected with the mesenchymal stem cell exosomes prepared in Example 2 via the tail vein, with an injection volume of 200 μg / mouse. After 8 weeks, they were killed and samples were collected for subsequent experiments.

[0027] After dissecting the mice, 100 mg of mouse skin tissue was taken, washed with PBS to remove blood stains, cut into small pieces and placed in a homogenizer, 1 mL of PBS was added to make a homogenate, and then placed in a -20°C refrigerator overnight. The next day, the cell membrane was destroyed by repeated freezing and thawing twice, and the supernatant was taken after centrifugation at 4°C and 5000g for 5 minutes. The hydroxyproline content of mouse skin was determined according to the instructions of the mouse hydroxyproline (Hyp) enzyme-linked immunosorbent assay kit (HypELISA Kit, USA), and finally the absorbance at 540nm~570nm was determined using an enzyme reader.

[0028] Skin, lung and other tissue samples were fixed with 4% paraformaldehyde, dewaxed with xylene, dehydrated with gradient ethanol, embedded in paraffin and sliced, and then HE staining and Masson staining were performed on 4 μm thick sections using HE staining kit and Masson trichrome staining kit.

[0029] The skin and lung tissue sections were stained with CHI3L1 antibody (CHI3L1, 1 / 400, Abcam, Britain) and α-SMA antibody (Cell Signaling Technology, 1 / 200, USA), respectively. The sections were then rinsed with tap water, dehydrated, made transparent, air-dried, and sealed. The local pathological changes of the skin were observed under a microscope. The dermis thickness of each group of mice was measured using Image J software, and the average value was calculated.

[0030] The spleen was placed in a grinder, 3 mL of PBS was added and ground thoroughly on ice, then transferred to a 15 mL centrifuge tube, centrifuged at 350 g for 5 min, the supernatant was discarded, 7 mL of red blood cell lysis buffer was added to resuspend, and lysed on ice for 5 min, mixed 2 to 3 times during the period, PBS was added to terminate the lysis, centrifuged at 350 g for 5 min, the supernatant was discarded, the cells were washed with PBS, the total number of spleen cells was calculated, and the cell concentration was adjusted to 1 × 10 with flow cytometry staining buffer. 7 / mL and perform immune cell staining. Add 100 μL of cell suspension and 1 μL of blocking antibody (Rat Anti-Mouse CD16 / CD32) to each tube, incubate at 4°C for 30 min, then wash with PBS and add 1 μL of flow cytometry dye (eBioscienc TM Fixable Viability Dye eFluor TM 780), incubated at 4°C in the dark for 30 min, washed and resuspended after incubation, and added with corresponding antibodies to label the target cell population, among which FITC-CD4, CD25-V450, PE-Foxp3 labeled regulatory T cells (Regulatory cells, Treg), CD11b + F4 / 80 +Markers: total macrophages, CD11c + F4 / 80 + M1 macrophages and CD163 + F4 / 80 + M2 macrophages were incubated in the dark at 4°C for 40 min, then washed twice with staining buffer, resuspended in 400 μL staining buffer, filtered through a 300-mesh filter, and tested on an analyzer.

[0031] The experimental results of this embodiment are as follows Figure 3-Figure 7 shown.

[0032] Figure 3 Figure A is a flowchart of constructing the SSc mouse model and intervening with MSC and MSC-exo, Figure B is a graph showing the results of HYP content in mouse skin tissue, Figure C is a graph showing the results of mouse dermal skin thickness, Figure D is a graph showing the results of HE staining of mouse skin and lung tissue, Figure E is a graph showing the results of Masson staining of mouse skin and lung tissue, Figure F is a graph showing the expression of CHI3L1 in mouse skin and lung tissue detected by IHC, and Figure G is a graph showing the expression of α-SMA in mouse skin and lung tissue.

[0033] Figure 4 CD4 infiltrating in mouse spleen + CD25 + FoxP3 + Treg cell ratio result chart, Figure 5 Mouse spleen infiltrating CD11b + F4 / 80 + Total macrophage results chart, Figure 6 Mouse spleen infiltrating CD11c + F4 / 80 + M1 macrophage ratio results, Figure 7 CD163 infiltrates in mouse spleen + F4 / 80 + Results of the proportion of M2 macrophages.

[0034] Depend on Figure 3 It can be seen that MSC and MSC-exo can significantly inhibit the infiltration of inflammatory cells in the lung and skin tissues of SSc, and inhibit the expression of fibrosis indicators HYP and α-SMA.

[0035] Depend on Figure 4-Figure 7 It can be seen that MSC and MSC-exo intervention can upregulate the CD4 + CD25 + Foxp3 + The proportion of T regulatory T lymphocytes (Treg) regulates the infiltrating CD11b in mouse spleen +F4 / 80 + Total macrophage differentiation, regulation of CD11c + F4 / 80 + M1 macrophages and CD163 + F4 / 80 + The balance of M2 macrophages significantly reduced inflammatory cell infiltration and fibrosis in the skin and lungs of SSc model mice.

[0036] The specific implementation of the present invention described above does not constitute a limitation on the protection scope of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A use of human umbilical cord mesenchymal stem cells and exosomes thereof in the preparation of a drug for treating systemic sclerosis, characterized in that: The method for preparing human umbilical cord mesenchymal stem cells comprises the following steps: S11. Obtain the umbilical cord under sterile conditions, clean the arterial and venous blood in the umbilical cord and soak it in sterile saline, use sterile scissors to cut the umbilical cord into 3-4 cm small segments, use a scalpel to peel off the outer membrane and scrape out Wharton's jelly; S12, cut Wharton's Jelly into 1~3mm tissue fragments, resuspend in 15mL F12 / DMEM medium, and add T-75cm 2 In the culture bottle, ensure that the cells are evenly distributed and occupy 40% to 50% of the bottom area of ​​the culture bottle. When the cell confluence reaches 80% to 90% of the bottom area of ​​the culture bottle, the cells can be subcultured. The F12 / DMEM medium contains 10% FBS and 1% double antibody; S13, discard the cell supernatant in the culture flask, wash with 1×PBS, then add 1 mL of trypsin to digest for 3 minutes, then add 1 mL of F12 / DMEM complete medium to terminate the digestion, collect the cells and place them in a 15 mL centrifuge tube, centrifuge at 12 rpm for 5 minutes, and after the centrifugation, collect the cell precipitate to obtain human umbilical cord mesenchymal stem cells; The human umbilical cord mesenchymal stem cell exosomes are prepared by ultra-high-speed centrifugation of human umbilical cord mesenchymal stem cells.

2. The use of human umbilical cord mesenchymal stem cells and exosomes thereof in the preparation of a drug for treating systemic sclerosis according to claim 1, characterized in that: The use of the mesenchymal stem cells and the exosomes thereof in preparing a drug for treating collagen deposition in skin tissue.

3. The use of human umbilical cord mesenchymal stem cells and exosomes thereof in the preparation of a drug for treating systemic sclerosis according to claim 1, characterized in that: The mesenchymal stem cells and exosomes thereof are used in preparing a drug for regulating the expression of chitinase 3-like protein 1 and α-smooth muscle actin in skin tissue.

4. The use of human umbilical cord mesenchymal stem cells and exosomes thereof in the preparation of a drug for treating systemic sclerosis according to claim 1, characterized in that: The use of the mesenchymal stem cells and the exosomes thereof in the preparation of a drug for regulating the proportion of spleen-infiltrating regulatory T cells, wherein the regulatory T cells are CD4 + CD25 + FoxP3 + Treg cells.

5. The use of human umbilical cord mesenchymal stem cells and exosomes thereof in the preparation of a drug for treating systemic sclerosis according to claim 1, characterized in that: Mesenchymal stem cells and exosomes thereof are used in the preparation of CD11b + F4 / 80 + Application of macrophage differentiation drugs.

6. The use of human umbilical cord mesenchymal stem cells and exosomes thereof in the preparation of a drug for treating systemic sclerosis according to claim 1, characterized in that: The mesenchymal stem cells and exosomes thereof are used in preparing a drug for regulating hydroxyproline in skin tissue.

7. The use of human umbilical cord mesenchymal stem cells and exosomes thereof in the preparation of a drug for treating systemic sclerosis according to claim 1, characterized in that: The specific steps of the method for preparing human umbilical cord mesenchymal stem cell exosomes are as follows: S21, taking out the cell cryopreservation tube of human umbilical cord mesenchymal stem cells from the liquid nitrogen tank, bathing in a constant temperature water bath at 37°C for 3 minutes, transferring to a 15 mL centrifuge tube, washing with 1×PBS, centrifuging at 1200 rpm for 5 minutes, and discarding the supernatant to obtain the mesenchymal stem cells; S22. The mesenchymal stem cells were resuspended in 1 mL of Baso serum-free cell culture medium and added to a T-75 culture flask. After three days of culture, the supernatant was collected and exosomes were obtained by ultrahigh-speed centrifugation.

8. The use of human umbilical cord mesenchymal stem cells and exosomes thereof in the preparation of a drug for treating systemic sclerosis according to claim 7, characterized in that: The specific steps of the ultracentrifugation method are as follows: at 4°C, the supernatant is ultracentrifuged at a speed of 10,000 g for 30 min, then ultracentrifuged at a speed of 120,000 g for 70 min, the supernatant is discarded, and then resuspended with 1×PBS and ultracentrifuged at a speed of 120,000 g for 70 min to obtain human umbilical cord mesenchymal stem cell exosomes.

9. The use of human umbilical cord mesenchymal stem cells and exosomes thereof in the preparation of a drug for treating systemic sclerosis according to claim 7, characterized in that: The Baso serum-free cell culture medium contains 5% UltraGRO TM -Advanced and 1% double resistance.

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