Screening method of anti-fibrosis mesenchymal stem cells as well as exosome and application of anti-fibrosis mesenchymal stem cells

Abstract

The invention discloses a screening method of anti-fibrosis mesenchymal stem cells as well as derivatives and application of the anti-fibrosis mesenchymal stem cells, and relates to the technical field of biomedicine.The screening method comprises the steps that an MMP1 gene in the mesenchymal stem cells serves as an index for screening the anti-fibrosis capacity of the mesenchymal stem cells; and the P3 generation human umbilical cord mesenchymal stem cell strain MMP1 gene expression (FPMK value = 30) is used as the quantitative standard of the anti-fibrosis capability. The anti-fibrosis mesenchymal stem cells and the exosome thereof are used for treating tissue fibrosis diseases. The invention provides a construction method for screening a high-quality umbilical cord mesenchymal stem cell strain aiming at stem cell treatment of tissue fibrosis, establishes a pre-clinical effectiveness evaluation system of mesenchymal stem cells, and provides a cell selection standard for basic research and clinical transformation of tissue fibrosis treatment by mesenchymal stem cells, so that the treatment effect is improved, and the clinical application prospect is wide. The clinical application of the mesenchymal stem cells is quickly promoted.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a screening method for anti-fibrosis mesenchymal stem cells, exosomes thereof, and applications thereof. Background Art

[0002] Fibrosis is the result of disordered tissue damage and repair during chronic inflammation. Its further development will lead to decreased organ function, even failure, and even death, which seriously threatens human health and life. Fibrosis can occur in any part of the body, such as the liver, lungs, kidneys, heart, blood vessels, eyes, pancreas, skin, bones, etc. Among various types of fibrosis, the morbidity and mortality of liver, lung and kidney fibrosis are particularly high, which seriously threatens human health. The pathological microenvironment of tissue fibrosis is very complex. When tissue fibrosis occurs, the damaged part heals excessively, resulting in a large amount of extracellular matrix (ECM) deposition, in which collagen is covalently cross-linked, making the fibrotic matrix more stable and difficult to degrade and penetrate; at the same time, fibroblasts and macrophages are activated, releasing a variety of inflammatory factors, aggravating the inflammatory response.

[0003] Existing anti-fibrosis research usually focuses on promoting the proliferation of tissue cells or inhibiting the activation of fibroblasts, while in anti-fibrosis treatment, it is necessary to consider the obstruction of tissue repair by deposited ECM. Cells with the ability to degrade ECM have become an important emerging therapeutic tool for depositing ECM. After reaching the fibrotic organ, the cell continuously releases proteases to degrade ECM, promote the recovery of fibrosis, and promote the regeneration and repair of damaged tissues. There are three main types of enzymes in the body that can degrade ECM: matrix metalloproteinase (MMP), plasmin and cathepsin, among which MMP plays a major role. MMP can degrade multiple ECM components such as collagen, elastin, laminin, and fibronectin.

[0004] Mesenchymal stem cells (MSCs) are a type of adult stem cell derived from the early mesoderm of development, with self-replication and multidirectional differentiation potential, and are widely found in tissues such as bone marrow, fat, umbilical cord, placenta, and dental pulp. A large number of studies have shown that MSCs can exert anti-fibrotic effects through paracrine cytokines.

[0005] So far, there are more than 1,500 clinical studies on MSCs for the treatment of related diseases registered in the US Clinical Trials Database (www.clincaltrial.gov), but the treatment effects are uneven. We analyzed these clinical studies and found that the tissue sources (such as umbilical cord, placenta, dental pulp, fat, amniotic membrane, etc.), donor sources (different gender, age and genetic background, etc.), and cell generations (such as P1, P3, P5 and P10, etc.) of the mesenchymal stem cells used in these clinical studies are different. Based on this, we studied human umbilical cord mesenchymal stem cells (hUC-MSCs) from different donor sources and found that although hUC-MSCs from different donor sources have similar cell morphology and cell surface markers, they have large functional differences in cell viability, tri-lineage differentiation potential, paracrine cytokines and immunoregulatory ability. It is precisely because of this functional difference in mesenchymal stem cells that their clinical treatment effects vary greatly, which ultimately hinders their widespread clinical application.

[0006] Therefore, for tissue fibrosis diseases, based on the molecular mechanism of ECM degradation, biomarkers of hUC-MSCs with high therapeutic value are established, and high-quality exosomes are obtained, thereby improving the therapeutic effect and solving the important problems that need to be solved in this field. In addition, most of the therapeutic effects of mesenchymal stem cells depend on their paracrine effects. The discovery that exosomes can replace their parent cells provides researchers with important research ideas. Exosomes have great advantages: they do not replicate themselves, which largely avoids the risk of tumorigenesis; compared with cell therapy, exosomes are safer, and as nanoparticles, they are also biocompatible and low immunogenic, allowing them to cross protective biological barriers such as the blood-brain barrier. Therefore, they have broad and promising therapeutic applications. Summary of the invention

[0007] In order to solve the above technical problems, the present invention designs a method for screening anti-fibrosis mesenchymal stem cells.

[0008] In order to achieve the above technical effects, the present invention is implemented by the following technical scheme: A method for screening anti-fibrotic mesenchymal stem cells is characterized in that it comprises the following steps:

[0009] S1. Screen several umbilical cord tissues from healthy donors, conduct sterility, mycoplasma and virus tests, and if qualified, separate primary human umbilical cord mesenchymal stem cells by tissue block suspension method, and subculture them to P3 generation in serum-free medium;

[0010] S2. Testing the P3 generation human umbilical cord mesenchymal stem cells. After passing the test, they are placed in a temporary master cell bank;

[0011] S3. Collect an appropriate amount of P3 human umbilical cord mesenchymal stem cells, add 1 mL of Trizol, gently pipette 3 times, and mix well; extract RNA, and perform transcriptome sequencing after passing quality control;

[0012] S4. Analyze the sequencing data using bioinformatics technology to obtain different gene expression matrices of human umbilical cord mesenchymal stem cells; calculate the expression level of the MMP1 gene of all human umbilical cord mesenchymal stem cell lines based on the gene expression matrix;

[0013] S5. Evaluate the anti-fibrotic ability of hUC-MSCs from different donors in S4;

[0014] S6, correlation analysis was performed between the MMP1 gene expression levels in hUC-MSCs from different donors in S4 and the corresponding anti-fibrosis ability in S5;

[0015] S7. The expression level of MMP1 gene in hUC-MSCs is used as a screening index for its anti-fibrosis ability, i.e., a biomarker for the anti-fibrosis ability of human umbilical cord mesenchymal stem cells;

[0016] S8. Anti-fibrosis mesenchymal stem cells can be obtained by screening according to the above indicators;

[0017] Furthermore, in S1, the sources of the mesenchymal stem cells include but are not limited to various tissues (such as umbilical cord, placenta, amnion, dental pulp, fat, etc.) and mesenchymal stem cells derived from induced pluripotent stem cells.

[0018] Furthermore, in S2, the detection is one or more of sterility detection, mycoplasma, endotoxin, virus, morphology, surface marker, differentiation ability, cytokine detection, telomere activity, STR, and soft agar cloning.

[0019] Furthermore, in S7, the MMP1 gene in hUC-MSCs was used as a screening index for its anti-fibrosis ability: the FPMK value of MMP1 gene expression in the P3 generation human umbilical cord mesenchymal stem cell line = 30 was used as a quantitative standard for the anti-fibrosis ability evaluation index MMP1 of human umbilical cord mesenchymal stem cells.

[0020] Another object of the present invention is to provide an anti-fibrotic mesenchymal stem cell derived exosome, characterized in that it comprises the following steps:

[0021] The mesenchymal stem cell lines with strong anti-fibrosis ability obtained by the above screening are further subjected to three-dimensional large-scale culture in vitro to obtain a large amount of culture supernatant, and the culture supernatant is concentrated and ultracentrifuged to obtain high-purity mesenchymal stem cell-derived exosomes with strong anti-fibrosis ability.

[0022] Another object of the present invention is to provide an application of anti-fibrosis mesenchymal stem cells, characterized in that the anti-fibrosis mesenchymal stem cells and exosomes derived therefrom are used in the treatment of fibrotic diseases; the fibrotic diseases include but are not limited to liver fibrosis, pulmonary fibrosis and renal fibrosis.

[0023] The beneficial effects of the present invention are:

[0024] The present invention provides a stem cell therapy for liver fibrosis, establishes a screening method and evaluation index for high-quality mesenchymal stem cell lines, so as to solve the problem of mesenchymal stem cell heterogeneity in the prior art, and provides cell selection standards for basic research and clinical transformation of mesenchymal stem cell therapy for tissue fibrosis, so as to improve the treatment effect and rapidly promote the clinical application of mesenchymal stem cells.

[0025] At the same time, the present invention performs serum-free three-dimensional large-scale in vitro culture on the mesenchymal stem cells screened based on the above method to obtain a large amount of culture supernatant, and concentrates and ultracentrifuges the culture supernatant to obtain mesenchymal stem cell-derived exosomes with strong anti-fibrosis ability, which are verified by animal experiments to have good therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0027] Figure 1 This is a diagram showing the results of identifying human umbilical cord mesenchymal stem cells in Example 1 of the present invention;

[0028] Figure 2 This is a bar graph of the MMP1 gene expression levels of human umbilical cord mesenchymal stem cells from different donors in Example 1 of the present invention;

[0029] Figure 3 The anti-fibrosis ability of human umbilical cord mesenchymal stem cells from different donors in Example 1 of the present invention;

[0030] Figure 4 This is the correlation analysis between the expression level of MMP1 and the anti-fibrosis ability of human umbilical cord mesenchymal stem cells from different donors in Example 1 of the present invention;

[0031] Figure 5 is the MMP1 gene expression level of human umbilical cord mesenchymal stem cells from different donors in Example 2 of the present invention;

[0032] Figure 6 The rat liver function test results in Example 3 of the present invention;

[0033] Figure 7 The results of rat liver fibrosis level in Example 3 of the present invention;

[0034] Figure 8 This is the HE staining result of rat liver in Example 3 of the present invention;

[0035] Fig. 9 The Masson staining result of rat liver in Example 3 of the present invention;

[0036] Fig.10 This is the identification result of high-quality exosomes in Example 4 of the present invention;

[0037] Fig.11 The rat liver function test results in Example 4 of the present invention;

[0038] Fig.12 The results of rat liver fibrosis level in Example 4 of the present invention;

[0039] Fig.13 This is the HE staining result of rat liver in Example 4 of the present invention;

[0040] Fig.14 This is the Masson staining result of rat liver in Example 4 of the present invention. DETAILED DESCRIPTION

[0041] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] A method for screening anti-fibrosis mesenchymal stem cells comprises the following steps:

[0043] Step 1: Screen and isolate primary human umbilical cord mesenchymal stem cells from several human umbilical cords, and subculture them to P3 generation using serum-free medium.

[0044] The specific operations are:

[0045] (1) This study was approved by the Stem Cell Ethics Committee of Xi'an High-tech Hospital;

[0046] (2) Screen healthy pregnant women who meet the requirements for the establishment of the biobank for sample collection and obtain informed consent from the pregnant women;

[0047] (3) Under sterile conditions, the umbilical cord is collected and the length of the umbilical cord between the two ligature sites is not less than 20 cm. The umbilical cord is placed in a pre-prepared umbilical cord storage and transportation bottle and transported to the cell culture room through a cold chain for primary cell isolation of umbilical cord mesenchymal stem cells;

[0048] (4) Place the umbilical cord in a 15 cm culture dish with an appropriate amount of physiological saline, remove the ligatures at both ends, cut into 2-3 cm segments, and transfer to a 250 mL wide-mouth bottle with 75% alcohol for disinfection for 3 min;

[0049] (5) The sterilized umbilical cord was transferred to a 250 mL wide-mouth bottle with normal saline and washed 2-3 times to remove any residual blood on the surface;

[0050] (6) Transfer the umbilical cord to a 15 cm culture dish with saline solution, use forceps to peel the umbilical cord along the umbilical vein, remove one umbilical vein and two umbilical arteries, separate the Wharton's jelly into a 15 mL centrifuge tube with saline solution, wash the Wharton's jelly 2-3 times to remove residual blood, and cut it into 1 mm 3 Left and right sized tissue blocks;

[0051] (7) Inoculate the tissue block into a culture bottle that has been pre-filled with serum-free medium and culture it in a 37°C, 5% incubator;

[0052] (8) Performing the first half-volume medium change on the 7th day and the second half-volume medium change on the 10th day, harvesting primary human umbilical cord mesenchymal stem cells when the cell confluence reaches 60%-70%;

[0053] (9) After counting the primary human umbilical cord mesenchymal stem cells, the cells were counted at 9000 cells / cm 2 Inoculate in T175 culture flasks, place in a 37°C, 5% CO2 incubator and culture until the cells are 80-90% confluent, then digest and passage until P3 human umbilical cord mesenchymal stem cells are obtained;

[0054] (10) P3 generation human umbilical cord mesenchymal stem cells were subjected to relevant tests and entered into the cell bank after passing the tests, including but not limited to sterility testing, mycoplasma, endotoxin, virus, morphology, surface markers, trilineage differentiation ability, telomere activity, STR, soft agar cloning and immunomodulatory ability. The results are as follows Figure 1As shown in the figure, in terms of cell morphology, the hUC-MSCs we obtained were small in size, short spindle-shaped, and grew in a whirlpool shape, with good cell morphology. In terms of cell phenotype, the results of flow cytometry identification showed that the hUC-MSCs we obtained highly expressed CD44, CD73, CD90 and CD105, and did not express CD34, CD19, CD11b, CD45 and HLA-DR. In terms of differentiation ability, the results of three-lineage induction differentiation showed that after osteogenic induction and alizarin red staining, the hUC-MSCs we obtained had a large number of red-stained calcium nodules; after adipogenic induction and oil red O staining, red-stained lipid droplets appeared; after chondrogenic induction and Alcian blue staining, a large number of blue-stained proteoglycans synthesized by chondrocytes appeared. The above results all meet the ISCT identification standards for MSCs, indicating that the separation and culture of hUC-MSCs were successful.

[0055] Step 2: Use common transcriptome sequencing technology to quantitatively analyze the MMP1 (FPKM) gene expression level of human umbilical cord mesenchymal stem cells (P3 generation) from different donors;

[0056] The specific operations are:

[0057] (1) When the human umbilical cord mesenchymal stem cells are 80-90% confluent, remove the culture medium, add physiological saline to wash twice, add an appropriate amount of TrypLE, digest for 2 minutes, add the stop solution, and repeatedly blow the cells. Transfer an appropriate amount of the cell suspension to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes;

[0058] (2) Remove the supernatant, add 1 mL of Trizol, and mix by gently pipetting up and down three times;

[0059] (3) RNA was extracted and transcriptome sequencing was performed after passing quality control;

[0060] (4) Analyze the sequencing data using bioinformatics techniques to obtain different gene expression matrices;

[0061] (5) Based on the gene expression matrix, the expression level of MMP1 gene in human umbilical cord mesenchymal stem cells from different donors was calculated.

[0062] Step 3: According to the MMP1 gene expression level (FPKM value) in the sequencing results, the results are as follows Figure 2As shown, three human umbilical cord mesenchymal stem cell lines with low MMP1 expression (FPKM value <1) (UCMSCs34, UCMSCs66, UCMSCs70), three medium MMP1 expression (28>FPKM value <32) (UCMSCs05, UCMSCs25, UCMSCs41) and three high MMP1 expression (FPKM value >60) (UCMSCs03, UCMSCs14, UCMSCs20) were screened and their anti-fibrosis abilities were evaluated.

[0063] The specific operations are:

[0064] 1) In order to prepare the conditioned medium, the 9 hUC-MSCs selected above were cultured in a culture flask to a confluence of 50%, and then the medium was replaced with new medium and cultured for another 24 hours, and the culture supernatants were collected respectively.

[0065] 2) A549 cells were cultured in Ham's F-12K medium containing 10% fetal bovine serum to 70% confluence, and treated with 10 ng / mL TGF-β1 or 10 ng / mL TGF-β1+conditioned medium, and cultured for 24 h at 37°C and 5% CO2.

[0066] 3) Discard the supernatant, collect the A549 cells, add appropriate amount of Trizol, and gently pipette three times to mix;

[0067] 4) RNA was extracted and after passing quality control, the expression level of the fibrosis indicator Collagen I gene was detected by QPCR.

[0068] Step 4: Quantitative analysis of the anti-fibrosis ability of hUC-MSCs from different donors in step 4 was performed to detect the expression level of the fibrosis indicator Collagen I gene. Figure 3 As shown, hUC-MSCs from different donors have different anti-fibrotic abilities.

[0069] Step 5: Correlation analysis was performed between the expression of MMP1 gene in hUC-MSCs from different donors in step 2 and the expression of Collagen I in the corresponding groups in step 4. The results are as follows: Figure 4 As shown, we found that the expression of MMP1 gene in hUC-MSCs was inversely proportional to the expression of Collagen I, that is, the expression of MMP1 gene in hUC-MSCs was proportional to its anti-fibrosis effect.

[0070] Step 6: The expression level of MMP1 gene in hUC-MSCs is used as a screening index for its anti-fibrosis ability, that is, a biomarker of the anti-fibrosis ability of mesenchymal stem cells.

[0071] Example 2

[0072] This embodiment provides a method for constructing a quantitative standard for the anti-fibrosis ability of human umbilical cord mesenchymal stem cells, comprising the following steps:

[0073] Step 1: Isolate and culture primary mesenchymal stem cells in human umbilical cord Wharton's jelly, and subculture to P3 generation.

[0074] The specific operations are:

[0075] (1) This study was approved by the Stem Cell Ethics Committee of Xi'an High-tech Hospital;

[0076] (2) Screen healthy pregnant women who meet the requirements for the establishment of the biobank for sample collection and obtain informed consent from the pregnant women;

[0077] (3) Under sterile conditions, the umbilical cord is collected and the length of the umbilical cord between the two ligature sites is not less than 20 cm. The umbilical cord is placed in a pre-prepared umbilical cord storage and transportation bottle and transported to the cell culture room through a cold chain for primary cell isolation of umbilical cord mesenchymal stem cells;

[0078] (4) Place the umbilical cord in a 15 cm culture dish with an appropriate amount of physiological saline, remove the ligatures at both ends, cut into 2-3 cm segments, and transfer to a 250 mL wide-mouth bottle with 75% alcohol for disinfection for 3 min;

[0079] (5) The sterilized umbilical cord was transferred to a 250 mL wide-mouth bottle with normal saline and washed 2-3 times to remove any residual blood on the surface;

[0080] (6) Transfer the umbilical cord to a 15 cm culture dish with saline, use forceps to peel the umbilical cord along the umbilical vein, remove one umbilical vein and two umbilical arteries, separate the Wharton's jelly into a 15 mL centrifuge tube with saline, wash the Wharton's jelly 2-3 times to remove residual blood, and cut into tissue blocks of about 1 mm3 in size;

[0081] (7) Inoculate the tissue block into a culture bottle that has been pre-filled with serum-free medium and culture it in a 37°C, 5% incubator;

[0082] (8) Performing the first half-volume medium change on the 7th day and the second half-volume medium change on the 10th day, harvesting primary human umbilical cord mesenchymal stem cells when the cell confluence reaches 60%-70%;

[0083] (9) After counting the primary human umbilical cord mesenchymal stem cells, the cells were counted at 9000 cells / cm 2 The cells were inoculated into T175 culture flasks and cultured in a 37°C, 5% CO2 incubator until the cells reached 80-90% fusion, and then digested and passaged until P3 human umbilical cord mesenchymal stem cells were obtained;

[0084] (10) P3 human umbilical cord mesenchymal stem cells undergo relevant tests and enter the cell bank after passing the tests, including but not limited to sterility testing, mycoplasma, endotoxin, virus, morphology, surface markers, trilineage differentiation ability, telomere activity, STR, soft agar cloning and immune regulation ability.

[0085] Step 2: Use common transcriptome sequencing technology to quantitatively analyze the MMP1 gene expression level of human umbilical cord mesenchymal stem cells (P3 generation) from different donors.

[0086] The specific operations are:

[0087] (1) When the human umbilical cord mesenchymal stem cells are 80-90% confluent, remove the culture medium, add physiological saline to wash twice, add an appropriate amount of TrypLE, digest for 2 minutes, add the stop solution, and repeatedly blow the cells. Transfer an appropriate amount of the cell suspension to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes;

[0088] (2) Remove the supernatant, add 1 mL of Trizol, and mix by gently pipetting up and down three times;

[0089] (3) RNA was extracted and transcriptome sequencing was performed after passing quality control;

[0090] (4) Analyze the sequencing data using bioinformatics techniques to obtain different gene expression matrices;

[0091] (5) Based on the gene expression matrix, the expression level of MMP1 gene in all human umbilical cord mesenchymal stem cell lines was calculated.

[0092] Step 3: The gene expression level of MMP1 (FPMK value) of human umbilical cord mesenchymal stem cell (P3 generation) cell lines from different donors in step 2 ( Figure 5 ), according to the results in Example 1, the MMP1 gene FPMK value = 30 was used as a quantitative standard for the anti-fibrosis ability of human umbilical cord mesenchymal stem cells.

[0093] Example 3

[0094] This embodiment provides an application of anti-fibrotic human umbilical cord mesenchymal stem cells in a rat model of liver fibrosis.

[0095] 1. MSCs treatment of liver fibrosis rat model:

[0096] (1) Establishment of rat model of liver fibrosis.

[0097] 1) 6-8 weeks old healthy SD rats were randomly divided into a normal control group and a liver fibrosis model group, 15 rats in each group, a total of 45 rats, namely the control group (corn oil), 1μL / g CCl4 group, and 2μL / g CCl4 group. The rats were given drugs by gavage according to the grouping, once every 5 days for 40 consecutive days. The samples (including blood and liver) were collected on the 10th, 20th, 30th, and 40th days to detect the degree of liver fibrosis, so as to determine the induction conditions of liver fibrosis.

[0098] (2) MSCs treatment. After successful modeling, the rats with liver fibrosis were randomly divided into three groups: a saline group, a hUC-MSCs cell group with low MMP1 expression (UC-MSCs66), and a hUC-MSCs cell group with high MMP1 expression (UC-MSCs14), with 10 rats in each group. hUC-MSCs were injected into the tail vein for treatment, with 1×10 6 cells / kg, 1mL / rat, injected once. 4 weeks after the treatment, 7% chloral hydrate was injected intraperitoneally for anesthesia, and blood and liver of the rats were collected for detection and observation.

[0099] 2. Detection of related indicators after MSCs treatment of rats with liver fibrosis model:

[0100] 1) Liver function test: 4 weeks after cell transplantation, blood was collected from the eyeballs of experimental rats and immediately placed in the prepared dry EP tube. After standing for 15 minutes, the blood was centrifuged at 3000 rpm for 15 minutes. The supernatant was taken as plasma after standing. After labeling, the plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) levels were detected by automatic biochemical analyzer. The results are as follows Figure 6 As shown, compared with the UC-MSCs66 treatment group, the UC-MSCs14 treatment group had lower plasma ALT, AST and TBIL levels, indicating a better therapeutic effect.

[0101] 2) Detection of liver fibrosis indicators: 4 weeks after cell transplantation, rats in each group were anesthetized and killed, liver tissue was taken, 100 mg of tissue blocks were accurately weighed, liver tissue homogenate was prepared, centrifuged, and the supernatant was retained. ELISA was used to detect the levels of hyaluronic acid (HA), laminin (LAMA5) and type III procollagen (PCIII) in the supernatant. The levels of hyaluronic acid, laminin and type III procollagen in liver tissue can reflect the degree of extracellular matrix deposition. The results are as follows Figure 7 As shown in the figure, compared with the UC-MSCs66 treatment group, the levels of hyaluronic acid, laminin and type III procollagen in the plasma of the UC-MSCs14 treatment group were lower, indicating that the degree of fibrosis was significantly reduced after UC-MSCs14 treatment, and it had a better therapeutic effect.

[0102] 3) Pathological changes of liver: 4 weeks after cell transplantation, rats in each group were anesthetized and killed, and liver tissues were taken and cut into small pieces of about 1.5 cm × 1.5 cm × 0.3 cm. They were fixed with 10% formaldehyde for 72 h, sliced, and stained with hematoxylin-eosin to observe the pathological changes of liver tissue. Figure 8 ) showed that the liver tissue and liver cell structure of the normal control group were normal, without inflammatory cell infiltration and fibrous tissue hyperplasia; in the model group, a large range of liver cell degeneration and necrosis, fibrous tissue hyperplasia, and a large number of inflammatory cells infiltrated around the portal area and central vein were observed, showing typical pathological changes of liver fibrosis; compared with the UC-MSCs66 treatment group, the liver cell degeneration and necrosis, inflammatory cell infiltration and fibrous tissue hyperplasia of rats in the UC-MSCs14 treatment group were improved to varying degrees. Masson staining results showed that the liver tissue of the model group showed typical manifestations of liver fibrosis, and the improvement of liver fibrosis in the UC-MSCs14 treatment group was better than that in the UC-MSCs66 treatment group ( Fig. 9 ).

[0103] Example 4

[0104] This embodiment provides an application of anti-fibrotic human umbilical cord mesenchymal stem cell-derived exosomes in a rat model of liver fibrosis.

[0105] 1. MSCs treatment of liver fibrosis rat model:

[0106] (1) Preparation of exosomes from human umbilical cord mesenchymal stem cells.

[0107] 1) Preparation of mesenchymal stem cell supernatant

[0108] ① After the above mesenchymal stem cells were revived, 5×10 5 cells / dish, inoculated in a 10 cm culture dish and cultured for 60 h.

[0109] ② Weigh 1 g of microcarrier into a 100 mL glass bottle, add deionized water to 60 mL, sterilize at 121°C for 20 min, cool to room temperature and place in a biosafety cabinet for later use.

[0110] ③Take out 500mL of cell culture spinner flask, add 80mL of deionized water, cover the top cap, loosen the side cap, sterilize at 121℃ for 40min, cool to room temperature and place in a biosafety cabinet for use.

[0111] ④ Aspirate the deionized water in the glass bottle containing the microcarriers, add 80 mL of culture medium, mix by pipetting, and wait for the microcarriers to settle naturally before aspirating the culture medium. Add 100 mL of culture medium again, mix by pipetting, and transfer to the cell culture spinner bottle. Place the cell culture bottle in the corresponding position on the magnetic stirrer, turn on the magnetic stirrer, 50 rpm, and balance for 1 hour.

[0112] ⑤Take out the cell culture spinner bottle, wait for the microcarriers to settle naturally, discard the culture supernatant, and take the cell culture medium containing 2×10 7 Add the cell suspension of cells to the rotating bottle, and then add the culture medium to 150mL, place it on a magnetic stirrer for intermittent stirring. The intermittent stirring condition is 25rpm, stir for 10 minutes, stop for 1 hour, and cycle 24 times. After 24 hours, add 150mL of culture medium, adjust the magnetic stirrer parameters to 25rpm for continuous stirring. On the 4th day, add 200mL of culture medium, and adjust the magnetic stirrer parameters to 35rpm for continuous stirring; on the 6th day, adjust the magnetic stirrer parameters to 45rpm for continuous stirring; on the 7th day, adjust the magnetic stirrer parameters to 50rpm for continuous stirring.

[0113] ⑥ On the 7th day of culture, take the cell culture spinner bottle out of the incubator, wait for the microcarriers to settle naturally, and then suck out the culture supernatant from the side of the bottle. Add 500 mL of freshly prepared serum-free culture medium.

[0114] The culture supernatant was subsequently harvested every 4 days until the end of the culture.

[0115] 2) Preparation of high-quality exosomes.

[0116] ①Ultrafiltration and concentration of the supernatant of the conditioned medium. Collect the supernatant culture medium of the 3D culture of mesenchymal stem cells, centrifuge at 2000g for 10 minutes under low temperature conditions, and remove the cells, cell debris and microcarriers in the culture medium. Collect the supernatant solution after centrifugation, add it to the sample cup in the ultrafiltration tube to remove excess water, and centrifuge it at 3500g for 40 minutes at 4°C. After the centrifugation, pour out the flow-through in the filtrate collection cup, cover the concentrate collection cup on the sample filter cup, and centrifuge it at 2000g for 10 minutes at 4°C. Collect the concentrate in the collection cup and place it in a refrigerator at 4°C.

[0117] ②High-purity exosome concentration. Soak the centrifuge tubes used in the ultra-high-speed centrifuge in 75% alcohol for disinfection in advance, dry them, and wipe the centrifuge cup and cup cover with 75% alcohol. Take the collected concentrate and add it to the dried centrifuge tube, balance it, slowly place it in the centrifuge cup, cover the cup cover and tighten it. Place the centrifuge cup on the pre-cooled rotor and centrifuge it at 100,000g for 90 minutes. After the centrifugation, remove the supernatant and resuspend the precipitate with PBS buffer. Add the precipitate resuspended in PBS buffer back to the centrifuge tube, balance it and centrifuge it at 100,000g for 90 minutes. After the end, remove the supernatant and resuspend the precipitate with PBS buffer. After testing the solution resuspended in PBS to determine the concentration of exosomes, aliquot it and store it at -80°C. Take it as needed to avoid repeated freezing and thawing.

[0118] According to the above scheme, high-purity exosomes (such as Fig.10 shown).

[0119] (2) Establishment of rat model of liver fibrosis.

[0120] 1) 6-8 weeks old healthy SD rats were randomly divided into a normal control group and a liver fibrosis model group, 15 rats in each group, a total of 45 rats, namely the control group (corn oil), 1μL / g CCl4 group, and 2μL / g CCl4 group. The rats were given drugs by gavage according to the grouping, once every 5 days for 40 consecutive days. Samples (including blood and liver) were collected on the 30th day to detect the degree of liver fibrosis.

[0121] (3) Exosome treatment. After successful modeling, the rats with liver fibrosis were randomly divided into three groups, namely, the normal saline group, the exosomes derived from hUC-MSCs cells (UC-MSCs66) with low expression of MMP1 (Exo66) group, and the exosomes derived from hUC-MSCs cells (UC-MSCs14) with high expression of MMP1 (Exo14) group, with 10 rats in each group. Exosome treatment was performed by tail vein injection, 200 μg / rat, once. Four weeks after the completion of treatment, 7% chloral hydrate was used for intraperitoneal overdose anesthesia, and blood, liver, etc. of the rats were collected for detection and observation.

[0122] 2. Detection of related indicators after exosome treatment of rats with liver fibrosis model:

[0123] (1) Liver function test: 4 weeks after cell transplantation, blood was collected from the eyeballs of experimental rats and immediately placed in the prepared dry EP tube. After standing for 15 minutes, the blood was centrifuged at 3000 rpm for 15 minutes. After standing for 15 minutes, the supernatant was collected as plasma. After labeling, the plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) levels were detected by an automatic biochemical analyzer. The results are as follows: Fig.11As shown, compared with the Exo66 treatment group, the Exo14 treatment group had lower plasma ALT, AST and TBIL levels, indicating a better therapeutic effect.

[0124] (2) Detection of liver fibrosis indicators: 4 weeks after cell transplantation, rats in each group were anesthetized and killed, liver tissue was obtained, 100 mg of tissue blocks were accurately weighed, liver tissue homogenate was prepared, centrifuged, and the supernatant was retained. The ELISA method was used to detect the levels of hyaluronic acid (HA), laminin (LAMA5) and type III procollagen (PCIII) in the supernatant. The levels of hyaluronic acid, laminin and type III procollagen in liver tissue can reflect the degree of extracellular matrix deposition. The results are as follows Fig.12 As shown in the figure, compared with the Exo66 treatment group, the levels of hyaluronic acid, laminin and type III procollagen in the plasma of the Exo14 treatment group were lower, indicating that the degree of fibrosis was significantly reduced after Exo14 treatment, with better therapeutic effect.

[0125] (3) Pathological changes of liver tissue: 4 weeks after cell transplantation, rats in each group were anesthetized and killed, and liver tissue was removed and cut into small pieces of approximately 1.5 cm × 1.5 cm × 0.3 cm. The pieces were fixed with 10% formaldehyde for 72 h, sliced, and stained with hematoxylin-eosin to observe the pathological changes of liver tissue. Fig.13 ) showed that the liver tissue and liver cell structure of the normal control group were normal, without inflammatory cell infiltration and fibrous tissue hyperplasia; in the model group, a large range of liver cell degeneration and necrosis, fibrous tissue hyperplasia, and a large number of inflammatory cells infiltrated around the portal area and central vein were observed, showing typical pathological changes of liver fibrosis; compared with the Exo66 treatment group, the liver cell degeneration and necrosis, inflammatory cell infiltration and fibrous tissue hyperplasia of the rats in the Exo14 treatment group were improved to varying degrees. Mass staining results showed that the improvement of liver fibrosis in the Exo14 treatment group was better than that in the Exos66 treatment group ( Fig.14 ).

Claims

1. A method for screening anti-fibrotic mesenchymal stem cells, characterized in that: The following steps are involved: S1. Screen several umbilical cord tissues from healthy donors, conduct sterility, mycoplasma and virus tests, and if qualified, separate primary human umbilical cord mesenchymal stem cells by tissue block suspension method, and subculture them to P3 generation in serum-free medium; S2. Testing the P3 generation human umbilical cord mesenchymal stem cells. After passing the test, they are placed in a temporary master cell bank; S3. Collect an appropriate amount of P3 human umbilical cord mesenchymal stem cells, add 1 mL of Trizol, and gently pipette and beat three times to mix well; extract RNA and perform transcriptome sequencing; S4. Analyze the sequencing data using bioinformatics technology to obtain different gene expression matrices of human umbilical cord mesenchymal stem cells; calculate the expression level of the MMP1 gene of all human umbilical cord mesenchymal stem cell lines based on the gene expression matrix; S5. Evaluate the anti-fibrotic ability of hUC-MSCs from different donors in S4; S6, correlation analysis was performed between the MMP1 gene expression levels in hUC-MSCs from different donors in S4 and the corresponding anti-fibrosis ability in S5; S7. The expression level of MMP1 gene in hUC-MSCs is used as a screening index for its anti-fibrosis ability, i.e., a biomarker for the anti-fibrosis ability of human umbilical cord mesenchymal stem cells; S8. Mesenchymal stem cells with strong anti-fibrosis properties can be obtained by screening according to the above indicators; the anti-fibrosis mesenchymal stem cells are further cultured, and after concentration, ultracentrifugation and purification, exosomes derived from human umbilical cord mesenchymal stem cells are obtained.

2. The method for screening anti-fibrosis mesenchymal stem cells according to claim 1, characterized in that: In S1, the sources of the mesenchymal stem cells include umbilical cord, placenta, amniotic membrane, dental pulp, fat and induced pluripotent stem cells.

3. The method for screening anti-fibrosis mesenchymal stem cells according to claim 1, characterized in that: In S2, the detection is one or more of sterility detection, mycoplasma, endotoxin, virus, morphology, surface marker, differentiation ability, cytokine detection, telomere activity, STR, and soft agar cloning.

4. The method for screening anti-fibrosis mesenchymal stem cells according to claim 1, characterized in that: In S7, the MMP1 gene in hUC-MSCs was used as a screening index for its anti-fibrosis ability: the FPMK value of MMP1 gene expression in the P3 generation human umbilical cord mesenchymal stem cell line = 30 was used as a quantitative standard for the anti-fibrosis ability evaluation index MMP1 of human umbilical cord mesenchymal stem cells.

5. An anti-fibrotic mesenchymal stem cell-derived exosome, characterized in that: The following steps are involved: The mesenchymal stem cell lines with strong anti-fibrosis ability obtained by the above screening are subjected to three-dimensional large-scale culture in vitro to obtain a large amount of culture supernatant, and the culture supernatant is concentrated and ultracentrifuged to obtain high-purity mesenchymal stem cell-derived exosomes with strong anti-fibrosis ability.

6. An application of anti-fibrosis mesenchymal stem cells, characterized in that: Application of the anti-fibrosis mesenchymal stem cells and exosomes thereof in treating fibrotic diseases.

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