MiR-148-based mesenchymal stem cell as well as preparation method and application thereof

Abstract

The invention provides a mesenchymal stem cell based on miR-148 as well as a preparation method and application of the mesenchymal stem cell. The preparation method comprises the following steps: inserting a miR-148 sequence into a lentiviral vector pLVX-Puro to construct a pLVX-miR-148 recombinant vector, packaging the pLVX-miR-148 recombinant vector in a 293T cell, and transferring the pLVX-miR-148 recombinant vector into a mesenchymal stem cell to obtain the mesenchymal stem cell based on miR-148. The mesenchymal stem cells based on miR-148 are umbilical cord mesenchymal stem cells with high expression of miR-148, the number of MSCs planted in the intestinal tract can be remarkably increased, the abundance of intestinal microorganisms and the number of lactobacillus reuteri are increased, and therefore the inflammatory bowel disease is improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a mesenchymal stem cell based on miR-148 and a preparation method and application thereof. BACKGROUND

[0002] Inflammatory bowel diseases (IBDs) include ulcerative colitis (UC) and Crohn's disease (CD), which are chronic, recurrent, non-specific, inflammatory diseases involving the ileum, colon and rectum, and seriously affect the quality of life of patients. The exact cause of IBDs is still uncertain, and studies have shown that abnormal activation of the immune system, genetic susceptibility and changes in intestinal flora caused by mucosal barrier defects may play an important role in the pathogenesis of IBDs. IBDs are currently difficult to cure, and more treatment options are needed for different patients.

[0003] In recent years, although some new biological agents have been developed for the treatment of IBDs, traditional drugs such as 5-aminosalicylates acid (5-ASA), immunomodulators and anti-TNF-α are still the main clinical drugs for the treatment and maintenance of IBDs due to their effectiveness, safety and low cost. Among them, 5-ASA is mainly used for the treatment of mild to moderate IBDs, glucocorticoids and sterol drugs are mainly used in the treatment of moderate to severe IBDs, and TNF-α monoclonal antibody has a certain therapeutic effect on refractory moderate to severe IBDs, but glucocorticoids, sterol drugs and TNF-α monoclonal antibody cannot be used for a long time and have certain side effects. Therefore, finding new methods to assist or replace traditional therapy has important clinical significance for the treatment of IBDs.

[0004] Mesenchymal stem cells (MSCs) have strong proliferation ability and multi-directional differentiation potential, and are easy to obtain, abundant in source, low in immunogenicity, and convenient for autologous transplantation. Based on its strong immune regulation and tissue repair function, it is expected to become a promising stem cell product for the treatment of IBDs. MSCs mainly induce lymphocyte cell cycle arrest and apoptosis by releasing various soluble factors (such as cytokines, chemokines and growth factors), thereby achieving immune regulation and inflammation control, and promoting tissue regeneration. Liang et al. retrospectively analyzed 7 cases of IBDs (3 cases of UC and 4 cases of CD), and found that allogeneic transplantation of MSCs could alleviate symptoms and reduce inflammatory response. In the treatment of fistulizing CD, local injection or transplantation of MSCs mixed with silk fibroin glue can secrete growth factors to promote wound healing and regulate epithelial-mesenchymal transition of cells at the fistula site, promoting wound healing. Since the first report of successful healing of fistula after injection of MSCs, several clinical trials have proven the safety and effectiveness of this new treatment for fistulizing CD.

[0005] The therapeutic effect of MSCs on IBDs has been confirmed in preclinical studies of many animal models of IBDs. Intravenous injection of MSCs can reach the site of intestinal injury and colonize the intestinal mucosa, control the development of local inflammation, improve local microcirculation, and repair damaged tissue. Patients with IBDs often exhibit symptoms such as microvascular dysfunction, endothelial barrier damage, and chronic inflammation of the colon, leading to poor healing of the colon tissue. MSCs promote angiogenesis and improve local blood supply by differentiating into vascular endothelial cells, secreting VEGF, FGF, IGF, and EGF, and exerting effects at the level of tissue repair. In a rat model of UC, the number of transplanted MSCs that migrated to the site of injury increased with increasing intestinal injury and colonized the site of injury to promote repair of the injury. MSCs that migrated to the site of the lesion can locally regulate the secretion of inflammatory factors such as IL-1β, TNF-a, and IFN-γ by immune-related cells, thereby reducing the inflammatory response. MSCs exhibit strong immunomodulatory effects in preclinical models of various inflammatory diseases in a paracrine manner, and exosomes are one of the main paracrine substances. MSC-Exo can polarize macrophages to the M2 type, thereby down-regulating the inflammatory response and maintaining the integrity of the intestinal mucosal barrier, reducing the expression of oxidative stress factors such as MPO and MDA, and increasing the expression of antioxidant factors such as GSH and SOD to inhibit oxidative stress, thereby alleviating the symptoms of patients with IBDs. In addition, MSC-Exo can inhibit the proliferation and differentiation of T cells, promote the apoptosis of activated T cells, and aggregate Tregs cells, ultimately leading to a decrease in the expression of pro-inflammatory cytokines such as TNF-a and IFN-γ and an increase in the expression of anti-inflammatory cytokines such as IL-10 and TGF-β, thereby promoting the repair of damaged intestinal tissue. Studies have shown that MSCs can express PD-L1, which can bind to the PD-1 receptor on the surface of activated T cells, B cells, and NK cells to inhibit the activity of T and B cells, which is expected to provide a new direction for the treatment of IBDs.

[0006] There are mainly two administration methods for MSCs to treat IBDs, namely local and systemic administration, and both methods have certain therapeutic effects, but there is still no unified standard for the specific administration method. Although MSCs have been extensively studied in preclinical studies and clinical trials, the cellular and molecular mechanisms of MSCs for in vivo therapeutic effects are still largely unknown.

[0007] With the development of metabolomics and bacterial genetics, many L. reuteri metabolites have been discovered, such as Reuterin, histamine, Exopolysaccharide (EPS), SCFAs, etc., which are involved in L. reuteri antibacterial and immunomodulatory processes. L. reuteriMetabolism of glycerol produces Reuterin and 3-hydroxypropionaldehyde (3-HPA) and exists in the form of a mixture of dimers, which can inhibit the proliferation of gram-positive and gram-negative bacteria, protozoa and fungi; L. reuteri It can also be metabolized from L-His in daily food intake to generate histamine, inhibit the MAPK signaling pathway of intestinal macrophages in CD children, and reduce the production of TNF; EPS is a key substance for bacterial biofilm formation and adhesion to the surface of epithelial cells, L. reuteri Synthetic EPS can inhibit E. coli Adhesion to epithelial cells and the production of E. coli , Salmonella Typhimurium TNF-a and IL-6 and other pro-inflammatory cytokines caused by infection; in addition, L. reuteri It can produce various SCFAs such as acetate, propionate and butyrate, which can improve intestinal microbial imbalance caused by mouse colitis and enhance gastrointestinal peristalsis. These results all show that, L. reuteri It can participate in the regulation of intestinal immune response and remodeling of intestinal microbiota through its metabolites. SUMMARY

[0008] The technical problem to be solved: In view of the above existing technical problems, the purpose of the present application is a mesenchymal stem cell based on miR-148 and its preparation method and application. The mesenchymal stem cell (MSCs) with high expression of miR-148 can significantly increase the number of MSCs planted in the intestinal tract, increase the abundance of intestinal microorganisms and the proliferation of Lactobacillus reuteri, Limosilactobacillus reuteri , L. reuteri And improve inflammatory bowel disease.

[0009] Technical scheme: The application of a mesenchymal stem cell based on miR-148 in the preparation of a drug for treating inflammatory bowel disease, the preparation method of the mesenchymal stem cell based on miR-148 comprises the following steps: S1. Add BamH I and EcoR I restriction sites at both ends of the sequence of miR-148, respectively, perform double enzyme digestion of the lentiviral vector pLVX-Puro with BamH I and EcoR I, prepare linearized pLVX-Puro empty vector, and link, transform and screen the obtained pLVX-Puro empty vector and miR-148 sequence with added restriction sites to obtain a recombinant vector pLVX-miR-148; S2. Package the recombinant vector pLVX-miR-148 in 293T cells to obtain lentiviral particles; S3. Transfer the lentiviral particles into mesenchymal stem cells to obtain mesenchymal stem cells based on miR-148.

[0010] The application of the mesenchymal stem cell based on miR-148 in increasing the abundance of intestinal microorganisms.

[0011] The application of the mesenchymal stem cell based on miR-148 in promoting the proliferation of Lactobacillus reuteri in the intestine.

[0012] The inflammatory bowel disease includes ulcerative colitis and Crohn's disease.

[0013] The nucleotide sequence of the miR-148 is UCAGUGCACUACAGAACUUUGU.

[0014] Preferably, the mesenchymal stem cell is umbilical cord mesenchymal stem cell.

[0015] Beneficial effects: the mesenchymal stem cell based on miR-148 prepared by the application has better migration ability than ordinary mesenchymal stem cells, can be more planted in the intestine, can increase the abundance of intestinal flora and the number of beneficial bacteria in the body, reduce the weight loss and intestinal inflammation caused by inflammatory bowel disease, and can better treat inflammatory bowel disease. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Figure 1 is the influence of mesenchymal stem cells MSCs on the intestinal flora of mice; in the figure: A: analysis of the change of intestinal flora from the level of genus; B: analysis of the change of intestinal flora from the level of species; data is expressed as mean ± SD (n = 5 in each group); Figure 2 Figure 2 is the influence of microRNAs in the exosomes of mesenchymal stem cells MSCs on the proliferation of Lactobacillus reuteri; in the figure: A: after 1 h of treatment with 3 µM miR-148, the bacterial solution is spread on the plate, and the number of bacteria on the plate is detected after 24 h; B: continuous tracking of the OD of each treatment group after treatment with different microRNAs L. reuteri L. reuteri 600 ; Figure 3 Figure 3 is the 2% dextran sulfate sodium (DSS) induced colitis of mice; in the figure: A: after 7 days of DSS administration, the colon tissue of the mice is taken out, and H&E staining is used to detect the intestinal inflammation of the mice; B: after 7 days of DSS administration, the colon tissue of the mice is taken out, and the colon length of the mice is measured to evaluate whether the modeling is successful.

[0017] Figure 4 L. reuteri Figure 4 is the mesenchymal stem cell based on miR-148 can reduce intestinal inflammation; in the figure: A: the mice are continuously administered with 1×10 10 L. reuteri ​​​After 4 weeks of oral administration, mice with colitis induced by 2% DSS were taken out of the colon tissue for H&E staining after 7 days of induction to evaluate the inflammation of the intestinal tissue of mice; the body weight of mice (B) was measured after 7 days of DSS induction, the disease activity index (Disease Activity Index, DAI, C) was evaluated, the histological score was made according to H&E (D), the expression of calprotectin in peripheral blood was detected (E), and the healthy group was standardized, *p<0.05, with statistical difference, the data were expressed as mean±SD (n=5 in each group).

[0018] Figure 5 For the expression efficiency of lentivirus pLVX-miR-148 in MSCs; in the figure: A: GFP positive rate after MSCs were infected with lentivirus pLVX-miR-148 for 72 h under fluorescence microscope; B: total RNA was extracted from MSCs infected with lentivirus pLVX-miR-148 for 72 h, qRT-PCR was used to detect the expression of miR-148 in cells, and NC was used for standardization, *p<0.05, with statistical difference, the data were expressed as mean±SD (n=5 in each group).

[0019] Figure 6 For the promotion of MSCs migration by miR-148; in the figure: A: after MSCs were infected with lentivirus pLVX-miR-148 for 72 h, MSCs (1×10 5 ) were added to the Transwell chamber, serum-free medium was added to the lower chamber, and after 4 h of migration, the chamber was taken out and fixed with 4% paraformaldehyde, and then stained with crystal violet, and the MSCs migrated to the lower chamber were photographed under phase contrast microscope. The migration ability of MSCs was detected by Transwell experiment; B: the number of cells migrated to the membrane was counted and standardized with NC, *p<0.05, with statistical difference, the data were expressed as mean±SD (n=5 in each group).

[0020] Figure 7 For the promotion of MSCs migration by miR-148; in the figure: A: after MSCs were infected with lentivirus pLVX-miR-148 for 72 h, MSCs (1×10 6 ) were added to the Transwell chamber, serum-free medium was added to the lower chamber, and after 4 h of migration, the chamber was taken out and fixed with 4% paraformaldehyde, and then stained with crystal violet, and the MSCs migrated to the lower chamber were photographed under phase contrast microscope. The migration ability of MSCs was detected by Transwell experiment; B: the number of cells migrated to the membrane was counted and standardized with NC, *p<0.05, with statistical difference, the data were expressed as mean±SD (n=5 in each group).

[0021] Figure 8 The MSCs with high expression of miR-148 can better treat inflammatory bowel disease; in the figure: A: mice with colitis induced by 2% DSS, cell transplantation was performed at 1, 3, 5 days after successful modeling, 100 μl of cell suspension (1×10 6 After 2 weeks of MSCs transplantation, the body weight of the mice was measured (B), the disease activity index (Disease Activity Index, DAI) was evaluated (C), the histological score was evaluated according to H&E (D), the expression of calprotectin in peripheral blood was detected (E), and the healthy group was standardized, * indicates p<0.05, with statistical difference, the data is expressed as mean±SD (n=5 in each group).

[0022] Figure 9 The MSCs with high expression of miR-148 can increase the abundance of intestinal probiotics and L. reuteri the number of mice. In the figure: A: mice with colitis induced by 2% DSS, cell transplantation was performed at 1, 3, 5 days after successful modeling, 100 μl of cell suspension (1×10 6 After 2 weeks of MSCs transplantation, the body weight of the mice was measured (B), the disease activity index (Disease Activity Index, DAI) was evaluated (C), the histological score was evaluated according to H&E (D), the expression of calprotectin in peripheral blood was detected (E), and the healthy group was standardized, * indicates p<0.05, with statistical difference, the data is expressed as mean±SD (n=5 in each group). L. reuteri the number of mice. In the figure: A: mice with colitis induced by 2% DSS, cell transplantation was performed at 1, 3, 5 days after successful modeling, 100 μl of cell suspension (1×10 L. reuteri the number of mice. In the figure: A: mice with colitis induced by 2% DSS, cell transplantation was performed at 1, 3, 5 days after successful modeling, 100 μl of cell suspension (1×10 DETAILED DESCRIPTION

[0023] The application will be further described below in conjunction with examples, and the following examples are an explanation of the application, and the application is not limited to the following examples: Example 1

[0024] This example is a method for isolating and culturing mesenchymal stem cells MSCs, which comprises the following steps: S1. With the informed consent of the puerpera, the human umbilical cord sample is aseptically collected after the puerpera gives birth, and then placed in pre-cooled physiological saline and transported to a biological safety cabinet under low temperature; S2. After sterilization of the surgical knife and tissue scissors using high-pressure steam sterilization method, the outer layer of the umbilical cord and the arterial structure and venous structure are removed under aseptic operation, and then the Wallton glue is used for mechanical digestion; S3. Using aseptic operation, the tissue is cut into 1 mm x 1 mm x 1 mm slices and placed in the prepared culture medium, and then placed in a 37°C, 5% CO2 cell incubator for culture; the culture medium is prepared by adding 10% FBS, 1% penicillin streptomycin, and 2 mM L-glutamine to DMEM / F12 culture medium; daily monitoring is performed using an inverted phase contrast microscope, and when the cells are 50% confluent, the tissue blocks are removed after the cells are 50% confluent, and the cells are subcultured when the confluence reaches 80-90%, and the cells are subcultured to P3 for subsequent experiments; S4. The mesenchymal stem cells fused to 80-90% are separated using trypsin-EDTA digestion solution (0.25%), and subcultured at a ratio of 1:2, and the mesenchymal stem cells MSCs used in this application are in good condition from the 3rd to the 10th passage. Example 2

[0025] This embodiment is a method for constructing, packaging and titering a high-expression miR-148 lentivirus vector, comprising the following steps: S1. Constructing a lentivirus vector: according to the NCBI database (NR_029597.1), the core sequence of miR-148 is: 5'-UCAGUGCACUACAGAACUUUGU-3', and BamH I and EcoR I restriction enzyme sites are added at both ends of the miR-148 sequence, and the designed sequence is synthesized by a company; the lentivirus vector pLVX-Puro is double-enzyme cut by BamH I and EcoR I, and the enzyme cutting product is subjected to agarose gel electrophoresis, and the enzyme cutting product is recovered by a gel recovery kit. The enzyme cutting product is a linearized pLVX-Puro empty vector, and the obtained pLVX-Puro empty vector and miR-148 are connected by T4 ligase (4°C connection overnight), and the connection system is shown in Table 1; since the pLVX-Puro vector contains Amp + resistance, therefore, Amp +The connection product cultured on the resistant LB plate was transformed into the bacteria of stable 3 (37°C overnight culture), and the colonies grown on the plate were observed, three independent, round-shaped positive colonies were picked from the plate and placed into a test tube containing 5 mL of LB liquid medium, and labeled for incubation on a shaker; three bacterial liquids were subjected to small-scale plasmid extraction, and the extracted plasmids were subjected to agarose gel electrophoresis, the pLVX-Puro vector was 8000 bp in size, the suspected correct plasmid was subjected to Kpn I enzyme cutting identification, and the enzyme cutting system 2 was as shown in the table; the plasmid with correct position was sent to the company for sequencing, and the sequencing results were compared with the primer sequence to obtain the successfully constructed lentiviral vector pLVX-miR-148, and the correct plasmid was stored in a-80°C refrigerator; Table 1 T4 ligase connection system

[0026] Table 2 Kpn I enzyme cutting system

[0027] S2. Culture 293T cells: 293T cells use DMEM (Dulbecco's Modified Eagle Medium) as culture medium, with 10% FBS, 1% penicillin / streptomycin, when the cells reach 80~90% density, subculture, wash the culture medium in the cell culture bottle with sterile PBS once, then dissolve the cells with 0.25% trypsin / EDTA solution for 30 s, after dissolution, add an equal amount of culture medium to the trypsin / EDTA solution, suspend the cells gently, and move them to a new culture bottle to reach the appropriate cell density; S3. Virus packaging: pLVX-miR-148 lentivirus is packaged in 293T cells, and when the confluence of 293T cells reaches about 60%, the lentivirus packaging is performed; pLVX-miR-148, pMD2.G, pSPAX2 and PEI are added to a 1.5 mL EP tube containing 1 mL H-DMEM at a ratio of 4:3:2:2, and room temperature is kept for 10 min; the above solution is added to the 293T culture dish, and cultured for 6 h, and then 10 mL of medium containing 10% FBS and 1% sodium pyruvate is used for medium replacement; after 24 h, the first batch of virus supernatant is collected, and the medium (H-DMEM+10% FBS+1% sodium pyruvate) is replaced; 48 h later, the virus supernatant is collected again; the obtained virus supernatant is centrifuged in a refrigerated centrifuge for 10 min at 3000 g; the centrifuged virus supernatant is collected and filtered into a pre-cooled high-speed centrifuge tube using a 0.45 μm PES filter head; the centrifuge tube is transferred to a high-speed refrigerated centrifuge (the whole process is operated on ice), and centrifuged (after centrifugation, a white precipitate can be seen at the bottom of the tube, which is the concentrated virus); 200 μL of 4°C pre-cooled phosphate buffer solution is added to the centrifuge tube, and the centrifuge tube is inclined in the 4°C refrigerator to ensure that the liquid in the tube can cover the virus precipitate, so that the virus can be completely dissolved in the liquid; 24 h later, the precipitate is fully dissolved by repeatedly blowing the tube wall with a pipette; according to 200 μL per tube, it is divided and stored in a -80°C ultra-low temperature refrigerator for standby; the virus supernatant and high-speed centrifuge tube must be strictly treated according to the sterilization operation, first soaked in 50% 84 disinfectant overnight, then soaked in 5% SDS solution for 12 h, and finally soaked in 75% alcohol for at least 2 h before disposal; S4. Titer determination: select 293T cells with good growth conditions, aspirate the old culture medium in the culture dish, wash once with 37°C preheated phosphate buffer solution, digest with 0.05% trypsin for 1 min, then terminate with complete culture medium, centrifuge to obtain cells, resuspend the cells in complete culture medium and count; further dilute the cell suspension to a final concentration of 10 5 cells / mL, inoculate the above cell suspension into a 96-well plate, inoculate 100 μL of cells per well, and incubate in a 37°C, 5% CO2 cell incubator for 24 h; the above packaged adenovirus is 10 -4 , 10 -5 , 10 -6 , 10 -7Dilution, 100 μL of each dilution of virus liquid was inoculated into the 96-well plate containing 293T described above, 12 holes were inoculated at each concentration, and then placed in a 37°C, 5% CO2 cell incubator for 18 h. The virus-infected 293T cells were observed under a fluorescence microscope and the number of fluorescent cells was counted, and the number of fluorescent cells x 10 / dilution was considered as the virus titer (pfu / mL). Example 3

[0028] This example is a method for preparing MSCs with high expression of miR-148, comprising the following steps: S1. The mesenchymal stem cells MSCs prepared in Example 1 were inoculated in a six-well plate at a cell number of 5×10 6 cells per hole, and when the cell confluence reached about 70%, 200 μL of the lentivirus liquid prepared in Example 2 was added to the complete culture medium containing 4 μL / mL polybrene to prepare MSCs with high expression of miR-148; S2. Centrifugation at 800xg for 30 min, and after 24 h, the fresh culture medium was replaced, and after 48 h, green fluorescence was observed in the MSCs. The total RNA in the MSCs was extracted, and the expression level of miR-148 in the MSCs was detected by fluorescence quantitative PCR. Example 4

[0029] This example is a method for Transwell migration assay experiment, comprising the following steps: S1. The upper and lower chambers of the Transwell were separated by a 8 μm pore size sieve, and the MSCs or MSCs with high expression of miR-148 cell suspension was added to the upper chamber, and DMEM / F12 was added to the lower chamber. The cells in the upper chamber adhered to the lower side by passing through the membrane pores, and the degree of cell migration was determined by counting the number of cells under the membrane; S2. MSCs were inoculated in the upper chamber at a cell number of 5×10 5 cells per chamber, and the basal medium was added to the lower chamber. After incubation in the incubator for 4 h, fixation was performed using 4% paraformaldehyde, and then staining was performed in 5% crystal violet. After rinsing with tap water, the cells in the upper chamber were wiped off and photographed under an inverted phase contrast microscope. The number of cells migrated to the lower chamber was counted. For each group, the holes with uniform cell migration were selected, and 5 corners of each hole were photographed under a phase contrast microscope. The number of cells in the 4 corners was counted by Image J, and the relative migration ratio was obtained by comparing with the number of cells in the upper sample. Example 5

[0030] This example is the effect of MSCs with high expression of miR-148 on mouse colitis, comprising the following steps: S1. Establishing a DSS-induced mouse colitis model: Healthy male C57 mice aged 5-8 weeks, weighing 19-21 g, were randomly grouped and labeled. Mice were allowed to drink 2.5% DSS aqueous solution freely for 7 days. During the modeling period, weight loss, stool consistency, bleeding and death were recorded and scored according to the Disease Activity Index (DAI) (Table 3). S2. After establishing the model, mouse colon tissue was removed, fixed, and embedded in paraffin. Hematoxylin and eosin (H&E) staining was then performed, with the following steps: Sections were dewaxed in xylene for 5-10 min, repeated twice. They were then passed sequentially through 100%, 95%, 85%, and 70% ethanol for 2-5 min each, and finally rinsed with distilled water. Hematoxylin staining was applied for 5-15 min, adjusting the time according to the tissue type; rinsing with running water for 15-30 min, or briefly alkalizing with 0.75% ammonium chloride solution to make the cell nuclei blue, followed by dehydration through 70%, 85%, and 95% ethanol for 2-3 min each. Eosin staining was applied for 1-5 min; if staining was difficult, glacial acetic acid could be added. After dehydration with 95% and 100% ethanol, the tissue was cleared twice with xylene for 5 min each time. Excess xylene was wiped away, neutral resin was added, and a coverslip was placed to avoid air bubbles. Histological changes were observed and histopathological scoring was performed (Table 4). S3. Treatment of DSS-induced colitis in mice by transplantation of MSCs expressing high miR-148: One week after DSS modeling, MSCs expressing high miR-148 prepared in Example 3 were transplanted intraperitoneally. The transplantation was performed three times, on days 1, 3, and 5, with a single transplantation of 1 × 102 cells. 6 (Control group injected with PBS) Mouse weight was recorded at 1 week and 2 weeks after transplantation. Fecal calprotectin and colon length were detected. Fecal samples from the colon of mice were separated and 16S RNA was sequenced to obtain changes in the intestinal flora of mice. S4. Immunohistochemistry: Paraffin sections of 4 µM mouse colon tissue were dewaxed and dehydrated. Heat-induced epitope repair was obtained by heating with Tris-EDTA (pH 8) at 98°C for 40 min. Non-specific antigens were blocked with 5% bovine serum and incubated at 37°C for 10 min. The sections were then stained with rabbit anti-human MHC polyclonal class I antibody (1:400, CST) at 4°C overnight. After washing with phosphate-buffered saline, 50 µL of horseradish peroxidase-conjugated goat anti-rabbit antibody (1:1000, CST) was added and incubated at room temperature for 20 min. Then, 100 µL of DAB was added for color development. S5. Fecal calprotectin detection and intestinal flora detection: the mice were sacrificed at 7 d, 14 d, 21 d and 28 d after treatment, and the residual feces in the intestines were collected for calprotectin determination. The reagent used was mouse Calprotectin ELISA kit (S100A8 / S100A9), item number: ab263885. Part of the mouse feces was subjected to 16S RNA high-throughput sequencing to detect the changes of the intestinal flora of the mice.

[0031] Table 3 Disease activity index (DAI) score of ulcerative colitis model mice

[0032] Table 4 Histological activity score of ulcerative colitis

[0033] As shown in Figure 1 , the inventors found in the previous study of the therapeutic effect of MSCs on inflammatory bowel disease that MSCs can increase the abundance of intestinal flora in mice and change the ecological niche of intestinal flora, and the number of probiotic Lactobacillus reuteri (L. reuteri) significantly increases. Limosilactobacillus reuteri, L. reuteri L. reuteri is a gram-positive facultative anaerobic bacterium of the genus Lactobacillus, which widely exists in the intestines of vertebrates. It can ferment sugars into lactic acid, acetic acid, ethanol, hydrogen peroxide, bacteriocins and other antagonistic substances, inhibit the growth and reproduction of harmful bacteria, and help restore the balance of intestinal flora. In addition, L. reuteri L. reuteri can colonize the intestinal mucosa, form a biological barrier, block the adhesion of pathogenic bacteria to the gastrointestinal mucosa, compete for nutrients to inhibit the growth of pathogens, neutralize bacterial toxins, promote the expansion of CD4+CD8αα+ double-positive intraepithelial T lymphocytes in the intestinal mucosa, and reduce intestinal inflammation in IBD patients. L. reuteri L. reuteri is one of the few probiotics that can grow in the presence of gastric acid and bile. Due to its strong antibacterial, immunomodulatory and anti-inflammatory activity, as well as almost no safety risks, it can be used as a candidate drug for the treatment of digestive system diseases. L. reuteri

[0034] As shown in Figure 2 , high-throughput sequencing of MSC exosome miRNAs found that more than 300 known miRNAs and more than 100 unknown miRNAs were expressed in the exosomes. Among them, the expression levels of miR-21, miR-143, miR-100, let-7 family, miR-221, miR-222, miR-146, miR-24, miR-125, miR-151, miR-148, miR-10a, miR-92a, etc. are higher. In order to further clarify which miRNA regulates L. reuteri ​Proliferation, treated with the above miRNAs respectively L. reuteri , detected OD 600 Absorbance, the results showed that miR-148 can significantly promote L. reuteri Proliferation.

[0035] As Figure 3 , Figure 4 shown, oral L. reuteri can inhibit weight loss in mice, improve 2% DSS-induced inflammatory bowel disease mouse DAI score, significantly reduce intestinal inflammation in mice, reduce the expression of calprotectin in peripheral blood, thereby improving inflammatory bowel disease, indicating L. reuteri can promote the recovery of intestinal inflammation in mice.

[0036] As Figures 5-7 shown, in order to further clarify that miR-148 plays a crucial role in MSCs regulating L. reuteri treatment of inflammatory bowel disease, the inventors constructed MSCs with high expression of miR-148 (MSCs-miR-148) Figure 5 ). The migration ability of MSCs was detected by Transwell experiment, and it was found that the migration ability of MSCs with high expression of miR-148 was significantly higher than that of the control group (MSCs-miR-148) Figure 6 ). Select 5~8 weeks healthy male C57 mice, body weight 19~21 g, randomly grouped and labeled, using 2.5% DSS aqueous solution, let the mice drink freely for 7 d, after inducing mice to develop ulcerative colitis, transplant MSCs with high expression of miR-148, the control group transplanted MSCs infected with empty virus. After 2 weeks of MSCs transplantation, the mouse colon was taken out and immunohistochemistry was performed to detect human MHC I expression, and it was found that the number of MSCs with high expression of miR-148 planted in the intestinal tract of mice was significantly higher than that of the control group (MSCs-miR-148) Figure 7 ). The above results show that miR-148 can promote the colonization of MSCs in the intestinal tissue of inflammatory bowel disease.

[0037] As Figure 8 shown, compared with the control group, the mice transplanted with MSCs with high expression of miR-148 had significantly reduced DAI score, histological score and peripheral blood calprotectin concentration, indicating that high expression of miR-148 in MSCs can better promote the recovery of inflammatory bowel disease.

[0038] As Figure 9 shown, after 2 weeks of transplantation of MSCs with high expression of miR-148, the intestinal feces of mice were collected and high-throughput sequencing was performed, and it was found that the intestinal flora abundance of mice transplanted with MSCs with high expression of miR-148 increased, and the number of L. reuteri significantly increased, indicating that MSCs with high expression of miR-148 can regulate intestinal flora.

[0039] In summary, miR-148 in MSCs exosomes can improve intestinal inflammation of mice and treat inflammatory bowel disease by increasing the number of goblet cells in the intestinal tract of mice. L. reuteri In summary, miR-148 in MSCs exosomes can improve intestinal inflammation of mice and treat inflammatory bowel disease by increasing the number of goblet cells in the intestinal tract of mice.

[0040] The above description is only the preferred embodiment of the present application, and does not limit the present application in any form. Any person skilled in the art can make many possible changes and modifications to the technical solutions of the present application, or modify equivalent embodiments, without departing from the spirit and technical solutions of the present application, by using the disclosed methods and technical contents. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present application, without departing from the technical solutions of the present application, still falls within the scope of protection of the technical solutions of the present application.

Claims

1. A use of miR-148-based mesenchymal stem cells in the preparation of a medicament for treating inflammatory bowel disease, characterized in that: The preparation method of the mesenchymal stem cell based on miR-148 comprises the following steps: S1. Adding BamH I and EcoR I enzyme cutting sites at both ends of the sequence of miR-148, carrying out double enzyme cutting of the lentivirus vector pLVX-Puro by using BamH I and EcoR I, preparing a linearized pLVX-Puro empty vector, and connecting, transforming and screening the obtained pLVX-Puro empty vector and the miR-148 sequence with enzyme cutting sites to obtain a recombinant vector pLVX-miR-148; S2. Carrying out packaging of the recombinant vector pLVX-miR-148 in 293T cells to obtain lentivirus particles; S3. Transferring the lentivirus particles into mesenchymal stem cells to obtain mesenchymal stem cells based on miR-148.

2. Use according to claim 1, characterized in that: The mesenchymal stem cell based on miR-148 is applied to increasing the abundance of intestinal microorganisms.

3. Use according to claim 1, characterized in that: The mesenchymal stem cell based on miR-148 is applied to promoting the proliferation of Lactobacillus reuteri in the intestine.

4. Use according to claim 1, characterized in that: The inflammatory bowel disease comprises ulcerative colitis and Crohn's disease.

5. The use according to claim 1, characterized in that: The nucleotide sequence of the miR-148 is UCAGUGCACUACAGAACUUUGU.

6. Use according to claim 1, characterized in that: The mesenchymal stem cell is an umbilical cord mesenchymal stem cell.

Images