Gene edited human umbilical cord mesenchymal stem cell for treating hepatic fibrosis and liver cirrhosis and application of gene edited human umbilical cord mesenchymal stem cell

Abstract

The invention relates to application of mesenchymal stem cells of overexpressed Numb genes in preparation of drugs for treating chronic liver diseases. The invention also provides a mesenchymal stem cell modified by genetic engineering, and the mesenchymal stem cell modified by genetic engineering is a mesenchymal stem cell of an overexpressed Numb gene. The invention provides a novel human umbilical cord mesenchymal stem cell (hUC-MSC) for treating chronic and acute hepatic failure, hepatic fibrosis and liver cirrhosis. The hUC-MSCs overexpressing the Numb gene can inhibit liver inflammatory response and hepatic fibrosis progress in a dose-dependent manner; biliary duct reaction is inhibited in a dose-dependent manner, and cholestasis is improved; the liver cell proliferation can be promoted in a dose-dependent manner, and the content of serum albumin is increased. And the treatment effects are better than those of the same dose of hUC-MSCs. The hUC-MSCsNumb-OE can be directly used as a cell for treating chronic and acute hepatic failure, hepatic fibrosis and cirrhosis, and also can be used as a tool for screening biological or chemical medicines for treating hepatic fibrosis and cirrhosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and liver disease prevention and treatment, and specifically, to gene-edited human umbilical cord mesenchymal stem cells for treating liver fibrosis and cirrhosis and applications thereof. Background Art

[0002] Cirrhosis is a pathological stage in which various chronic liver diseases progress to diffuse fibrosis, pseudolobule formation, regenerative nodules, and intrahepatic and extrahepatic vascular proliferation. Compensated cirrhosis has no obvious clinical symptoms, while decompensated cirrhosis is characterized by portal hypertension and severe liver function damage. Patients often die from multiple organ failure due to complications such as ascites, gastrointestinal bleeding, hepatic encephalopathy, hepatorenal syndrome, or cancer. At present, liver transplantation is internationally recognized as the best treatment for end-stage cirrhosis, but it is limited by donor shortage, rejection, and toxic side effects of immunosuppressants. Therefore, it is particularly urgent to explore alternative therapies for liver transplantation. In recent years, stem cell transplantation has become a new hot spot in the treatment of cirrhosis, especially human umbilical cord mesenchymal stem cells (hUC-MSCs), which have the advantages of easy access, no rejection, and no ethical issues. It has shown initial results in the treatment of hepatitis B cirrhosis and alcoholic cirrhosis, but how to regulate the precise differentiation of hUC-MSCs after transplantation to improve the efficacy remains to be further studied.

[0003] Numb is an evolutionarily highly conserved stem cell fate determinant that controls the fate of stem cells through asymmetric allocation during mitosis. In recent years, Numb has received extensive attention as a tumor suppressor. For example, increasing the expression level of Numb can inhibit liver cancer, breast cancer, prostate cancer, melanoma, etc., but little is known about its role in the treatment of liver cirrhosis. Shu et al. reported that after Numb knockout mice were fed a 3,5-diethoxycarbonyl-1,4-dihydropyridine (DDC) diet for 6 weeks, extensive periportal hepatic progenitor cell proliferation and bile duct reaction were observed, and the degree of liver fibrosis was more severe than that of wild-type mice, indicating that Numb deficiency can promote the progression of experimental liver fibrosis. Our previous study found that the level of Numb protein in liver biopsy tissues of patients with autoimmune hepatitis, hepatitis B, and primary biliary cholangitis-related cirrhosis was significantly decreased, which was 51.9%, 25.1%, and 25.3% of that in healthy people, respectively, indicating that the level of Numb protein in the liver of patients with cirrhosis was significantly insufficient. According to our results, hUC-MSCs overexpressing Numb gene (hUC-MSCs Numb-OE ) can play an important role in the treatment of liver fibrosis and cirrhosis. And after verification, there is no patent related to the content of this application, and no literature reports on the application of gene-edited cells in the field of liver disease that are the same as this patent application have been found. Summary of the invention

[0004] The purpose of the present invention is to provide a gene-edited human umbilical cord mesenchymal stem cell and its application for treating liver fibrosis and cirrhosis in view of the deficiencies in the prior art.

[0005] In a first aspect, the present invention provides the use of mesenchymal stem cells overexpressing the Numb gene in the preparation of a drug for treating chronic liver disease.

[0006] As a preferred example, the pathological manifestations of chronic liver disease are one or more of disordered arrangement of liver cells, extensive necrosis, structural destruction of liver lobules, infiltration of inflammatory cells, bile duct cell proliferation, and fibrous septa, and the mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0007] As another preferred example, the chronic liver disease is hepatitis, acute-on-chronic liver failure, liver fibrosis, and liver cirrhosis.

[0008] As another preferred example, the hUC-MSCs overexpressing the Numb gene reduce bile duct cell proliferation and bile duct reaction, improve serum liver function and cholestasis, inhibit the progression of liver fibrosis, and promote liver tissue repair.

[0009] As another preferred example, the drug is a biotherapeutic drug, a chemical drug or a herbal drug.

[0010] In a second aspect, the present invention provides a genetically modified mesenchymal stem cell, wherein the genetically modified mesenchymal stem cell is a mesenchymal stem cell that overexpresses the Numb gene.

[0011] As a preferred example, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0012] In a third aspect, the present invention provides mesenchymal stem cells overexpressing the Numb gene, which are prepared by the following method:

[0013] Step A, obtaining primary mesenchymal stem cells;

[0014] Step B, subculture of primary mesenchymal stem cells;

[0015] Step C: Construction of Numb gene overexpression lentivirus;

[0016] Step D: transfecting mesenchymal stem cells with Numb gene overexpressing lentivirus, and screening stable transfected cell lines to obtain mesenchymal stem cells overexpressing Numb gene.

[0017] In a fourth aspect, the present invention provides a pharmaceutical composition for treating chronic liver disease, comprising mesenchymal stem cells overexpressing the Numb gene as the only active ingredient.

[0018] As a preferred example, the chronic liver disease is hepatitis, acute-on-chronic liver failure, liver fibrosis, and liver cirrhosis.

[0019] The invention has the advantages that: the invention provides a new hUC-MSC for treating liver fibrosis and liver cirrhosis: hUC-MSCs (hUC-MSCs) overexpressing Numb gene Numb-OE ) can dose-dependently inhibit liver inflammation and liver fibrosis progression; dose-dependently inhibit bile duct reaction and improve cholestasis; and dose-dependently promote liver cell proliferation and increase serum albumin content. The above therapeutic effects are better than the same dose of hUC-MSCs. This shows that hUC-MSCs Numb-OE The cells can be used directly to treat liver fibrosis and cirrhosis, and can also be used as a tool to screen biological or chemical drugs for the treatment of liver fibrosis and cirrhosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 :Identification of purity and proliferation ability of hUC-MSC Note: (a) Microscopic morphological observation of hUC-MSC (×40); (b) Cell cycle detection of hUC-MSC; (c) Flow cytometry identification of hUC-MSC surface antigens; (d) Osteogenic induction of hUC-MSC (Alizarin red staining, ×40); (e) Adipogenic induction of hUC-MSC (Oil Red O staining, ×400).

[0021] Figure 2 :Detection of Numb protein and gene expression levels in hUC-MSC after lentiviral transfection Note: (a) 48hEGFP green fluorescence expression after lentiviral transfection (×40); (b) Numb immunoblot and grayscale integral ratio; (c) Numb mRNA expression level.MSC, hUC-MSC group; NUMB-EV, hUC-MSC NUMB-EV Group; NUMB-OE, hUC-MSC NUMB-OE Group. **p<0.01.

[0022] Figure 3 :Numb-Exon3 OE Can inhibit the progression of liver fibrosis Note: Sham, sham operation group; BDL, bile duct ligation group; EV-L / -M / -H, hUC-MSC NUMB-EV(L / M / H) Group: OE-L / -M / -H, hUC-MSC NUMB-OE(L / M / H) Group. *p<0.05, **p<0.01.

[0023] Figure 4 :hUC-MSC Numb-OEinjection inhibits liver inflammation and fibrosis progression Note: (a) HE staining (×200) and Sirius red collagen staining (×100); (b) α-SMA, Col-1, Col-4 immunohistochemical staining (×200); (c) Hyp content in liver tissue; (d) Semi-quantitative analysis of Sirius red collagen staining; (e) TNF-α, TGF-β1, α-SMA, Col-1, Col-4 mRNA expression levels in liver tissue. Sham, sham operation group; BDL, bile duct ligation group; EV-EV-L / -M / -H, hUC-MSC NUMB-EV(L / M / H) Group: OE-L / -M / -H, hUC-MSC NUMB-OE(L / M / H) Group. *p<0.05, **p<0.01.

[0024] Figure 5 :hUC-MSC Numb-OE Injection to inhibit bile duct reaction Note: (a) CK7, CK19 immunohistochemical staining (×200); (b) CK7, CK19 mRNA expression levels in liver tissue. Sham, sham operation group; BDL, bile duct ligation group; EV-L / -M / -H, hUC-MSC NUMB-EV(L / M / H) Group: OE-L / -M / -H, hUC-MSC NUMB-OE(L / M / H) Group. *p<0.05, **p<0.01.

[0025] Figure 6 :hUC-MSC Numb-OE Transplantation can promote hepatocyte proliferation in a dose-dependent manner. Note: (a) Immunohistochemical staining of Alb and HNF-4α (×200); (b) mRNA expression levels of Alb and HNF-4α in liver tissue. Sham, sham operation group; BDL, bile duct ligation group; EV-L / -M / -H, hUC-MSC NUMB-EV(L / M / H) Group: OE-L / -M / -H, hUC-MSC NUMB-OE(L / M / H) Group. *p<0.05, **p<0.01.

[0026] Figure 7 :hUC-MSC Numb-OE Transplantation can inhibit Notch signaling activation in a dose-dependent manner. Note: (a) Numb, RBP-Jκ, Hes1 immunohistochemical staining (×200); (b) Numb, RBP-Jκ, Hes1 mRNA expression levels in liver tissue. Sham, sham operation group; BDL, bile duct ligation group; EV-L / -M / -H, hUC-MSC NUMB-EV(L / M / H) Group: OE-L / -M / -H, hUC-MSC NUMB -OE(L / M / H) Group. *p<0.05, **p<0.01. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0028] Example 1

[0029] 1 Materials and methods

[0030] 1.1 Materials

[0031] Primary human umbilical cord derived mesenchymal stem cells (hUC-MSC) were obtained from Shanghai Essar Biotechnology Co., Ltd. Umbilical cord mesenchymal stem cell serum-free medium (NC0103), stem cell mild digestion enzyme (NC1004.1), and serum-free cell freezing medium (NC1001.1) were purchased from Beijing Yocon Company. Human umbilical cord mesenchymal stem cell osteogenic differentiation medium (PD-017) and human umbilical cord mesenchymal stem cell adipogenic differentiation medium (PD-019) were purchased from Wuhan Pricella Company. Cell cycle detection kit (CCS012) was purchased from Lianke Biotechnology Company. Numb gene overexpression lentiviral vector LV-NUMB (96140-1) and empty lentiviral control vector (CON335) were designed and constructed by Shanghai Genechem Company. α-smooth muscle actin antibody (α-SMA, ab124964), collagen type I alpha1 (Col-1, ab270993), collagen type IV alpha1 (Col-4, ab6586), hepatocyte nuclear factor 4alpha antibody (HNF-4α, ab201460), Numb antibody (ab220362), RBP-Jκ antibody (ab180588), Hes1 antibody (ab108937) were all purchased from Abcam. Glyceraldehyde-3-phosphate dehydrogenase antibody (GAPDH, 60004-1-Ig), cytokeratin 7 antibody (CK7, 155391-1-AP), and CK19 antibody (10712-1-AP) were all purchased from Proteintech. Albumin antibody (Alb, sc271605) was purchased from Stancruz Biotechnology.Flow staining buffer (00-4222-57), CD166-PE antibody (12-1668-41), CD44-FITC antibody (11-0441-81), HLA-DR-FITC antibody (11-9952-41), mouse IgG1-PE antibody (12-4714-81), CD105-PE antibody (12-1057-41), CD45-PE antibody (12-0459-42), rat IgG2b-FITC antibody (11-403 1-81), mouse IgG2a-FITC antibody (11-4724-81), CD19-PE antibody (12-0199-42), CD34-PE antibody (12-0349-41), CD73-FITC antibody (11-0739-41), mouse IgG1-FITC antibody (11-4714-81), CD90-FITC antibody (11-0909-41), CD11b-FITC antibody (11-0118-41) were purchased from Thermo Fisher Scientific. Hydroxyproline assay kit (A030-2-1) was purchased from Nanjing Jiancheng Bioengineering Institute. RNA extraction solution (G3013) and chloroform substitute (G3014) were purchased from Wuhan Saiweier Biotechnology Co., Ltd. RIPA lysis buffer (P0013B) and BCA protein concentration assay kit (P0009) were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Protease inhibitor (B14001) and phosphatase inhibitor (B15001) were purchased from Selleck, Inc., reverse transcription kit (RR047A) was purchased from TAKARA, Inc., SYBR fluorescent dye premix (QPK-201) was purchased from TOYOBO.

[0032] 1.2 Cell recovery, subculture and cryopreservation

[0033] Remove the frozen cells from liquid nitrogen, thaw in a 37℃ water bath, and mix the cell suspension with 5 times the volume of culture medium to dilute the freezing solution. Centrifuge at 1000rpm for 5min, retain the precipitate, add culture medium to resuspend the cells, and gently blow to mix. Transfer to a cell culture dish, shake horizontally, and place in an incubator. Under the microscope, it is observed that the cell fusion degree reaches more than 80%, and it can be passaged. Collect the culture supernatant, add sterile PBS to wash twice, add digestive enzymes, observe under the microscope that the cell morphology shrinks and becomes round, aspirate the culture supernatant and blow the cells, collect them in a centrifuge tube, centrifuge at 1000rpm for 5min. Discard the supernatant, add culture medium to resuspend, and then passage to a new culture dish at 1:8. If you want to freeze the cells, after discarding the supernatant, add pre-cooled freezing solution to resuspend the cells, mix thoroughly, and transfer to a cryopreservation tube and place at -80℃. After 24h, transfer to liquid nitrogen for long-term storage.

[0034] 1.3 Identification of mesenchymal stem cell differentiation potential

[0035] Cells cultured to the third generation (P3) were taken and inoculated into 6-well plates, divided into osteogenic induction group and adipogenic induction group, with three replicates in each group. When the cells are in good growth state and have high fusion, they are replaced with the corresponding induction differentiation medium. The medium was changed every 3 days in the osteogenic induction group. After 20 days of induction, the cell morphology was observed under the microscope and a large number of calcified nodules were found, and then stained with Alizarin Red. After adding induction solution A for 3 days in the adipogenic induction group, it was replaced with induction solution B. After maintaining solution B for 1 day, it was replaced with solution A, and so on. When intracellular lipid droplets were observed under the microscope, the induction was stopped and stained with Oil Red O dye.

[0036] 1.4 Cell cycle and surface antigen flow cytometry

[0037] Collect 1×10 6 P3 cells, centrifuge and discard the supernatant. Wash twice with pre-cooled PBS, centrifuge and discard the supernatant. Add 1ml DNA staining solution and 10μl membrane permeabilization agent, vortex for 5-10s to mix, and incubate at room temperature in the dark for 30min. Select the lowest loading speed and detect on the flow cytometer. Wash the digested cells three times with flow cytometry buffer, divide the cell suspension into multiple 1.5ml ep tubes according to the number of detection indicators, centrifuge and discard the supernatant. The cells were stained with FITC- or PE-conjugated antibodies (anti-CD19-PE, anti-CD34-PE, anti-CD45-PE, anti-CD105-PE, anti-CD166-PE, anti-CD11b-FITC, anti-CD44-FITC, anti-CD73-FITC, anti-CD90-FITC, and anti-HLA-DR-FITC). The antibodies were mixed with the flow buffer according to the ratio specified in the official website instructions, and the cells in the corresponding EP tubes were resuspended. The cells were stained with FITC- or PE-conjugated host immunoglobulin antibodies as isotype controls, incubated on ice in the dark for 30 minutes, centrifuged and the supernatant was discarded, washed three times with buffer, mixed by pipetting, and then tested on the machine.

[0038] 1.5 Lentiviral transfection

[0039] After counting under a microscope, the cells were inoculated in a 6-well plate, with a total of about 5×10 3cells / well, set up a normal group (MSC), an empty lentiviral transfection group (Numb-EV) and an overexpression lentiviral transfection group (Numb-OE), with 4 replicates in each group. After 24 hours of plating, the cell fusion was observed under a microscope at 30%-40%, and lentiviral transfection could be performed. According to the results of the previous pre-experiment, the multiplicity of infection (MOI) = 10 was selected for transfection, and the normal culture medium was replaced 6 hours after transfection to continue culturing the cells; the EGFP green fluorescence expression of the cells was photographed under an inverted fluorescence microscope at 48 hours; the total protein and RNA of each group of cells were extracted at 72 hours to detect the expression of the Numb gene.

[0040] 1.6 Experimental animals

[0041] SD rats, half male and half female, totaling 48, SPF grade, weighing 120-140 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., license number: SCXK (Beijing) 2016-0006. All animals were raised, modeled, and observed at the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine, with the license number of SYXK (Shanghai) 2014-0008. The experimental animal protocol was approved by the Experimental Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine, ethics number: PZSHUTCM2310200001.

[0042] 1.7 Model preparation and group intervention

[0043] After one week of adaptive feeding, the experimental animals were randomly divided into (1) sham operation group (Sham); (2) bile duct ligation group (BDL); (3) empty lentiviral vector transfected hUC-MSC group (hUC-MSC Numb-EV ) Low dose group (hUC-MSC NUMB -EV(L) ), medium dose group (hUC-MSC NUMB-EV(M) ), high dose group (hUC-MSC Numb-EV(M) ); (4) hUC-MSC cells were transfected with lentiviral vectors overexpressing Numb gene (hUC-MSC Numb-OE ) Low dose group (hUC-MSC Numb-OE(L) ), medium dose group (hUC-MSC Numb-OE(M) ), high dose group (hUC-MSC Numb-OE(H) ). Six animals in each group. The experimental animals were anesthetized by intraperitoneal injection of 3% sodium pentobarbital 1.8 ml / kg body weight, and the midline of the abdomen was incised to separate the common bile duct and the left and right hepatic ducts. The left and right hepatic ducts and the common bile duct were ligated respectively to prepare a cholestatic rat cirrhosis model. Each cell injection group was injected with the corresponding dose of cells through the spleen and then the abdomen was closed. The sham operation group only separated the common bile duct and the left and right hepatic ducts and then the abdomen was closed. Samples were collected at the end of the fourth week after modeling, and serum and liver tissue samples were collected.

[0044] 1.8 Serum biochemistry test for liver function

[0045] After the experimental animals were anesthetized, blood was collected through the inferior vena cava, and after standing at room temperature for 4 hours, the blood was centrifuged at 4°C and 3500rpm for 30 minutes, and the upper serum was aspirated. The activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and γ-glutamyl transferase (GGT) and the contents of total bilirubin (TBil), direct bilirubin (DBil), indirect bilirubin (IBil), albumin (Alb) and total bile acid (TBA) were tested by the Laboratory Department of Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine.

[0046] 1.9 Pathological staining of liver tissue

[0047] The liver tissue was fixed, dehydrated and paraffin-embedded before sectioning with a thickness of 4 μm. HE staining or Sirius red collagen staining was performed, and the inflammatory damage and collagen deposition of the liver tissue were observed under a microscope. The collagen positive area ratio was calculated using ImageScope software.

[0048] 1.10 Determination of hydroxyproline (Hyp) content in liver tissue

[0049] Accurately weigh 50±2 mg of liver tissue and determine the Hyp content in liver tissue using the alkaline hydrolysis method according to the kit instructions.

[0050] 1.11 Immunohistochemical staining

[0051] The tissue sections were dewaxed into water, immersed in citric acid (pH = 6.0) or Tris-EDTA (pH = 9.0) solution for antigen heat repair, cooled to room temperature, incubated in 3% H2O2 at room temperature in the dark for 15 minutes, blocked with 10% goat serum at room temperature for 30 minutes, and added with primary antibodies α-SMA (1: 3000), Col-1 (1: 500), Col-4 (1: 200), CK7 (1: 1000), CK19 (1: 1000), Alb (1: 50), HNF-4α (1: 1000), Numb (1: 1000), RBP-Jκ (1: 500), Hes1 (1: 1000) at 4°C overnight. The secondary antibodies were incubated at room temperature for 30 minutes, DAB was used for color development, hematoxylin was used for contrast staining, the sections were sealed with neutral gum, and scanned and analyzed with a digital pathology information scanner.

[0052] 1.12 Real-time fluorescence quantitative polymerase chain reaction (RT-PCR)

[0053] The total RNA of liver tissue was extracted by Trizol method, and the mRNA expression was detected by SYBR Green PCR Master Mix reaction system. 1 μl cDNA was added to 384-well plate, and ViiA TM 7real-time PCR System was used for detection. The quantitative method used the ΔΔCT method, and the reaction conditions were as follows: 42°C for 15 min, 92°C for 2 min, 95°C for 15 s, 45 cycles, and 60°C for denaturation, annealing and extension for 1 min. The detection indicators and primer sequences are as follows:

[0054]

[0055] 1.13 Statistical methods

[0056] SPSS26.0 statistical software was used to analyze the experimental results. The quantitative data were expressed as mean ± SD. For comparisons among multiple groups, if the data were normally distributed and the variances were equal, one-way ANOVA was used for comparison; LSD method was used for pairwise comparisons among groups. All experiments were repeated 3 times or more, and P < 0.05 indicated that the difference was statistically significant.

[0057] 2 Results

[0058] 2.1 hUC-MSC culture and identification

[0059] After subculture, hUC-MSC cells can be seen to grow attached to the wall in a whirlpool shape with a long spindle shape ( Figure 1 a). Flow cytometry was used to detect cell cycle and phenotype, and the results showed CD11b(-), CD19(-), CD34(-), CD45(-), HLA-DR(-), CD44(+), CD73(+), CD90(+), and CD105(+); 60.4% of cells were in the G1 phase ( Figure 1 b,c). Obvious calcium nodule-like deposits stained with Alizarin red appeared around hUC-MSCs during osteogenic induction ( Figure 1 d), adipogenic induction showed the appearance of vacuolar lipid droplets in the cells, and Oil Red O staining showed red ( Figure 1 e).

[0060] 2.2 Construction of hUC-MSC cell line overexpressing Numb gene in vitro

[0061] hUC-MSCs in good growth state at P3 were selected and transfected with lentivirus carrying EGFP at MOI=10. After 48h, cells were marked with EGFP ( Figure 2 a). Western blot and RT-PCR results showed that hUC-MSC Numb-OECompared with hUC-MSC Numb-EV The expression levels of Numb gene protein and mRNA in the hUC-MSC group were significantly increased (p < 0.01). Numb-EV There was no significant difference in protein and mRNA expression levels between the hUC-MSC group and the simple hUC-MSC group (Figures b and c).

[0062] 2.3 hUC-MSC Numb-OE Transplantation can improve serum biochemical parameters in a dose-dependent manner

[0063] The results of serum biochemical tests showed that compared with the Sham group, the serum ALT, AST, ALP, GGT activities and TBIL, DBIL, IBIL, TBA levels of the rats in the BDL group were significantly increased (P < 0.01), and the Alb content was significantly decreased (P < 0.01). NUMB-EV(L) Except for the hUC-MSC group, the other cell injection groups could significantly reduce the activities of ALT, AST, ALP, GGT and the contents of TBIL, DBIL, IBIL and TBA (P < 0.01). Numb-OE The Alb content in the low, medium and high dose groups increased in a dose-dependent manner. In addition to ALT, hUC-MSC NUMB-EV(L) The improvement of other indicators in the hUC-MSC group was statistically significant compared with the BDL group (P<0.01). NUMB-EV(L) Group comparison, hUC-MSC Numb-OE(L) The hUC-MSC group could further reduce the activities of ALT, AST, ALP, GGT and the contents of TBIL, DBIL, IBIL, TBA, and increase the content of Alb (P<0.05 or P<0.01). NUMB-EV(M) Group comparison, hUC-MSC Numb-OE(M) The hUC-MSC group had a better effect in reducing the activities of ALT, AST, ALP, GGT and the contents of DBIL, IBIL, and TBA, and further increased the Alb content (P < 0.01). NUMB-EV(H) Group comparison, hUC-MSC Numb -OE(H) The hUC-MSC group had a better effect in improving all indicators (P < 0.01). Numb-OE hUC-MSCs can improve serum biochemical parameters in a dose-dependent manner. Numb-OE(M) hUC-MSC Numb-OE(L) The ALP activity (P<0.01) and TBA content (P<0.05) of the hUC-MSC group were further reduced, and there was a trend of improvement in the other indicators, but the differences were not statistically significant. Numb-OE(H) hUC-MSCNumb-OE(L) The hUC-MSC group had better improvement in all indicators (P<0.05 or P<0.01); Numb-OE(H) hUC-MSC Numb-OE(M) The group could further improve the indicators of ALT, AST, TBIL, DBIL, IBIL, etc. (P<0.05) Figure 3 ).

[0064] 2.4hUC-MSC Numb-OE Cocoa transplantation inhibits hepatic inflammation and fibrosis progression in a dose-dependent manner

[0065] HE and SR staining showed that the morphology and structure of the liver tissue of the rats in the Sham group were normal, with no dilatation of the hepatic sinusoids, no inflammatory cell infiltration, and no obvious collagen deposition in the tissue. Compared with the Sham group, the rats in the BDL group had disordered arrangement of liver tissue cells, extensive necrosis, and destruction of the liver lobule structure. A large number of inflammatory cell infiltrations and bile duct cell proliferation were observed in and around the portal area, forming multiple fibrous septa and a large amount of collagen deposition. Compared with the BDL group, all cell transplantation groups showed improvement in liver inflammatory cell infiltration, reduction in new bile ducts and collagen deposition, especially hUC-MSC. Numb-OE(H) The most significant improvement was found in the Figure 4 a).

[0066] The detection of Hyp content in liver tissue showed that compared with the Sham group, the Hyp content in liver tissue of rats in the BDL group was significantly increased (P < 0.01). Compared with the BDL group, the Hyp content in each cell treatment group was downregulated (P < 0.05 or P < 0.01), and hUC-MSC Numb -OE It can reduce Hyp content in a dose-dependent manner ( Figure 4 c), SR semi-quantitative results have the same trend ( Figure 4 d).

[0067] Immunohistochemical staining showed that the positive expression of α-SMA, Col-1, and Col-4 in liver tissues of each cell treatment group was significantly reduced, especially in hUC-MSC. Numb-OE The group is more obvious ( Figure 4 b).

[0068] RT-PCR results showed that compared with the Sham group, the mRNA levels of TNF-α, TGF-β1, α-SMA, Col-1, and Col-4 in the BDL group were significantly increased (P < 0.01). Compared with the BDL group, the mRNA expression levels of TNF-α, TGF-β1, α-SMA, Col-1, and Col-4 in each cell injection group were significantly downregulated (P < 0.05 or P < 0.01). Numb-OE(M) Group and hUC-MSCNUMB-EV(M) There was no significant difference in TGF-β1 mRNA level between the two groups. Numb-OE Each dose group compared with the same dose of hUC-MSC Numb-EV The other indicators of the group were significantly improved (P<0.05 or P<0.01). Numb-OE It can dose-dependently reduce the mRNA expression levels of TNF-α, TGF-β1, α-SMA, Col-1, and Col-4 ( Figure 4 e).

[0069] 2.5hUC-MSC Numb-OE Transplantation inhibits bile duct response in a dose-dependent manner

[0070] Immunohistochemical staining showed that compared with the Sham group, the positive expression of CK7 and CK19 in the liver tissue of the BDL group rats was significantly increased. Compared with the BDL group, the positive expression of CK7 and CK19 in each cell injection group was reduced, especially in hUC-MSC Numb-OE(H) The group with the best improvement ( Figure 5 a).

[0071] RT-PCR results showed that compared with the Sham group, the levels of CK7 and CK19 mRNA in the BDL group were significantly increased (P < 0.01). Compared with the BDL group, the levels of CK7 and CK19 mRNA in each cell injection group were significantly downregulated (P < 0.05 or P < 0.01). Numb-OE Different dose groups compared with the same dose of hUC-MSC Numb-EV The hUC-MSCs Numb-OE The mRNA expression levels of CK7 and CK19 were decreased in a dose-dependent manner. Figure 5 b).

[0072] 2.6 hUC-MSC Numb-OE Transplantation promotes hepatocyte proliferation in a dose-dependent manner

[0073] Immunohistochemical staining showed that compared with the Sham group, the positive expression of Alb and HNF-4α in the liver tissue of the BDL group rats was significantly reduced. Compared with the BDL group, the positive expression of HNF-4α in each cell injection group increased; and hUC-MSC Numb-OE The positive expression of Alb in each dose group increased significantly, especially in hUC-MSC Numb-OE(H) The most obvious increase was in the Figure 6 a).

[0074] RT-PCR results showed that compared with the Sham group, the levels of Alb and HNF-4α mRNA in the BDL group were significantly decreased (P < 0.01). NUMB-EV(L) Except for the hUC-MSC group, the other cell injection groups could significantly upregulate the HNF-4α mRNA level (P<0.01); NUMB-EV(H) and hUC-MSC Numb-OE Each dose group could significantly upregulate the level of Alb mRNA (P<0.05 or P<0.01). Numb-OE Each dose group compared with the same dose of hUC-MSC Numb-EV The hUC-MSCs Numb-OE Each dose group could dose-dependently upregulate the expression levels of Alb and HNF-4α mRNA ( Figure 6 b).

[0075] 2.7 hUC-MSC Numb-OE Transplantation inhibits Notch signaling pathway activation in a dose-dependent manner

[0076] Activation of Notch signaling pathway is a key mechanism for promoting biliary liver fibrosis. Immunohistochemical staining showed that compared with the Sham group, the positive expression of Numb in the liver of rats in the BDL group was significantly reduced, and the positive expression of RBP-Jκ and Hes1 was significantly increased. Compared with the BDL group, the positive expression of Numb in each cell injection group was significantly increased, and the positive expression of RBP-Jκ and Hes1 was significantly reduced, especially in hUC-MSC. Numb-OE(H) The most obvious change was in the Figure 7 a).

[0077] RT-PCR results showed that compared with the Sham group, the level of Numb mRNA in the BDL group was significantly decreased (P < 0.01), while the levels of RBP-Jκ and Hes1 mRNA were significantly increased (P < 0.01). NUMB-EV(L) Except for the hUC-MSC group, the mRNA levels of Numb in the other cell injection groups were significantly increased (P<0.05 or P<0.01); the mRNA levels of RBP-Jκ and Hes1 were significantly decreased (P<0.01). Numb-OE(L) hUC-MSC NUMB-EV(L) There was no significant difference in RBP-Jκ mRNA level between the two groups. Numb-OE Compared with the same dose of hUC-MSC, the medium and high groups Numb-EVThe mRNA levels of Numb and Hes1 were further improved in the hUC-MSC group (P<0.05 or P<0.01). Numb-OE Each dose group could dose-dependently up-regulate Numb mRNA level, and dose-dependently down-regulate RBP-Jκ and Hes1 mRNA levels ( Figure 7 b).

[0078] The present invention uses a classic animal model experiment to confirm that the gene can significantly inhibit the progression of liver fibrosis and cirrhosis, and its intervention effect is equivalent to supplementing the full-length Numb gene, but can avoid the tumorigenic risk caused by supplementing the full-length Numb gene. The present invention can be used to develop new target drugs or biological drugs for treating liver fibrosis and cirrhosis, and for treating liver fibrosis, cirrhosis and related complications caused by various chronic hepatitis.

[0079] The hUC-MSCs of the present invention are transformed into hUC-MSCs (hUC-MSCs) overexpressing Numb gene by using lentiviral transfection technology. Numb-OE ) were injected into rats with liver fibrosis to observe their effects. The results confirmed that the hUC-MSCs of the present invention Numb-OE After injection, it can improve liver function and liver tissue pathology in a dose-dependent manner, inhibit liver inflammation and the progression of liver fibrosis. Its main mechanism of action is to effectively promote liver regeneration and reduce bile duct cell proliferation and bile duct reaction.

[0080] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. Application of mesenchymal stem cells overexpressing Numb gene in the preparation of drugs for the treatment of chronic liver disease.

2. The use according to claim 1, characterized in that: The pathological manifestations of chronic liver disease are one or more of disordered arrangement of liver cells, extensive necrosis, structural destruction of liver lobules, infiltration of inflammatory cells, bile duct cell proliferation, and fibrous septa. The mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

3. The use according to claim 1 or 2, characterized in that: The chronic liver disease is hepatitis, acute-on-chronic liver failure, liver fibrosis, and liver cirrhosis.

4. The use according to claim 1 or 2, characterized in that: The hUC-MSCs overexpressing the Numb gene reduce bile duct cell proliferation and bile duct reaction, improve serum liver function and the degree of cholestasis, inhibit the progression of liver fibrosis, and promote liver tissue repair.

5. The use according to claim 1 or 2, characterized in that: The drug is a biotherapeutic drug, a chemical drug or a herbal drug.

6. A genetically engineered mesenchymal stem cell, characterized in that: The genetically engineered mesenchymal stem cells are mesenchymal stem cells that overexpress the Numb gene.

7. The genetically engineered mesenchymal stem cells according to claim 6, characterized in that: The mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

8. The mesenchymal stem cells overexpressing the Numb gene are prepared by the following method: Step A, obtaining primary mesenchymal stem cells; Step B, subculture of primary mesenchymal stem cells; Step C: Construction of Numb gene overexpression lentivirus; Step D: transfecting mesenchymal stem cells with Numb gene overexpressing lentivirus, and screening stable transfected cell lines to obtain mesenchymal stem cells overexpressing Numb gene.

9. A pharmaceutical composition for treating chronic liver disease, comprising the mesenchymal stem cells overexpressing the Numb gene as claimed in claim 6 or 7 as the only active ingredient.

10. The pharmaceutical composition according to claim 9, characterized in that The chronic liver disease is hepatitis, acute-on-chronic liver failure, liver fibrosis, and liver cirrhosis.

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