CTLA4. FasL modified liver oval cell and bone marrow mesenchymal stem cell co-transplantation composition and application thereof in hepatopathy treatment

Abstract

The invention belongs to the technical field of regenerative medicine and cell therapy, and particularly relates to a CTLA4. FasL modified liver oval cell and bone marrow mesenchymal stem cell co-transplantation composition and application thereof in liver disease treatment. The invention provides a co-transplantation composition of CTLA4. FasL modified liver oval cells combined with bone marrow mesenchymal stem cells. The T cell rejection reaction is inhibited through the hexamer synergistic effect of the fusion protein CTLA4. FasL, and meanwhile the liver microenvironment is rapidly improved through the paracrine effect of BMSCs. Experiments prove that the composition significantly improves the transplanted cell colonization rate and the liver function recovery speed, and provides a new scheme for treatment of hepatic failure.

Description

Technical Field

[0001] This application belongs to the field of regenerative medicine and cell therapy technology, specifically relating to a co-transplantation composition of CTLA4.FasL-modified hepatic oval cells and bone marrow mesenchymal stem cells and its application in the treatment of liver diseases. Background Technology

[0002] Hepatic oval cell transplantation shows great promise for treating liver failure and warrants extensive and in-depth research. In acute liver necrosis and cirrhosis, the proliferation of mature hepatocytes is suppressed, while portal oval cells are activated, proliferate, and differentiate into hepatocytes and bile duct epithelial cells, promoting the repair of damaged liver. This suggests that hepatic oval cell transplantation may be a highly promising approach for treating acute and chronic liver failure. Hepatic oval cells possess strong proliferative capacity and bidirectional differentiation potential, and their small size—only 1 / 3 to 1 / 2 the diameter of mature hepatocytes—improves implantation efficiency. In recent years, significant progress has been made in the isolation, purification, and culture of hepatic oval cells, laying the foundation for their transplantation applications.

[0003] Hepatic oval cell transplantation for end-stage liver disease and metabolic liver disease presents two major challenges. First, recipient lymphocyte-mediated rejection hinders the survival and colonization of transplanted hepatic oval cells. Up to 80% of the transplanted cells are cleared by the recipient in the early stages (48 hours post-transplantation). Second, hepatic oval cells require a relatively long time window to further differentiate into functional progeny hepatocytes before they can perform their synthetic and metabolic functions; therefore, they cannot immediately exert their effects after cell transplantation to support the improvement of recipient liver function. In clinical application, immunosuppressants (high-dose methylprednisolone pulse followed by oral tacrolimus and mycophenolate mofetil) are needed to suppress lymphocyte-mediated rejection. However, this approach is not effective against CD8+. + The suppression of T cells is not ideal and also brings risks such as kidney toxicity and potential infection. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a co-transplantation composition of CTLA4.FasL-modified hepatic oval cells and bone marrow mesenchymal stem cells and its application in the treatment of liver diseases. Specifically, the following technical solution is adopted: In a first aspect, the present invention provides a cell composition for the treatment of liver diseases, the cell composition comprising hepatic oval cells modified with the CTLA4.FasL fusion gene and bone marrow mesenchymal stem cells.

[0005] The CTLA4.FasL gene-modified hepatic oval cells combined with BMSCs transplantation provided by this invention can significantly improve the liver function of recipients. CTLA4.FasL gene-modified hepatic oval cells combined with BMSCs transplantation offer a novel treatment modality for cell therapy in acute and chronic liver failure / metabolic liver disease. A literature search, both domestically and internationally, has revealed no studies on CTLA4.FasL promoting xenogeneic hepatic oval cell colonization in rat spleens, nor on CTLA4.FasL gene-modified hepatic oval cells combined with bone marrow mesenchymal stem cell transplantation promoting liver injury repair. The co-transplantation modality of gene-modified hepatic oval cells combined with BMSCs is highly innovative and has significant application value and prospects in the treatment of acute and chronic liver failure / metabolic liver disease and regenerative medicine research.

[0006] As a further preferred embodiment, the ratio of hepatic oval cells modified with the CTLA4.FasL fusion gene to bone marrow mesenchymal stem cells is 1:1 to 1:5.

[0007] Secondly, the present invention provides a method for preparing the above-mentioned cell composition for treating liver diseases, comprising the following steps: CTLA4.FasL fusion gene was transfected into hepatic oval cells using a lentiviral vector to obtain hepatic oval cells modified with the CTLA4.FasL fusion gene; The cell composition for treating liver disease was obtained by mixing the liver oval cells modified with the CTLA4.FasL fusion gene with bone marrow mesenchymal stem cells.

[0008] This invention modifies mouse hepatic oval cells with the immunomodulatory fusion gene CTLA4.FasL via lentiviral transfection, and then co-transplants them with bone marrow mesenchymal stem cells into a rat model of acute liver failure via the spleen. The study investigates the survival and colonization of hepatic oval cells in the xenogeneic spleen microenvironment, evaluates the inhibitory effect of CTLA4.FasL on xenogeneic lymphocyte response, and compares the effects of co-transplantation on improving liver function and survival rate in rats with acute liver failure.

[0009] Thirdly, this invention provides the use of the above-described cell composition for treating liver diseases in the preparation of drugs for treating liver diseases.

[0010] As a further preferred embodiment, the liver disease includes functional failure or metabolic liver disease.

[0011] The beneficial effects of this invention are as follows: This invention provides a co-transplantation composition of CTLA4.FasL-modified hepatic oval cells combined with bone marrow mesenchymal stem cells. The hexamer of the fusion protein CTLA4.FasL synergistically inhibits T-cell rejection, while the paracrine effect of BMSCs rapidly improves the liver microenvironment. Experiments have demonstrated that this composition significantly improves the colonization rate of transplanted cells and the speed of liver function recovery, providing a new treatment option for liver failure. Detailed Implementation

[0012] The technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this application. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0013] This invention aims to specifically inhibit rejection responses and even induce immune tolerance by targeting both T cell activation and apoptosis. It modifies hepatic oval cells by combining co-stimulatory pathways and apoptosis-related genes CTLA4 and FasL. CTLA4 (cytotoxic T lymphocyte associated antigen 4) competitively binds to B7 molecules on the surface of antigen-presenting cells with CD28, inhibiting the activation of co-stimulatory signaling pathways and inducing T cell dysfunction. FasL (Fas Ligand) can bind to its receptor in a trimeric form, triggering downstream apoptotic signals and inducing apoptosis of activated T cells. While the use of single CTLA4 and FasL genes for transplant immunization has been reported, the effects have been limited. Therefore, our experiment attempts to combine the two genes, fusing the extracellular coding regions of CTLA4 and FasL to express a fusion protein that simultaneously induces T cell dysfunction and apoptosis. Furthermore, the fusion protein can form a hexamer, exerting a synergistic effect, and its immunosuppressive efficacy is far superior to that of a single immunoregulatory protein.

[0014] BMSCs can secrete various cytokines and growth factors, inhibiting excessive intrahepatic inflammation and promoting liver function recovery. Studies by Banas et al. found that transplantation of BMSCs into a CCl4-induced fulminant hepatic failure mouse model significantly improved recipient survival. Growth factors in the recipient's liver tissue, including interleukin-8 (IL-8), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), monocyte chemoattractant protein-1 (MCP-1), nerve growth factor (NGF), and hepatocyte growth factor (HGF), were upregulated. Parekada et al. found that injection of BMSCs into conditioned medium stimulated hepatocyte regeneration and inhibited hepatocyte apoptosis, suggesting that BMSCs promote liver repair through paracrine secretion of liver regeneration factors.

[0015] Example 1 Research on the Co - transplantation of CTLA4.FasL - Modified Hepatic Oval Cells and Bone Marrow Mesenchymal Stem Cells for Liver Diseases 1. Materials and Methods Cytological experiments were designed for observation, and one - way ANOVA and LSD - t test were used for comparing the means among multiple groups.

[0016] 1.1 Experimental cells and main reagents: Mouse hepatic oval cell line; Recombinant lentivirus Lv - CTLA4.FasL carrying CTLA4.FasL gene was cryopreserved by the General Surgery Research Institute of the First Affiliated Hospital of Nanchang University; DMEM, RMPI 1640 and fetal bovine serum (Hyclone); Tissue genomic DNA extraction kit (Beijing TianGen Biochemical Technology Co., Ltd.); Taq polymerase (Takara); Rabbit anti - mouse CK - 19 (Abcam); SP immunohistochemistry kit and DAB chromogenic reagent (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.); Lymphocyte separation reagent (Tianjin Haoyang); sFasL ELISA detection kit (Raybiotech); Cell proliferation ELISA, Brdu (Roche); Trypan blue, Concanavalin A (ConA), and Mitomycin C were all purchased from Sigma. 1.2 Experimental animals: 80 healthy female SD rats, body weight 200 - 300 g; Male C57BL / 6 mice, body weight 20 - 30 g, all provided by the Experimental Animal Center of the Medical Department of Nanchang University, license number: SYXK(Gan)2015 - 0001. They were raised in a SPF - level laboratory with free access to food, water and feed.

[0017] 1.3 Culture and identification of hepatic oval cells Culture medium: DMEM culture solution containing 10% volume fraction of FBS; Added materials: 10 mg / L insulin; Cultivation time: It can be cultured in DMEM culture solution containing 10% volume fraction of fetal bovine serum and 10 mg / L insulin for a long time; Cultivation time: It can be cultured in DMEM culture solution containing 10% volume fraction of fetal bovine serum and 10 mg / L insulin for a long time; Identification: Mouse hepatic oval cells highly express hepatic oval cell - related genes such as CK19, Thy - 1, c - Kit, and do not express Alb 1.4 Infection efficiency of Lv / CTLA4.FasL and CTLA4.FasL level in culture supernatant: Hepatic oval cells in logarithmic growth phase were inoculated into 96-well plates at 5 × 10³ cells per well. After 4 h of adhesion, the lentiviral vector Lv-CTLA4.FasL and polybrene (5 mg / L) were added, with a multiplicity of infection (MOI) of 10. Uninfected hepatic oval cells and blank lentivir-infected hepatic oval cells served as controls. Five days after infection, the viability of hepatic oval cells and the expression of the red fluorescent protein lentiviral vector were observed under a fluorescence microscope. The CTLA4.FasL level in the supernatant and the CTLA4.FasL level after continuous passage of CTLA4.FasL-infected hepatic oval cells were measured by ELISA.

[0018] 1.5 Hepatic oval cells transplanted into the spleen: In SD rats that underwent 2 / 3 hepatectomy, 0.5 mL of PBS containing 5 × 10⁻⁶ cells was slowly and evenly injected into the spleen via a 1 mL syringe. 6 One hepatic oval cell was used. Based on the type of transplanted cells, experimental rats were randomly divided into four groups of 20 rats each: Lv-CTLA4.FasL-infected hepatic oval cell transplantation group (CTLA4.FasL-hepatic oval cell group); Lv-mCherry-infected hepatic oval cell transplantation group (blank lentivirus-hepatic oval cell group); hepatic oval cell transplantation group (hepatic oval cell group); and a PBS group without hepatic oval cells. Whole blood and spleen tissue samples were collected from the four groups of rats at four time points: 1, 5, 14, and 21 days after hepatectomy, with 5 rats at each time point. Serum CTLA4.FasL concentration was measured by ELISA, and spleen tissue samples were collected for mixed lymphocyte reaction and immunohistochemical analysis.

[0019] 1.6 ELISA and Immunohistochemistry: Hepatic oval cell culture supernatant and recipient rat serum were collected. Serum CTLA4.FasL levels were detected according to the ELISA kit instructions using a Bio-Tec Elx 800 microplate reader, and the absorbance value at 450 nm was recorded. Paraffin-embedded sections of spleen tissue and hepatic oval cell slides from recipient rats were fixed in 40 g / L paraformaldehyde for 1 h, and CK-19 expression was detected using the SP immunohistochemical method.

[0020] 1.7 Single-channel mixed lymphocyte reaction was used to isolate splenic lymphocytes from C57BL / 6 mice, and 100 μL of cells were collected, with a cell count of 1×10⁻⁶. 5Cells were seeded into 96-well plates and incubated with 25 mg / L mitomycin C at 37 °C for 30 min to inhibit lymphocyte proliferation; these cells were used as stimulating cells. Splenic lymphocytes were isolated from CTLA4.FasL-hepatic oval cell transplanted rats, blank lentivirus-hepatic oval cell transplanted rats, hepatic oval cell transplanted rats, and PBS-treated rats; these rat lymphocytes were used as reacting cells. 100 μL of stimulating cells and 100 μL of reacting cells were seeded into each well of the 96-well plate (cell count 1 × 10⁻⁶). 5 The cells were cultured in an incubator at 37 ℃, 5% CO2 (volume fraction), and saturated humidity (mixed lymphocyte culture group). In the mixed lymphocyte reaction, liver oval cells were cultured; or CTLA4.FasL-liver oval cell culture supernatant was added (CTLA4.FasL-liver oval cells were inoculated with blank lentivirus-liver oval cells, and the concentrations were 10, 50, and 100 μg / L), blank lentivirus-liver oval cell culture supernatant, and liver oval cell culture supernatant. After culturing for 96 h, the proliferation of rat lymphocytes in each group was determined by 5-Brdu assay.

[0021] Splenic lymphocytes were isolated from rats with CTLA4.FasL-hepatic oval cell transplantation, rats with blank lentivirus-hepatic oval cell transplantation, rats with hepatic oval cell transplantation, and rats in the PBS group. Lymphocyte viability was detected by trypan blue exclusion assay, and cell counts were performed, with the cell concentration adjusted to 1×10⁹ L⁻¹. Rat lymphocytes were used as reaction cells, and mitomycin C-treated mouse lymphocytes were used as stimulation cells. 4 mg / L concanavalin A, a mitogen, was added to the rat lymphocyte culture medium. Reaction cells were seeded in 96-well culture plates and cultured in an incubator at 37 ℃, 5% CO₂, and saturated humidity.

[0022] 1.8 BrdU Assay for Rat Lymphocyte Proliferation: The mixed lymphocyte co-culture system was cultured at 37 ℃, 5% CO2 (v / v) and saturated humidity for 96 h, and the proliferation of rat lymphocytes in each group was measured. BrdU-labeled solution was added to each well of the mixed lymphocyte reaction system and incubated for 6 h. After treatment with fixative and anti-Brdu antibody working solution, the reaction substrate was added, and absorbance values ​​were measured at 370 nm and 492 nm. The difference between the two values ​​was calculated. The rat lymphocyte culture group alone served as a negative control. The stimulation index (SI) was used to represent rat lymphocyte proliferation: SI = A492 nm - A370 nm (experimental group) / A492 nm - A370 nm (negative control group). Each group had 3 replicates, and the experimental results were repeated 3 times.

[0023] 1.9 Main observation indicators: ① Lv / CTLA4.FasL infection efficiency and CTLA4.FasL level in culture supernatant; ② Proliferation of rat lymphocytes cultured with xenogeneic mixed lymphocytes and stimulation index of different concentrations of CTLA4.FasL; ③ Serum CTLA4.FasL level and stimulation index of rats in each group after transplantation of hepatic oval cells; ④ Colonization results of hepatic oval cells in spleen.

[0024] 1.10 Statistical analysis was performed using SPSS 22.0 software. Quantitative data were expressed as -x±s. One-way ANOVA was used for comparisons of means among multiple groups, and the LSD-t test was used for multiple comparisons. A p-value < 0.05 was considered statistically significant.

[0025] 2.0 Results Infection efficiency and CTLA4.FasL levels in culture supernatant: Hepatic oval cells cultured in vitro were infected with Lv / CTLA4.FasL and polybrene after 4 h of adhesion. Five days later, the viability of hepatic oval cells and the expression of red fluorescent protein in the lentiviral vector were observed under a fluorescence microscope, showing an infection efficiency of 80%. After infection with the lentiviral vector Lv / CTLA4.FasL, the concentration of CTLA4.FasL in the culture supernatant reached (3.87±0.70) mg / L. CTLA4.FasL was not detected in either uninfected hepatic oval cells or the supernatant of cells infected with blank lentivirus. The concentration of CTLA4.FasL gradually decreased with continuous passage of hepatic oval cells, stabilizing after 10 passages.

[0026] The embodiments of this application have been described above. Specific examples have been used to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the core ideas of this application. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A cell composition for the treatment of liver disease, characterized in that, The cell composition comprises hepatic oval cells modified with the CTLA4.FasL fusion gene and bone marrow mesenchymal stem cells.

2. The cell composition for treating liver disease according to claim 1, characterized in that, The ratio of hepatic oval cells modified with the CTLA4.FasL fusion gene to bone marrow mesenchymal stem cells was 1:1 to 1:

5.

3. A method for preparing the cell composition for treating liver disease according to any one of claims 1-2, characterized in that, Includes the following steps: CTLA4.FasL fusion gene was transfected into hepatic oval cells using a lentiviral vector to obtain hepatic oval cells modified with the CTLA4.FasL fusion gene; The cell composition for treating liver disease was obtained by mixing the liver oval cells modified with the CTLA4.FasL fusion gene with bone marrow mesenchymal stem cells.

4. The use of the cell composition for treating liver disease according to claim 1 in the preparation of a liver disease medicament.

5. The application according to claim 4, characterized in that, The liver diseases mentioned include liver failure or metabolic liver disease.