Liver precursor cell and construction method therefor and use thereof

Abstract

Provided are a liver precursor cell and a construction method therefor and the use thereof. The method for constructing liver precursor cells comprises the steps of: S0, providing liver precursor cells, a mixed solution and a reprogramming culture medium, wherein the mixed solution comprises a serum-free culture medium, polyethyleneimine and a CLP plasmid; S1, carrying out a transfection treatment on the liver precursor cells and the mixed solution to obtain transfected liver precursor cells; and S2, placing the transfected liver precursor cells into the reprogramming culture medium for subculture to obtain immortalized liver precursor cells. The method solves the problems that in the prior art, liver precursor cells cannot be transformed into immortalized cell strains by means of immortalizing genes and cannot be continuously passaged in vitro.

Description

Hepatic progenitor cells and their establishment methods and applications Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a hepatic progenitor cell and a method for establishing a cell line and an application thereof. Background Art

[0002] Cell therapy is a relatively safe, effective and promising treatment method. Primary hepatocytes are the earliest cell source used in the clinical treatment of liver disease. Between 1999 and 2006, in 8 clinical cases of primary hepatocyte transplantation for the treatment of Crigler-Najjar Syndrome (CNS), the overall serum bilirubin of all patients decreased by more than 50%. In 2019, the British A. Dhawan team used microencapsulated primary hepatocytes for intraperitoneal transplantation to treat 8 children with acute liver failure, of which 4 children avoided liver transplantation and 3 children successfully bridged to liver transplantation after the acute phase. However, cell therapy and disease simulation based on hepatocytes have been limited by the ability of primary hepatocytes to expand in vitro.

[0003] Cell immortalization refers to the process in which cells, due to genetic changes or external stimuli such as viral infection, avoid the normal aging and death process during in vitro culture, acquiring the ability to proliferate indefinitely. This allows for long-term subculture and unlimited division and proliferation in vitro. Immortalizing primary hepatocytes in vitro may be a solution to address the limitations of primary hepatocyte cell numbers in vitro.

[0004] There are three main methods for immortalizing primary hepatocytes:

[0005] The first type is the immortalization of primary hepatocytes related to viral genes, such as simian virus 40T antigen (SV40T), adenovirus EIA and EIB genes, human papillomavirus E6 / E7 genes, and Epstein-Barr virus immortalization. Various mechanisms can inactivate the cell cycle regulatory proteins pRB and p53, allowing cells to bypass senescence and continue to be passaged. Kobayashi et al. transfected 21-week-old human fetal liver cells with a PSV3neo plasmid encoding the SV40T tumor antigen to obtain the immortalized liver cell line OUMS-29. Different viral immortalization methods have different adaptability to different types of cells. The E6 / E7 genes of the HPV16 virus strain can efficiently establish immortalized cell lines. At the same time, a large number of studies have shown that HPV16-E6 / E7 has good applicability for the immortalization of hepatocytes. Professor Yan Hexin's team used the HPV16-E6 / E7 gene to immortalize hepatic progenitor cells, which can be continuously passaged in vitro for more than 40 generations and express surface markers related to hepatic progenitor cells.

[0006] The second type involves hepatocyte immortalization related to telomerase and its catalytic subunit. hTERT, the catalytic subunit of telomerase, binds to related proteins to activate telomerase, prolonging the lifespan of cells in vitro and acting as a positive regulator of telomerase. Wege et al. reported that hTERT gene transfection into human fetal hepatocytes elongated telomeres, allowing them to transcend replicative senescence. These cells were able to survive for over 300 generations in vitro and maintain liver-specific functions.

[0007] The third type is reversible immortalization. Kobayashi et al. in Japan introduced a recombinant SSR69 retrovirus carrying the SV40T antigen, herpes simplex virus thymidine kinase (HSV-TK), and the resistance gene for hygromycin (an aminoglycoside antibiotic produced by Streptomyces) and flanked by loxP sites into primary adult human hepatocytes, successfully establishing a reversible immortalized hepatocyte cell line, NKTK-3. By transfecting hepatocytes with a recombinant adenovirus vector expressing Cre recombinase, the SV40T gene was effectively removed, and the NKTK-3 cells no longer expressed SV40T, restoring the hepatocyte line to its pre-immortalization state.

[0008] Currently, the immortalization technology of hepatic progenitor cells is not mature. Most immortalization genes cannot transform hepatic progenitor cells into immortalized cell lines, and cannot be continuously propagated in vitro.

[0009] Therefore, it is necessary to provide a hepatic progenitor cell and a method for establishing a line thereof and application thereof to solve the above-mentioned problems existing in the prior art.

[0010] Summary of the Invention

[0011] The purpose of the present invention is to provide a hepatic progenitor cell and its establishment method and application, so as to solve the problem that the immortalization gene in the prior art cannot transform hepatic progenitor cells into immortalized cell lines and cannot be continuously passaged in vitro.

[0012] To achieve the above objectives, the present invention provides a method for establishing a hepatic progenitor cell line, comprising the following steps:

[0013] S0: providing hepatic progenitor cells, a mixed solution, and a reprogramming medium, wherein the mixed solution comprises a serum-free medium, polyethyleneimine, and a CLP plasmid;

[0014] S1: performing transfection treatment on the hepatic progenitor cells and the mixed solution to obtain transfected hepatic progenitor cells;

[0015] S2: placing the transfected hepatic progenitor cells into the reprogramming medium for subculture to obtain immortalized hepatic progenitor cells.

[0016] The beneficial effect of the method for establishing a hepatic progenitor cell line described in the present invention is that: the present invention transfects the hepatic progenitor cells and the mixed solution to obtain transfected hepatic progenitor cells, and the transfected hepatic progenitor cells are placed in the reprogramming medium for subculture to obtain immortalized hepatic progenitor cells. The present invention uses CLP plasmids to transfect the hepatic progenitor cells, which does not produce cytotoxicity. Then, by subculture in the reprogramming medium, the transfected hepatic progenitor cells can be continuously passaged in vitro. Therefore, the present invention solves the problem in the prior art that immortalization genes cannot transform hepatic progenitor cells into immortalized cell lines and cannot be continuously passaged in vitro.

[0017] Preferably, the volume ratio of the serum-free culture medium to the polyethyleneimine is (500-1000):1.

[0018] Preferably, the final concentration of the CLP plasmid is (1-5) μg / μL.

[0019] Preferably, the step of providing hepatic progenitor cells comprises: placing hepatocytes in the reprogramming medium for expansion culture, and then replacing the reprogramming medium with a serum-reduced medium for continued culture to obtain hepatic progenitor cells.

[0020] Preferably, the reprogramming medium includes DMEM / F12 basal medium, and based on the content of DMEM / F12 basal medium: 15-25 ng / mL hepatocyte growth factor HGF, 15-25 ng / mL epithelial cell growth factor EGF, 5-15 μM ROCK kinase inhibitor Y27632, 1-5 μM Wnt signaling pathway agonist CHIR99021, 1-5 μM TGF-β signaling inhibitor A8301, 1-5 μM sphingosine-1-phosphate S1P and 5 μM indoleacetic acid LPA.

[0021] Preferably, the reprogramming medium further comprises N2 nutrient supplement, B27 nutrient supplement, sodium pyruvate and ascorbic acid.

[0022] Preferably, the reprogramming medium includes DMEM / F12 basal medium, and, based on the content of DMEM / F12 basal medium, the following: 1-10% N2 nutrient supplement, 1-10% B27 nutrient supplement, 1 mM sodium pyruvate, 5-15 μg / mL ascorbic acid, 15-25 ng / mL hepatocyte growth factor HGF, 15-25 ng / mL epithelial cell growth factor EGF, 5-15 μM ROCK kinase inhibitor Y27632, 1-5 μM Wnt signaling pathway agonist CHIR99021, 1-5 μM TGF-β signal inhibitor A8301, 1-5 μM sphingosine-1-phosphate S1P and 5 μM indoleacetic acid LPA.

[0023] The present invention also provides a hepatic progenitor cell, which is prepared by the hepatic progenitor cell establishment method.

[0024] The beneficial effects of the hepatic progenitor cells described in the present invention are that they are transfected with a CLP plasmid without producing cytotoxicity, and are then subcultured in a reprogramming medium to enable the transfected hepatic progenitor cells to be continuously passaged in vitro. Therefore, the present invention solves the problem in the prior art that immortalization genes cannot transform hepatic progenitor cells into immortalized cell lines and cannot be continuously passaged in vitro.

[0025] Preferably, the hepatic progenitor cells negatively express CD34, CD45 and HLA-DRPQ, and the hepatic progenitor cells positively express CD24 and CD326.

[0026] The present invention further provides a use of hepatic progenitor cells, wherein the hepatic progenitor cells are used to prepare a drug for treating acute liver failure.

[0027] The beneficial effect of the application of the hepatic progenitor cells of the present invention is that the injection containing the hepatic progenitor cells has a significant therapeutic effect on the mouse acute liver failure model induced by a D-gal inducer combined with an LPS inducer. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG1 is a schematic diagram of a plasmid according to an embodiment of the present invention;

[0029] FIG2 is a schematic diagram of the cell morphology of the cell line B42 according to an embodiment of the present invention;

[0030] FIG3 is a schematic diagram of the cell morphology of the fifteenth generation cell line B42 according to an embodiment of the present invention;

[0031] FIG4 is a schematic diagram of the flow cytometry detection results of the cell line B42 negatively expressing CD34 according to an embodiment of the present invention;

[0032] FIG5 is a schematic diagram of the flow cytometry detection results of the cell line B42 negatively expressing CD45 according to an embodiment of the present invention;

[0033] FIG6 is a schematic diagram of the flow cytometry detection results of the cell line B42 negative for HLA-DRPQ expression according to an embodiment of the present invention;

[0034] FIG7 is a schematic diagram of the flow cytometry detection results of the cell line B42 positively expressing CD24 according to an embodiment of the present invention;

[0035] FIG8 is a schematic diagram of the flow cytometry detection results of the cell line B42 positively expressing CD326 according to an embodiment of the present invention;

[0036] FIG9 is a comparative schematic diagram of the survival rates of the experimental group and the control group of mice in the acute liver failure model according to an embodiment of the present invention. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein should be the common meanings understood by people with ordinary skills in the field to which the invention belongs. The words "including" and similar words used in this article mean that the elements or objects appearing before the word cover the elements or objects listed after the word and their equivalents, without excluding other elements or objects.

[0038] Viral transfection, adenoviral transfection, and electroporation are commonly used methods for constructing immortalized cell lines.

[0039] Viruses can introduce foreign genes into cells, thereby modifying the cell genome. Through viral transfection, stable immortalized cell lines can be constructed for long-term cell culture and experimental research. Viral transfection can efficiently introduce foreign genes into cells, allowing them to express the target gene. However, lentiviral infection requires the use of large quantities of lentiviral vectors and reagents, which is costly. Furthermore, lentiviral infection requires strict control of the laboratory environment to avoid safety issues such as virus leakage. Lentiviral infection can only be used for specific cell types, and different cell types have different lentiviral infection efficiencies. Therefore, the applicability of lentiviral infection to construct immortalized cell lines is limited. Lentiviral infection may introduce foreign genes, causing cell mutations or even cancer. Furthermore, lentiviruses themselves may pose safety risks, such as cell death or inflammatory responses after viral infection.

[0040] Adenovirus infection is a non-pathogenic virus that can introduce exogenous genes into cells, thereby modifying the cell genome. Through adenovirus infection, stable immortalized cell lines can be constructed for long-term cell culture and experimental research. Adenovirus infection has the following defects: adenovirus infection can only be used for specific types of cells, and the infection efficiency of adenovirus varies among different types of cells. Therefore, the scope of application of adenovirus infection to construct immortalized cell lines is limited. Adenovirus infection requires the use of a large amount of adenovirus vectors and reagents, which is relatively expensive. In addition, adenovirus infection requires strict control of the laboratory environment to avoid safety issues such as virus leakage.

[0041] Electroporation uses high-voltage electrical pulses to introduce exogenous DNA into cells, thereby modifying the cell genome. This method can be used to create stable, immortalized cell lines for long-term cell culture and experimental research. However, electroporation requires the use of high-voltage electrical pulses, which can pose a safety hazard to researchers if performed improperly. Furthermore, electroporation can cause cell damage and death, affecting experimental results. Different cell types have varying adaptability to electroporation, and some cells may be harmed or die as a result of electroporation. Therefore, the applicability of electroporation for creating immortalized cell lines is limited. Cell lines created using electroporation can lose stability due to genomic instability, leading to inconsistent gene expression and affecting experimental results.

[0042] Existing technologies are not mature enough for the construction of immortalized liver progenitor cells and cannot be continuously propagated in vitro.

[0043] The present invention uses plasmid transfection to directly immortalize cells, obtain liver precursor cells (HepLPCs) with clear donor sources and traceable information, and transduce the plasmid CLP-ET carrying the exogenous gene HPV-16-E6 / E7-deltaTK gene into the liver precursor cell genome using a PEI transfection reagent transfection method to construct a stably transfected cell line. PEI transfection reagent is a type of cationic polymer transfection reagent that can bind to DNA to form complex microparticles and enter cells through endocytosis. After DNA enters the cell, the exogenous gene can be inserted into the cell genomic DNA by random integration. The transfection reagent does not need to introduce nucleic acid sequences such as viral sources or transposases into the inserted gene fragments, making it safer for subsequent clinical use of cell lines.

[0044] After cell transfection, two rounds of monoclonal screening were used to identify stably transfected cell lines. The functionality of these cell lines was verified through cell line surface marker detection, cell function verification, preliminary pharmacodynamic studies, cell line genome sequencing, and metabolomics sequencing. This cell line addresses the problem of immortalized hepatic progenitor cells, and because the cells are modified using only plasmids, there are no safety concerns.

[0045] The present invention provides a method for establishing a hepatic progenitor cell line, comprising the following steps:

[0046] S0: providing hepatic progenitor cells, a mixed solution, and a reprogramming medium, wherein the mixed solution comprises a serum-free medium, polyethyleneimine, and a CLP plasmid;

[0047] S1: performing transfection treatment on the hepatic progenitor cells and the mixed solution to obtain transfected hepatic progenitor cells;

[0048] S2: placing the transfected hepatic progenitor cells into the reprogramming medium for subculture to obtain immortalized hepatic progenitor cells.

[0049] Specifically, the present invention transfects the hepatic progenitor cells and the mixed solution to obtain transfected hepatic progenitor cells, and then subcultures the transfected hepatic progenitor cells in the reprogramming medium to obtain immortalized hepatic progenitor cells. The present invention uses a CLP plasmid to transfect the hepatic progenitor cells, which does not produce cytotoxicity. Then, by subculturing in the reprogramming medium, the transfected hepatic progenitor cells can be continuously passaged in vitro. Therefore, the present invention solves the problem that the immortalization gene in the prior art cannot transform hepatic progenitor cells into immortalized cell lines and cannot be continuously passaged in vitro.

[0050] In some embodiments of the present invention, the volume ratio of the serum-free culture medium to the polyethyleneimine is (500-1000):1.

[0051] In some embodiments of the present invention, the final concentration of the CLP plasmid is (1-5) μg / μL.

[0052] In some embodiments of the present invention, the step of providing hepatic progenitor cells comprises: placing the hepatocytes in the reprogramming medium for expansion culture, and then replacing the reprogramming medium with a serum-reduced medium for continued culture to obtain the hepatic progenitor cells.

[0053] In some embodiments of the present invention, the reprogramming medium includes DMEM / F12 basal medium, and, based on the content of DMEM / F12 basal medium, 15-25 ng / mL hepatocyte growth factor HGF, 15-25 ng / mL epithelial cell growth factor EGF, 5-15 μM ROCK kinase inhibitor Y27632, 1-5 μM Wnt signaling pathway agonist CHIR99021, 1-5 μM TGF-β signaling inhibitor A8301, 1-5 μM sphingosine-1-phosphate S1P and 5 μM indoleacetic acid LPA.

[0054] In some embodiments of the present invention, the reprogramming medium further comprises N2 nutrient supplement, B27 nutrient supplement, sodium pyruvate and ascorbic acid.

[0055] In some embodiments of the present invention, the reprogramming medium includes DMEM / F12 basal medium, and, based on the content of DMEM / F12 basal medium, the following: 1-10% N2 nutrient supplement, 1-10% B27 nutrient supplement, 1 mM sodium pyruvate, 5-15 μg / mL ascorbic acid, 15-25 ng / mL hepatocyte growth factor HGF, 15-25 ng / mL epithelial cell growth factor EGF, 5-15 μM ROCK kinase inhibitor Y27632, 1-5 μM Wnt signaling pathway agonist CHIR99021, 1-5 μM TGF-β signal inhibitor A8301, 1-5 μM sphingosine-1-phosphate S1P and 5 μM indoleacetic acid LPA.

[0056] The present invention also provides a hepatic progenitor cell, which is prepared by the hepatic progenitor cell establishment method.

[0057] Specifically, the present invention uses a CLP plasmid to transfect the hepatic progenitor cells without producing cytotoxicity. Subsequently, the transfected hepatic progenitor cells are subcultured in a reprogramming medium to enable continuous passage in vitro. Therefore, the present invention solves the problem in the prior art that immortalization genes cannot transform hepatic progenitor cells into immortalized cell lines and cannot be continuously passaged in vitro.

[0058] In some embodiments of the present invention, the hepatic progenitor cells negatively express CD34, CD45 and HLA-DRPQ, and positively express CD24 and CD326.

[0059] The present invention further provides a use of hepatic progenitor cells, wherein the hepatic progenitor cells are used to prepare a drug for treating acute liver failure.

[0060] Specifically, the injection solution containing hepatic progenitor cells has a significant therapeutic effect on the mouse acute liver failure model induced by D-gal inducer combined with LPS inducer.

[0061] Example

[0062] 1. Plasmid transfection into hepatic progenitor cells

[0063] FIG1 is a schematic diagram of a plasmid according to an embodiment of the present invention.

[0064] The insertion site of E6E7 was provided by the inventors and synthesized by Yunzhou Biotechnology (Guangzhou) Co., Ltd. The obtained E6E7 was inserted into the vector by enzyme digestion and ligation. The constructed CLP plasmid is shown in Figure 1.

[0065] The reprogramming medium consists of: DMEM / F12 basal medium, and, based on the content of DMEM / F12 basal medium, 1% N2 nutrient supplement (100X), 1% B27 nutrient supplement (50X), 1 mM sodium pyruvate, 10 μg / mL ascorbic acid, 20 ng / mL hepatocyte growth factor HGF, 20 ng / mL epithelial cell growth factor EGF, 10 μM ROCK kinase inhibitor Y27632, 3 μM Wnt signaling pathway agonist CHIR99021, 1 μM TGF-β signaling inhibitor A8301, 1 μM sphingosine-1-phosphate S1P and 5 μM indoleacetic acid LPA. DMEM / F12, N2 nutrient supplement, B27 nutrient supplement and sodium pyruvate were from Invitrogen; ascorbic acid was from Sigma-Aldrich; HGF and EGF were from Novoprotein; Y27632, CHIR99021, A8301, S1P and LPA were from TargetMol.

[0066] Unless otherwise specified, the culture medium used for subculture in the embodiments of the present invention is a reprogramming medium.

[0067] Resuscitate two lines of hepatic progenitor cells, seed 200,000 cells per well in a 6-well plate, and culture with 2 ml of reprogramming medium. Transfection is performed after the cells have adhered overnight. 30 minutes before transfection, replace the reprogramming medium with 1 ml of reduced serum medium and return the cells to the incubator for culture to obtain the hepatic progenitor cells to be transfected.

[0068] Take two 1.5 ml centrifuge tubes and mark them as the first centrifuge tube and the second centrifuge tube respectively, add 50 μl of serum-free culture medium and 6 μl of polyethyleneimine to the first centrifuge tube, and mix them by pipetting quickly 10 times to obtain a first mixed solution; add 50 μl of serum-free culture medium and 2 μg of CLP plasmid to the second centrifuge tube, mix them by pipetting, and let it stand for 10 minutes to obtain a second mixed solution, add the second mixed solution in the second centrifuge tube to the first mixed solution in the first centrifuge tube with a pipette, and mix them by pipetting quickly 10 times to obtain a mixed solution, add the mixed solution dropwise to a 6-well plate containing hepatic progenitor cells to be transfected, and then place the 6-well plate in a carbon dioxide incubator for transfection. After 8 hours of transfection, replace the serum-free culture medium in the 6-well plate with 2 ml of reprogramming medium for continuous subculture.

[0069] Resuscitated primary hepatic progenitor cells at passage Pr were seeded into 6-well plates, transfected with the CLP-ET plasmid, and continuously cultured in reprogramming medium. Subculture was performed when the primary hepatic progenitor cells reached a confluency greater than 80%. Each passage of transfected primary hepatic progenitor cells was designated P0+1 and cultured in 10 cm cell culture dishes. When the confluency of P0+5 primary hepatic progenitor cells exceeded 80%, they were seeded into 96-well plates using the limiting dilution method.

[0070] When the confluence of P0+5 primary hepatic progenitor cells reached 80%, they were subcultured and monoclonal cells were selected. The cell suspension was diluted to 70 cells / 20 ml by limiting dilution, and 200 μl of culture medium was added to each well of a 96-well plate, which is approximately equal to 0.7 cells / well / 200 μl. Four 96-well plates were plated. The clone numbers were named according to the order of the selected monoclonal cells. A total of 21 monoclonal cell lines were selected and recorded as P1 monoclonal cells.

[0071] P1 generation monoclonal cells were cultured in 96-well plates. Three days after plating the 96-well plates, wells containing single cells were selected from the 96-well plates. When the confluence of the P1 generation monoclonal cells reached more than 80%, subculture was performed, followed by routine digestion and subculture. All the P1 generation monoclonal cells in the 96-well plates were digested, centrifuged, and directly inoculated into 24-well plates. The plates were then cultured in a carbon dioxide incubator to obtain P1+1 generation monoclonal cells. The culture medium of the P1 generation monoclonal cells in the 24-well plates was replaced every 2-3 days.

[0072] The P1+1 generation monoclonal cells were cultured in 24-well plates. When the confluence of the P1+1 generation monoclonal cells reached more than 80%, they were subcultured, digested and subcultured as usual. All the P1+1 generation monoclonal cells in the 24-well plates were digested and centrifuged. The P1+1 generation monoclonal cells were directly inoculated into 6-well plates and cultured in a carbon dioxide incubator to obtain P1+2 generation monoclonal cells. The culture medium of the P1+1 generation monoclonal cells in the 6-well plates was replaced every 2-3 days.

[0073] P1+2 monoclonal cells were cultured in 6-well plates. When the confluence of P1+2 monoclonal cells reached more than 80%, they were subcultured and the culture supernatant of P1+2 monoclonal cells in the 6-well plates was collected and frozen at -20 degrees Celsius for the detection of AAT secretion and urea synthesis. All P1+2 monoclonal cells in the 6-well plates were digested, centrifuged and the cells were counted at 10,000 cells / cm 2 The cells were seeded into a 10 cm culture dish at a high density and cultured in a carbon dioxide incubator to obtain P1+3 generation monoclonal cells. The culture medium of the cells in the 10 cm culture dish was replaced every 2-3 days.

[0074] The P1+3 monoclonal cells were placed in a 10 cm culture dish for culture. When the confluence of the P1+3 monoclonal cells reached more than 80%, they were subcultured, digested and subcultured as usual. All the P1+3 monoclonal cells in the 10 cm culture dish were digested and centrifuged to count the cells. The number of cells was calculated as 10,000 cells / cm. 2 The cells were seeded into a 10 cm culture dish at a high density and cultured in a CO2 incubator to obtain P1+4 monoclonal cells. After confluence, the P1+4 monoclonal cells were harvested and cryopreserved. The culture medium of the P1+3 monoclonal cells in the 10 cm culture dish was replaced every 2-3 days.

[0075] After multiple cultures, 11 monoclonal cells survived out of the 21 monoclonal cell lines. The 11 monoclonal cell lines were labeled A, B, C, D, E, F, G, H, I, J, K, L, M, and N. AAT, ALB, and urea were detected in the 11 monoclonal cell lines. The test results are shown in Table 1.

[0076] Table 1

[0077] According to the AAT, ALB and urea synthesis test results in Table 1, 6 monoclonal cells were selected from the 11 P1+2 generation monoclonal cells for the second round of screening. The 6 monoclonal cells were recorded as 6 P2 generation monoclonal cells, and the 6 monoclonal cells were A, B, C, F, G, and H.

[0078] Resuspend six P2 monoclonal cell lines in reprogramming medium and plate them. Subculture the six P2 monoclonal cell lines when their confluence reaches 80%. Select the monoclonal cells and dilute them to 70 cells / 20 ml using limiting dilution. Add 200 μl of culture medium to each well of a 96-well plate, equivalent to approximately 0.7 cells / well / 200 μl.

[0079] 24 hours after plating, wells containing only single P2 monoclonal cells were selected from the 96-well plate and marked. When the confluence of the P2 monoclonal cells reached 80%, they were subcultured, digested, and passaged as usual. After all the P2 monoclonal cells in the 96-well plate were digested, the P2 monoclonal cells were directly inoculated into a 24-well plate after centrifugation and cultured in a CO2 incubator to obtain P2+1 monoclonal cells. The culture medium of the P2+1 monoclonal cells in the 24-well plate was replaced or passaged approximately every 3-4 days.

[0080] When the confluence of the P2+1 generation monoclonal cells in the 24-well plate reaches more than 80%, they are subcultured, digested and subcultured as usual. All the P2+1 generation monoclonal cells in the 24-well plate are digested, centrifuged and directly inoculated into a 6-well plate, which is then cultured in a carbon dioxide incubator to obtain P2+2 generation monoclonal cells. The culture medium of the P2+2 generation monoclonal cells in the 6-well plate is replaced or subcultured approximately every 3-4 days.

[0081] When the confluence of P2+2 monoclonal cells in the 6-well plate reached more than 80%, subculture was performed, and routine digestion and subculture were performed. All P2+2 monoclonal cells in the 6-well plate were digested and centrifuged to count the cells. The cells were counted according to 20,000 / cm 2 The cells were seeded into a 10 cm culture dish at a high density and cultured in a carbon dioxide incubator to obtain P2+3 generation monoclonal cells. The supernatant of the P2+3 generation monoclonal cells was collected and centrifuged at 200 g for 5 minutes to remove P2+3 generation monoclonal cell debris. The supernatant was stored at -80 degrees Celsius for urea and AAT synthesis detection.

[0082] When the confluence of P2+3 monoclonal cells in 10cm culture dish reaches more than 80%, subculture is performed, and routine digestion and subculture are performed. All P2+3 monoclonal cells in 10cm culture dish are digested and centrifuged to count the cells. The number of cells is 20,000 / cm 2 The cells were inoculated into T75 flasks at a high density and cultured in a carbon dioxide incubator to obtain P2+4 monoclonal cells.

[0083] Table 2 Test results

[0084] FIG2 is a schematic diagram of the cell morphology of the cell line B42 according to an embodiment of the present invention; FIG3 is a schematic diagram of the cell morphology of the cell line B42 according to an embodiment of the present invention after culture to the fifteenth generation.

[0085] Cell line B42 was selected as an immortalized hepatic progenitor cell through screening and subcultured to the fifteenth generation to obtain the fifteenth generation cell line B42. Cell morphology of cell line B42 and the fifteenth generation cell line B42 were photographed to produce Figures 2 and 3. Referring to Figures 2 and 3, the cell morphology of the fifteenth generation cell line B42 is nearly identical to that of cell line B42, indicating that cell line B42 retains the characteristics of hepatic progenitor cells after subculture, thus proving that cell line B42 is immortalized.

[0086] Flow cytometry was used to identify and analyze the cell line B42. The specific steps included:

[0087] Surface marker staining of cell line B42: cultured cell line B42 was taken, the culture medium was aspirated, and the cells were rinsed with 5 ml of sterile PBS buffer. 2 ml of trypsin digestion solution was then added dropwise to a culture dish to digest the cells to obtain a cell mixture. The cell mixture was placed in a 15 ml centrifuge tube, and the centrifuge tube was placed in a centrifuge and centrifuged at 200 g for 5 minutes. The supernatant of the centrifuged cell mixture was discarded to obtain a cell pellet.

[0088] Add 100 μL of staining buffer to the cell pellet to resuspend the cell pellet, and transfer the resuspended cell pellet to 6 1.5 ml centrifuge tubes with a specification of 100 μL / tube. Add 5 μL of the flow cytometry antibody to be tested to each of the above 6 1.5 ml centrifuge tubes and mix them by pipetting; place the centrifuge tubes in a 2-8 ° C refrigerator for 30 minutes, add sterile PBS buffer to each centrifuge tube at a dose of 800 microliters / tube, and then place the centrifuge tubes in a centrifuge and centrifuge at 300g for 5 minutes. After centrifugation, discard the supernatant, place the centrifuge tubes in a test tube rack and incubate at 37 degrees Celsius for 20 minutes, add 400 μL of staining buffer to each centrifuge tube after incubation to resuspend the cell pellet, and then transfer them to the flow tube. The resuspended cell mixture is subjected to flow cytometry detection of surface markers;

[0089] The names of the antibodies used in the flow cytometric detection of surface markers are: CD24, CD326, CD34, CD45 and HLA-DR / DP / DQ; among them, CD34 was purchased from abcam, catalog number ab81289; CD45 was purchased from abcam, catalog number ab40763; HLV-DR / DP / DQ was purchased from abcam, catalog number ab7856; CD326 was purchased from BD Biosciences, catalog number 565399; CD24 was purchased from abcam, catalog number ab290730; staining buffer was purchased from Biosharp, catalog number BL1136A, and trypsin digestion solution was purchased from Source Culture, catalog number S310JV.

[0090] Figure 4 is a schematic diagram of the flow cytometry detection results of the cell line B42 according to an embodiment of the present invention that negatively expresses CD34; Figure 5 is a schematic diagram of the flow cytometry detection results of the cell line B42 according to an embodiment of the present invention that negatively expresses CD45; Figure 6 is a schematic diagram of the flow cytometry detection results of the cell line B42 according to an embodiment of the present invention that negatively expresses HLA-DRPQ; Figure 7 is a schematic diagram of the flow cytometry detection results of the cell line B42 according to an embodiment of the present invention that positively expresses CD24; Figure 8 is a schematic diagram of the flow cytometry detection results of the cell line B42 according to an embodiment of the present invention that positively expresses CD326.

[0091] Flow cytometry was performed on the cell line B42. Referring to Figures 4, 5, 6, 7 and 8, the cell line B42 negatively expressed CD34, CD45, and HLA-DRPQ, and positively expressed CD24 and CD326.

[0092] 2. Treatment of liver failure with cell line B42

[0093] 1) Preparation of D-gal inducer: Weigh 500 mg of D-gal, add 10 mL of normal saline, and vortex to dissolve to obtain D-gal inducer at a concentration of 50 mg / mL. Store the D-gal inducer at 2-8°C and use within 2 hours. D-gal was obtained from Sigma.

[0094] 2) Preparation of LPS inducer: Weigh 6.9 mg of LPS, add 6.9 mL of NS, and vortex to dissolve to obtain a first solution. The concentration of the first solution is 1 mg / mL. The first solution is aliquoted into 100 μL tubes and stored at -80°C. On the day of the experiment, take one tube and add 0.9 mL of NS to obtain a second solution. The concentration of the second solution is 100 μg / mL. Take 0.4 mL of the second solution, add 9.6 mL of NS to the second solution, and mix well to obtain the LPS inducer. The final concentration of the LPS inducer is 4 μg / mL. The LPS inducer is stored at 2-8°C and used within 2 hours. LPS was obtained from Shanghai Maokang Biotechnology Co., Ltd.

[0095] 3) Modeling method: After acclimating to the environment for 2 days, 24 mice were randomly selected and weighed. D-gal inducer (500 mg / kg) was injected intraperitoneally at 10 mL / kg. 3 hours later, LPS inducer (40 μg / kg) was injected intraperitoneally at 10 mL / kg to establish a liver failure mouse model. Mouse species: C57BL / 6N mice; mouse grade: SPF; mouse sex: male; mouse age: 8 weeks; number of mice: 24; mouse source: Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.; mouse license number: SCXK (Beijing) 2021-0006; mouse qualification certificate number: 1100112301103117632.

[0096] 4) Drug Preparation: Remove several B42 cell lines from the liquid nitrogen tank and place them on dry ice for transport. Immediately immerse the B42 cell line in a 37°C water bath and gently shake until the liquid is completely melted. Shake well. Be careful not to immerse the hose connector in the water bath. Remove all the liquid with a pipette and add it to a 50mL sterile centrifuge tube. Centrifuge at 200g for 5 minutes. After centrifugation, discard the supernatant and dilute the B42 cell line to 5×10 with saline. 6 / mL, and filtered with a 40μm cell strainer before use to obtain an injection solution containing cell line B42.

[0097] 5) Animal grouping:

[0098] The liver failure mice were randomly divided into a control group and an experimental group, with 12 mice in each group. The control group was injected with 0.2 mL of normal saline through the tail vein 30 minutes before the injection of LPS inducer according to Table 3; the experimental group was injected with 0.2 mL of injection solution containing cell line B42 through the tail vein 30 minutes before the injection of LPS inducer according to Table 3:

[0099] Table 3 Animal grouping

[0100] 6) Test substance injection

[0101] The steps for tail vein injection are as follows:

[0102] a. Mouse acute liver failure model is placed in a holder;

[0103] b. Straighten and fix the tail of the mouse model of acute liver failure, and wipe it with alcohol for disinfection;

[0104] c. Pipette the cells evenly and draw 0.2 mL of injection solution containing cell line B42 into the syringe;

[0105] d. Insert the syringe needle into the tail vein of the mouse acute liver failure model and inject the injection solution containing the cell line B42 over a period of approximately 1 minute.

[0106] e. After the injection is complete, remove the syringe and apply pressure to the puncture site with a dry cotton ball to stop bleeding;

[0107] f. Mice with acute liver failure model were returned to cages for breeding.

[0108] 7) Clinical observation

[0109] After the liver failure mouse models in the control group and experimental group were injected with the injection solution containing the cell line B42: the liver failure mouse models in the control group showed no medication-related adverse reactions, while the liver failure mouse models in the experimental group mainly showed symptoms of decreased activity, mental depression and low body temperature.

[0110] 8) Survival rate

[0111] FIG9 is a comparative schematic diagram of the survival rates of the experimental group and the control group of mice in the acute liver failure model according to an embodiment of the present invention.

[0112] In the experimental group of mice with acute liver failure, survival was observed and death time was recorded after injection of the D-gal inducer (500 mg / kg) combined with the LPS inducer (40 μg / kg). Survival curves were generated using GraphPad Prism 8. Referring to Figure 9 , the survival rate of the mice in the experimental group with acute liver failure was 60%, while the survival rate of the mice in the control group was 0%, indicating a significant difference between the experimental groups. Therefore, hepatic progenitor cells have a significant therapeutic effect in the mouse model of acute liver failure induced by the D-gal inducer (500 mg / kg) combined with the LPS inducer (40 μg / kg).

[0113] While the embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations of these embodiments are possible. However, it should be understood that such modifications and variations are within the scope and spirit of the present invention as set forth in the claims. Furthermore, the invention described herein is susceptible to other embodiments and may be practiced or implemented in a variety of ways.

Claims

1. A method for establishing a hepatic progenitor cell line, characterized in that: The following steps are involved: S0: providing hepatic progenitor cells, a mixed solution and a reprogramming medium, wherein the mixed solution comprises a serum-free medium, polyethyleneimine and a CLP plasmid; S1: performing transfection treatment on the hepatic progenitor cells and the mixed solution to obtain transfected hepatic progenitor cells; S2: placing the transfected hepatic progenitor cells into the reprogramming medium for subculture to obtain immortalized hepatic progenitor cells.

2. The method for establishing a hepatic progenitor cell line according to claim 1, characterized in that: The volume ratio of the serum-free culture medium to the polyethyleneimine is (500-1000):

1.

3. The method for establishing a hepatic progenitor cell line according to claim 1, characterized in that: The final concentration of the CLP plasmid is (1-5) μg / μL.

4. The method for establishing a hepatic progenitor cell line according to claim 1, characterized in that: The step of providing hepatic progenitor cells comprises: placing hepatocytes in the reprogramming medium for expansion culture, and then replacing the reprogramming medium with a reduced serum medium for continued culture to obtain hepatic progenitor cells.

5. The method for establishing a hepatic progenitor cell line according to claim 1, characterized in that: The reprogramming medium includes DMEM / F12 basal medium, and based on the content of DMEM / F12 basal medium: 15-25 ng / mL hepatocyte growth factor HGF, 15-25 ng / mL epithelial cell growth factor EGF, 5-15 μM ROCK kinase inhibitor Y27632, 1-5 μM Wnt signaling pathway agonist CHIR99021, 1-5 μM TGF-β signaling inhibitor A8301, 1-5 μM sphingosine-1-phosphate S1P and 5 μM indoleacetic acid LPA.

6. The method for establishing a hepatic progenitor cell line according to claim 5, characterized in that: The reprogramming medium also includes N2 nutrient supplement, B27 nutrient supplement, sodium pyruvate and ascorbic acid.

7. The method for establishing a hepatic progenitor cell line according to claim 1, characterized in that: The reprogramming medium includes DMEM / F12 basal medium, and based on the content of DMEM / F12 basal medium: 1-10% N2 nutrient supplement, 1-10% B27 nutrient supplement, 1mM sodium pyruvate, 5-15μg / mL ascorbic acid, 15-25ng / mL hepatocyte growth factor HGF, 15-25ng / mL epithelial cell growth factor EGF, 5-15μM ROCK kinase inhibitor Y27632, 1-5μM Wnt signaling pathway agonist CHIR99021, 1-5μM TGF-β signal inhibitor A8301, 1-5μM sphingosine-1-phosphate S1P and 5μM indoleacetic acid LPA.

8. A hepatic progenitor cell, characterized in that The hepatic progenitor cell line is prepared by the method for establishing a hepatic progenitor cell line according to any one of claims 1 to 7.

9. The hepatic precursor cell according to claim 8, characterized in that The hepatic progenitor cells negatively express CD34, CD45 and HLA-DRPQ, and positively express CD24 and CD326.

10. A use of the hepatic precursor cells according to any one of claims 8 to 9, characterized in that: Used in preparing drugs for treating acute liver failure.

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