Monoclonal antibodies recognizing human liver progenitor cells and hybridoma cell line HLPC1

Abstract

The present invention discloses a monoclonal antibody for recognizing human liver progenitor cells and a hybridoma cell line HLPC1. Human liver progenitor cells are used as antigens and a hybridoma cell line HLPC1 is constructed by using monoclonal antibody technology, and the deposit number is CCTCC NO: C202253. The hybridoma cell line HLPC1 secretes a monoclonal antibody for recognizing human liver progenitor cells. Flow cytometry, immunohistochemistry, immunofluorescence and immunoblotting are established with this monoclonal antibody as a probe for detecting and analyzing human liver progenitor cells. The present invention provides a specific method for the study of human liver progenitor cells, and various detection techniques based on monoclonal antibodies of human liver progenitor cells can also be used in the study of human liver damage and regeneration.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a monoclonal antibody for recognizing human liver progenitor cells and a hybridoma cell line HLPC1. Background Art

[0002] The liver has the functions of synthesis, detoxification, metabolism, secretion, biotransformation and immune defense. It is mainly composed of hepatocytes and biliary epithelial cells, which are differentiated from liver progenitor cells (LPCs) during development. Hepatocytes have the functions of detoxifying various metabolites, regulating sugar and lipid metabolism, synthesizing serum proteins, and secreting bile. Bile duct epithelial cells form the bile duct to transport bile from hepatocytes to the gallbladder. When the liver is affected by toxic substances, drugs, hepatitis viruses, etc., liver damage will occur, liver inflammation and necrosis will occur, and even cirrhosis, liver cancer, liver failure and other serious liver diseases will occur. At present, liver transplantation is the best choice for the treatment of severe liver disease, but its application is limited by the shortage of donors and high costs. Therefore, patients with severe liver disease often die while waiting for a donated liver.

[0003] When acute liver failure (ALF) occurs, after extensive liver necrosis, liver progenitor cells play a variety of indispensable functions in systemic homeostasis, including the production of albumin, bile, and most coagulation factors, as well as carbohydrates, proteins, fats, hormones, bilirubin, etc. Pathologists believe that the maintenance of liver cell function after extensive liver necrosis in ALF patients mainly depends on the sufficient number and function of activated liver progenitor cells. Given that LPCs-driven liver regeneration is common in severe liver damage, enhancing this liver regeneration mode should be very promising in the clinical treatment of patients with severe liver disease.

[0004] Under normal conditions, hepatocyte-driven liver regeneration is achieved by the proliferation of pre-existing hepatocytes, while LPCs-driven liver regeneration is achieved by the proliferation and subsequent differentiation of LPCs. Therefore, LPCs-driven liver regeneration is very important in cases of severe liver injury accompanied by impaired hepatocyte proliferation. In LPCs-driven liver regeneration, after LPCs proliferate, hepatocytes or cholangiocytes first dedifferentiate into LPCs, and LPCs subsequently differentiate into hepatocytes.

[0005] LPCs are named oval cells or liver stem cells, etc. The nucleus is oval due to the large nuclear to cytoplasm ratio. Anatomically, LPCs are believed to be located in the Hering duct, also known as the intrahepatic bile duct, between the bile duct and the hepatocytes. LPCs express hepatocyte markers such as CK18 and albumin, as well as cholangiocyte markers such as CK19. In addition, LPCs also express markers such as CD34 and CD90. The different expression of these markers in LPCs indicates their liver progenitor cell characteristics and heterogeneity. It is generally believed that LPCs only appear in small numbers in the portal area in normal adult livers, and they appear in large numbers and proliferate in severe or chronic liver damage.

[0006] Therapeutic approaches based on the activation of LPCs represent a potential therapeutic approach for the future treatment of ALF. To date, stem cell therapy for ALF has become a rather hot area in clinical practice. Studies have shown that liver progenitor cells can take over key hepatocyte functions and, over time, differentiate into hepatocytes.

[0007] In the preliminary research of this invention, LPCs were isolated from a small amount of donated adult liver tissue. LPCs have the functions and characteristics of normal hepatocytes and bile duct cells, and are a type of liver stem cells with extensive research and application potential in various animal models of liver diseases. LPCs can differentiate into mature hepatocytes and play important physiological and repair functions, especially in the treatment of severe liver diseases such as liver regeneration and acute liver failure.

[0008] However, due to the lack of specific LPCs cell surface markers, LPCs isolation, culture, identification and tracing are key technologies and bottlenecks in liver disease research. At present, the identification of LPCs cells mainly relies on cell morphology observation and a series of combined determinations of non-specific cell surface antigens. These methods are often complicated to operate and difficult to achieve rapid and efficient cell detection, as well as cell sorting and purification. In addition, due to the differences in LPCs cell phenotypic characteristics among different animal individuals and treatment conditions, it is difficult to ensure the acquisition of high-purity LPCs cells using traditional methods, which limits the research and application of LPCs cells in liver injury and regeneration research.

[0009] Therefore, there is an urgent need to develop an efficient tool that can be used to identify human liver progenitor cells, as well as to trace or label liver progenitor cells. Summary of the invention

[0010] The purpose of the present invention is to provide a monoclonal antibody and a hybridoma cell line HLPC1 for recognizing human liver progenitor cells in view of the deficiencies of the prior art.

[0011] To achieve the above-mentioned purpose, the present invention provides the following technical solutions: As a first aspect, a monoclonal antibody for recognizing human liver progenitor cells is provided, which is deposited in the China Center for Type Culture Collection and secreted by the anti-human liver progenitor cell monoclonal antibody hybridoma cell line HLPC1 with the deposit number: CCTCC NO: C202253.

[0012] Furthermore, the monoclonal antibody is a mouse IgG2a monoclonal antibody.

[0013] As a second aspect, the present invention provides an anti-human liver progenitor cell monoclonal antibody hybridoma cell line HLPC1, which was deposited in the China Center for Type Culture Collection and Wuhan University, Wuhan, China on June 2, 2022, with the deposit number: CCTCC NO: C202253, and specifically secretes the above-mentioned monoclonal antibody.

[0014] As a third aspect, the present invention also provides an application of the monoclonal antibody for recognizing human liver progenitor cells in the detection of human liver progenitor cells, specifically: based on the monoclonal antibody as a primary antibody, or as a probe applied to flow cytometry, immunohistochemistry, immunofluorescence and immunoblotting, to detect and analyze human liver progenitor cells.

[0015] As a fourth aspect, the present invention also provides a use of the monoclonal antibody for recognizing human liver progenitor cells in the culture, passage and expansion of human liver tissue or human liver progenitor cells.

[0016] The beneficial effects of the present invention are as follows: the present invention has successfully developed a monoclonal antibody for recognizing human liver progenitor cells and its hybridoma cell line HLPC1, which has the advantages of high specificity and good repeatability, can specifically recognize the surface protein on the membrane of human liver progenitor cells, provides a specific method for the study of human liver progenitor cells, and provides an efficient tool for the rapid sorting, identification and tracing or labeling of human liver progenitor cells. Various detection technologies based on monoclonal antibodies of human liver progenitor cells can also be applied to the study of liver damage and regeneration of various severe liver diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the morphology of human liver progenitor cells obtained by the collagenase four-step perfusion method and density gradient centrifugation method, among which, Figure 1 (A) is the morphology of the sixth generation of cultured human liver progenitor cells (100X); Figure 1 (B) is the morphology of human liver progenitor cells at the 6th passage (200X);

[0018] Figure 2 This is a growth curve of human liver progenitor cells;

[0019] Figure 3This is a cytochemical staining diagram of human liver progenitor cells expressing AFP, Albumin, CK8 and CK19 cell markers; Figure 3 (A) is the AFP staining image of the 6th generation human liver progenitor cells; Figure 3 (B) is the Albumin staining image of the 6th generation human liver progenitor cells; Figure 3 (C) in the middle is the CK8 staining image of the 6th generation human liver progenitor cells; Figure 3 (D) in the middle is the CK19 staining image of the 6th generation human liver progenitor cells;

[0020] Figure 4 This is a diagram for analyzing the multidirectional differentiation ability of human liver progenitor cells, among which, Figure 4 (A) shows the morphology of the sixth generation of cultured human liver progenitor cells; Figure 4 (B) is an Oil Red O staining image of human liver progenitors differentiating into adipocytes; Figure 4 (C) is an Alizarin red staining image of human liver progenitor cells differentiating into osteoblasts; Figure 4 (D) is a glycogen staining image of human liver progenitor cells differentiating into hepatocytes;

[0021] Figure 5 This is the result of indirect immunofluorescence staining to detect human liver progenitor cells using HLPC1 monoclonal antibody as primary antibody;

[0022] Figure 6 This is the result of flow cytometry detection of human liver progenitor cells using HPLC1 monoclonal antibody as the primary antibody; Figure 6 (A) is the analysis result of flow cytometry of negative control. Figure 6 (B) is a flow cytometry analysis result diagram using monoclonal antibody HLPC1 as the primary antibody;

[0023] Figure 7 This is the result of detecting human liver progenitor cells by immunohistochemical staining using HLPC1 monoclonal antibody as primary antibody;

[0024] Figure 8 This is the result of Western blotting to detect human liver progenitor cells using HLPC1 monoclonal antibody as primary antibody. DETAILED DESCRIPTION

[0025] The present invention will be described in detail below through specific examples. These examples are provided in order to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art. As mentioned throughout the specification and claims, "including" or "comprising" is an open term and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present invention, but the description is for the purpose of the general principles of the specification and is not intended to limit the scope of the present invention.

[0026] The monoclonal antibody specifically recognizing human liver progenitor cells of the present invention can specifically bind to the membrane surface protein of human liver progenitor cells, and has no cross reaction with other cells such as rat bone marrow mesenchymal stem cells, mouse bone marrow mesenchymal stem cells, human adipose mesenchymal stem cells, human liver cell lines huh751 and HepG2, human liver mesenchymal stem cells, and human liver stellate cell line HSC-Li.

[0027] In addition, the present invention also provides a mouse hybridoma cell line HLPC1 that produces the above monoclonal antibody. The spleen cells of Balb / c mice immunized with human liver progenitor cells and mouse myeloma FO cells are obtained respectively, fused under the action of polyethylene glycol, selectively cultured in HAT and HT medium, screened positive cell clones by indirect immunofluorescence method, subcloned and screened for 3 consecutive times, selected positive cell clones, and finally obtained cells grown from single cell clones. After expanded culture, the mouse hybridoma cell line HLPC1 is successfully constructed and preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: C202253, and is classified and named as the monoclonal antibody hybridoma cell line HLPC1 against human liver progenitor cells. The cell line can stably secrete the above monoclonal antibody, harvest the cell culture supernatant, or inoculate the cells into the peritoneal cavity of Balb / c mice to obtain ascites, and separate and purify the above monoclonal antibody by various recognized technical methods.

[0028] Example 1: Isolation, culture, identification, cryopreservation and recovery of human liver progenitor cells.

[0029] (1) Isolation and culture of human liver progenitor cells

[0030] Obtain a small portion of donated adult donor liver tissue under sterile conditions, rinse the surface with 800 ml of PB buffer precooled to 4°C, treat the donated liver tissue with a four-step in vitro perfusion method using split enzymes and collagenase, and obtain human liver progenitor cells by density gradient centrifugation combined with adherent screening. Use DMEM culture medium (v / v) containing 10% fetal bovine serum, and culture the primary cultured cells in an incubator at 37°C and 5% (v / v) CO2. After 24-48 hours, the non-adherent cells are removed by replacing the DMEM culture medium, and adherent cells can be seen. After 3-5 days, clone-like growing cells can be observed, and they slowly proliferate to form dense clones. When the cells reach 80% to 90% fusion, they show a colony-like distribution, and are digested with 0.25% (w / v) trypsin, subcultured in T25 culture medium, and cultured and subcultured continuously. Figure 1 (A) shows the morphology of the sixth generation of cultured human liver progenitor cells (100 times). Figure 1 (B) shows the morphology of human liver progenitor cells at passage 6 (200 times).

[0031] (2) Identification of growth characteristics of human liver progenitor cells:

[0032] Human liver progenitor cells at the sixth passage were cultured and cultured at 2.0 × 10 4 The initial density of cells / well was inoculated into 6-well plates, and counting began after 24 hours of culture. Counting was performed every 24 hours, and cells in 3 wells were counted each time. The average value was obtained for kinetic analysis. The experimental results suggest that human liver progenitor cells have a good proliferation ability, such as Figure 2 As shown in the figure, P6 is the sixth generation of cultured human liver progenitor cells, and P10 is the tenth generation of cultured human liver progenitor cells.

[0033] (3) Identification of cell markers of human liver progenitor cells:

[0034] The sixth generation of human liver progenitor cells were taken, and mouse anti-human AFP, Albumin, CK18 and CK19 antibodies were used as primary antibodies to establish a cytochemical staining method to detect human liver progenitor cells, and to detect and analyze the positive expression of human liver progenitor cells after culture.

[0035] Well-growing human liver progenitor cells were selected for cell slide fixation, blocked at 37°C for 30 minutes, incubated at 37°C for 2 hours with monoclonal antibody as primary antibody, washed 3 times with PBS for 2 minutes each time, and then HRP-labeled anti-mouse immunoglobulin antibody was added as secondary antibody, incubated at 37°C for 1 hour, and washed with PBS. DAB color development, color development time was controlled under a microscope, washed with distilled water, counterstained with hematoxylin for 2 minutes, and the distilled water washing method was the same as before. The results are shown as follows Figure 3As shown in (A) to (D), human liver progenitor cells were positive for AFP, Albumin, CK18, and CK19.

[0036] (4) Identification of the multi-differentiation potential of human liver progenitor cells:

[0037] When the sixth generation cells were 80% to 90% confluent, they were digested with 0.25% (w / v) trypsin and plated at 5×10 4 The cells were seeded in 6-well culture plates at a density of 100 cells / well. The multipotent differentiation potential of human liver progenitor cells was evaluated under adipogenic, osteogenic and hepatogenic differentiation conditions, and undifferentiated 6th generation human liver progenitor cells were used as the control group. Figure 4 (A) shows the morphology of human liver progenitor cells at passage 6.

[0038] After 7 days of adipogenic induction, the cells were cultured for 21 days. The lipid droplets were not obvious after Oil Red O staining, which confirmed that the ability of human liver progenitor cells to differentiate into adipocytes was weak. Figure 4 As shown in (B) in the figure.

[0039] After induction culture in osteoblast medium, cells adhered to the wall and gradually grew larger, forming tighter intercellular connections. Calcified nodule-like structures appeared in the cells around 14 to 21 days. Alizarin red staining showed that red-stained cells aggregated in specific areas, reflecting that human liver progenitor cells can differentiate into osteoblasts, such as Figure 4 As shown in (C) in the figure.

[0040] After 5-7 days of induction culture into hepatocytes, as the induction time prolonged, the number of cells increased and formed tighter intercellular connections. After 21 days of culture, PAS staining (periodic acid Schiff staining) showed that human liver progenitor cells had a strong glycogen storage capacity, showing their potential to differentiate into functional mature hepatocytes, such as Figure 4 As shown in (D) in the figure.

[0041] (5) Cryopreservation and recovery of human liver progenitor cells:

[0042] Human liver progenitor cells were stored in DMEM culture medium containing 10% DMSO (v / v). After being frozen in liquid nitrogen for several months, the MSC cell characteristics and functions were still maintained after resuscitation culture.

[0043] Example 2: Preparation and purification of anti-human liver progenitor cell monoclonal antibodies.

[0044] (1) Animal immunization:

[0045] Human liver progenitor cells of passage 6 were selected and the cell concentration was 1.0×10 6Balb / c mice were intraperitoneally immunized once a week for three times. On the 20th day, blood was collected from the mice by eyeball blood sampling and serum was obtained by centrifugation. The antibody titer of the serum was detected by indirect immunofluorescence staining, and the mice corresponding to the serum samples with the highest titer were selected for booster immunization. The cell concentration for booster immunization was adjusted to 2.0×10 6 / , and cell fusion was performed 3 days later.

[0046] (2) Preparation of mouse hybridoma cell lines:

[0047] Peritoneal macrophages and spleen cells of normal mice without immunization were collected as feeder cells. The spleen cells of mice immunized with human liver progenitor cells were obtained aseptically and mixed with myeloma cells FO at a ratio of 10:1. Cell fusion was mediated by 50% (w / v) PEG in a 38°C water bath. The fused cell clumps were inoculated into the 96-well plate prepared the day before and cultured in a 37°C, 5% (v / v) CO2 incubator. HAT culture medium was replaced every 3 days, and HT culture medium was used to continue culturing hybridoma cells after two weeks.

[0048] When the hybridoma cells grow to 1 / 3~1 / 2 of the bottom area of ​​the well, the indirect immunofluorescence staining method is used to screen positive clones. The method is as follows: select well-growing human liver progenitor cells for climbing and fixing, take the culture supernatant of hybridoma cells as the primary antibody, incubate at 37°C for 1h, wash 3 times with PBS, 1 minute each time, add FITC-labeled secondary antibody, incubate at 37°C for 30min, and wash with PBS. The positive clones screened out are inoculated into a new 96-well plate containing feeder cells by limiting dilution method for subcloning culture. Repeat the above steps until the positive rate of cloned cell antibody detection reaches 100% for 3 consecutive times. The hybridoma cells are continuously passaged in vitro, repeatedly frozen and revived, and regularly screened and tested until the hybridoma cell line can continuously and stably secrete monoclonal antibodies.

[0049] (3) Preparation and purification of monoclonal antibodies:

[0050] Balb / c mice were pretreated with paraffin oil and injected intraperitoneally with 5×10 HLPC1 hybridoma cells per mouse. 6 Cells. The abdomen of the mouse was obviously swollen at about 2 weeks. The mouse was killed by bleeding from the eyeball and the ascites was collected. The ascites and saline were mixed at a ratio of 1:1 (v / v), and saturated ammonium sulfate was added while stirring until the final saturation of ammonium sulfate was 50%. The immunoglobulin was crudely extracted and purified by SPA (Staphylococcus aureus protein A method) to obtain the monoclonal antibody HLPC1 against human liver progenitor cells.

[0051] (4) Ascites titer and subtype identification of anti-human liver progenitor cell monoclonal antibody HLPC1:

[0052] The purified monoclonal antibody HLPC1 was diluted in equal proportions and used as the primary antibody. The FITC-labeled goat anti-mouse immunoglobulin antibody was used as the secondary antibody. The titer of the antibody was 1.2×10 5 The hybridoma cell culture supernatant was obtained, and the subtype of the monoclonal antibody was determined to be IgG2a using the ELISA method (Enzyme-Linked Immunosorbent Assays).

[0053] Example 3: Specificity identification of anti-human liver progenitor cell monoclonal antibodies.

[0054] (1) Tissue-specific detection of human liver:

[0055] Human tissue specimens such as stomach, heart, lung, liver, small intestine and fat were all paraffin blocks from surgically removed patient tissues. The paraffin sections of each tissue were detected using the anti-human liver progenitor cell monoclonal antibody HLPC1 by immunohistochemical staining. The results showed that scattered positive cells were visible in the human liver tissue sections, while the rest of the tissues were negative, which shows that the monoclonal antibody has the characteristics of specific binding and recognition of human liver progenitor cells.

[0056] (2) Species-specific detection:

[0057] Normal pig, rabbit, rat, mouse liver and other tissues were stained with immunohistochemistry, with human liver progenitor cells as the positive control and anti-human liver progenitor cell monoclonal antibody HLPC1 as the antibody to detect paraffin sections of pig and other animal tissues. The results showed that all sections of other species were negative, indicating that the monoclonal antibody HLPC1 has a strong specificity for human liver progenitor cells.

[0058] Example 4: Application of monoclonal antibodies in detecting human liver progenitor cells by immunofluorescence assay.

[0059] In this example, the monoclonal antibody against human liver progenitor cells was used as the primary antibody, and the fluorescent-labeled goat or sheep anti-mouse immunoglobulin antibody was used as the secondary antibody to establish an indirect immunofluorescence staining method to detect human liver progenitor cells, and observe and analyze the positive expression of human liver tissue or human liver progenitor cells in culture under a fluorescence microscope. Specifically:

[0060] Well-growing human liver progenitor cells were selected for cell climbing and cell fixation. The monoclonal antibody HLPC1 was used as the primary antibody, and the indirect immunofluorescence staining method was adopted (the specific method is the same as the preparation of the hybridoma cell line described in step (2) of Example 2). The positive rate was calculated under a fluorescence microscope. The results are as follows: Figure 5As shown, the results showed that 100% of human liver progenitor cells were positive and all were expressed on the cell membrane surface. The experiment showed that the monoclonal antibody HLPC1 can be used as a surface marker of human liver progenitor cells and can be effectively applied to immunofluorescence staining, which is conducive to further exploring the biological characteristics of the corresponding antigens of human liver progenitor cells.

[0061] Example 5: Application of monoclonal antibodies in flow cytometry detection of human liver progenitor cells.

[0062] In this example, a monoclonal antibody against human liver progenitor cells was used as the primary antibody, and a fluorescently labeled goat or sheep anti-mouse immunoglobulin antibody was used as the secondary antibody to establish a flow cytometer to detect human liver progenitor cells. The flow cytometer was used to detect and analyze the positive expression of human liver progenitor cells after culture. Specifically:

[0063] We selected the sixth generation of well-grown human liver progenitor cells and used the monoclonal antibody HLPC1 as a probe and FITC-labeled anti-mouse immunoglobulin antibody as a secondary antibody to detect human liver progenitor cells by flow cytometry. Figure 6 As shown in (B), the horizontal axis is the fluorescence intensity, the vertical axis is the number of cells, the positive rate of human liver progenitor cells is 88.6%, and the flow cytometry test results using mouse immunoglobulin as the primary antibody and as a negative control are shown in Figure 6 As shown in (A), the monoclonal antibody HLPC1 can efficiently bind to the surface protein of human liver progenitor cells and is suitable for the quantitative analysis of human liver progenitor cells.

[0064] Example 6: Application of monoclonal antibody HLPC1 in detecting human liver progenitor cells by immunohistochemistry.

[0065] In this example, a monoclonal antibody against human liver progenitor cells is used as the primary antibody, and an HRP-labeled goat or sheep anti-mouse immunoglobulin antibody is used as the secondary antibody to establish an immunohistochemical staining or cytochemical staining method to detect human liver or human liver progenitor cells, and to detect and analyze the positive expression of human liver tissue or human liver progenitor cells after culture. Specifically:

[0066] Well-growing human liver progenitor cells were selected for cell climbing and fixation. The cells were blocked at 37°C for 30 minutes, and monoclonal antibodies were used as primary antibodies. The cells were incubated at 37°C for 2 hours. The cells were washed with PBS three times for 2 minutes each time. HRP-labeled anti-mouse immunoglobulin antibodies were then added as secondary antibodies. The cells were incubated at 37°C for 1 hour and washed with PBS. DAB was used for color development, and the color development time was controlled under a microscope. After washing with distilled water, the cells were counterstained with hematoxylin for 2 minutes. The distilled water washing method was the same as before. The results were observed under a microscope, and the positive rate of human liver progenitor cells was 100%. Figure 7As shown. This shows that monoclonal antibodies have good specificity and sensitivity, and can be effectively used in cell or tissue immunochemical staining methods. The expression of corresponding antigens in human liver progenitor cells can be effectively observed under a microscope, which is helpful for the localization and quantitative analysis of human liver progenitor cells.

[0067] Example 7: Application of monoclonal antibodies in detecting human liver progenitor cells by western blot.

[0068] In this example, a monoclonal antibody against human liver progenitor cells was used as the primary antibody, and an HRP-labeled goat or sheep anti-mouse immunoglobulin antibody was used as the secondary antibody to establish an immunoblotting method to detect human liver or human liver progenitor cells, and analyze the positive expression of human liver tissue or human liver progenitor cells after culture. Specifically:

[0069] The in vitro expanded human liver progenitor cells were lysed with protein lysis buffer, and the total protein was extracted. SDS-PAGE electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was performed, and the membrane was blocked with 5% skim milk powder at room temperature for 1 hour after electrophoresis transfer. The monoclonal antibody HLPC1 was added as the primary antibody, and the cells were incubated at 4°C overnight. The cells were washed with TBST, where TBST was prepared by adding 0.5% (v / v) Tween-20 to TBS (Tris buffered saline). After washing, the secondary antibody was added, and the cells were reacted at room temperature for 2 hours. After washing with TBST, ECL was added for 1 minute, and the expression of the corresponding antigen was detected by exposure in a dark room. The results showed that the monoclonal antibody HLPC1 specifically bound to a protein band with a molecular weight of approximately 40KD (kilodaltons), such as Figure 8 The results show that the monoclonal antibody can be used in immunoblotting to qualitatively and semi-quantitatively analyze the corresponding antigens of human liver progenitor cells, and further extract the antigens and detect their expression and biological characteristics in human liver progenitor cells.

[0070] It should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Without making creative work, any modification, equivalent replacement, improvement, etc., shall be included in the protection scope of the present invention.

Claims

1. A monoclonal antibody that recognizes human liver progenitor cells, characterized in that It is secreted and produced by the hybridoma cell line HLPC1 with the deposit number: CCTCC NO: C202253.

2. The monoclonal antibody for recognizing human liver progenitor cells according to claim 1, characterized in that The monoclonal antibody is a mouse IgG2a monoclonal antibody.

3. Use of the monoclonal antibody for recognizing human liver progenitor cells according to claim 1 or 2 in the detection of human liver progenitor cells, characterized in that: The application is specifically: the monoclonal antibody is used as a probe in flow cytometry, immunohistochemistry, immunofluorescence or immunoblotting.

4. Use of the monoclonal antibody for recognizing human liver progenitor cells according to claim 1 or 2 in the culture, passage and expansion of human liver tissue or human liver progenitor cells.

5. A hybridoma cell line HLPC1, characterized in that It was deposited in the China Center for Type Culture Collection on June 2, 2022, with the accession number: CCTCC NO: C202253.

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