Monoclonal antibody and hybridoma cell line HLM1 that recognize human liver mesenchymal stem cells

Abstract

The present invention discloses a monoclonal antibody for identifying human liver mesenchymal stem cells and a hybridoma cell line HLM1. Human liver mesenchymal stem cells are used as antigens and a hybridoma cell line HLM1 is constructed by using monoclonal antibody technology, and the deposit number is CCTCC NO: C202250. The hybridoma cell line HLM1 secretes a monoclonal antibody for identifying human liver mesenchymal stem cells. Flow cytometry, immunohistochemistry, immunofluorescence and immunoblotting are established using this monoclonal antibody as a probe for detecting and analyzing human liver mesenchymal stem cells. The present invention provides a specific method for the study of human liver mesenchymal stem cells, and a variety of detection techniques based on monoclonal antibodies of human liver mesenchymal stem cells can also be used in the study of human liver injury and regeneration.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a monoclonal antibody for identifying human liver mesenchymal stem cells and a hybridoma cell line HLM1. Background Art

[0002] The liver has the functions of synthesis, detoxification, metabolism, secretion, biotransformation and immune defense. When affected by hepatotoxic substances, drugs, hepatitis viruses, etc., serious damage will occur, leading to abnormal liver function and even a large number of liver cell necrosis, thus causing liver failure. At present, liver transplantation is the best treatment for acute and chronic liver failure, but its application is limited by donor shortage and high cost. Mesenchymal stem cells (MSC) have self-renewal ability, multidirectional differentiation ability and low immunogenicity. It is considered to be an ideal cell source for research and treatment. At present, MSCs from various sources such as bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, etc. have been studied and applied in various liver diseases.

[0003] According to literature reports, Herrera et al. first isolated human liver mesenchymal stem cells (HLMSC) from adult liver tissue in 2006, and analyzed the biological characteristics of HLMSC. They found that its CD34, CD45, CD133 and other hematopoietic stem cell surface molecular markers were negatively expressed, while CD29, CD44, CD73, CD90 and other markers were positively expressed. In addition, HLMSC can secrete albumin, urea and other functions.

[0004] In the preliminary research of the present invention, HLMSCs were isolated and cultured from a small amount of donated adult liver tissues, and the HLMSCs were tested and analyzed. HLMSCs have normal MSC cell functions and characteristics, etc. It is a type of mesenchymal stem cells with extensive research and application potential in various animal models of liver diseases. HLMSCs can differentiate into mature hepatocytes and various other types of cells, especially showing their unique value in liver injury and regeneration, and the treatment of other liver diseases, and play an important physiological and repair function.

[0005] However, due to the lack of specific HLMSC cell surface markers, HLMSC isolation, culture, identification and tracing are key technologies and bottlenecks in liver disease research. At present, the identification of HLMSC 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 HLMSC cell phenotypic characteristics among different animal individuals and treatment conditions, it is difficult to ensure the acquisition of high-purity HLMSC cells using traditional methods, which limits the research and application of HLMSC cells in human liver injury and regeneration research.

[0006] Therefore, it is urgent to develop an efficient tool for identifying human liver mesenchymal stem cells. Summary of the invention

[0007] The purpose of the present invention is to provide a monoclonal antibody and a hybridoma cell line HLM1 for identifying human liver mesenchymal stem cells in view of the deficiencies in the prior art.

[0008] To achieve the above objectives, the present invention provides the following technical solutions: As a first aspect, a monoclonal antibody for recognizing human liver mesenchymal stem cells is provided, which is deposited in the China Center for Type Culture Collection and secreted by the hybridoma cell line HLM1 with the deposit number: CCTCC NO: C202250.

[0009] Furthermore, the monoclonal antibody is a mouse IgG1 monoclonal antibody.

[0010] As a second aspect, the present invention provides a hybridoma cell line HLM1, which was deposited in China Center for Type Culture Collection and Wuhan University, Wuhan, China on June 2, 2022, with the deposit number: CCTCC NO: C202250, and specifically secretes the above-mentioned monoclonal antibody. Classification name: anti-human liver mesenchymal stem cell monoclonal antibody hybridoma cell line HLM1.

[0011] As a third aspect, the present invention also provides an application of the monoclonal antibody for recognizing human liver mesenchymal stem cells in the detection of human liver mesenchymal stem cells, specifically: based on the monoclonal antibody as a primary antibody, or as a probe for application in flow cytometry, immunohistochemistry, immunofluorescence and immunoblotting.

[0012] As a fourth aspect, the present invention also provides a use of the monoclonal antibody that recognizes human liver mesenchymal stem cells in the culture, passage and expansion of human liver tissue or human liver mesenchymal stem cells.

[0013] The beneficial effects of the present invention are as follows:

[0014] The present invention has successfully developed a monoclonal antibody for recognizing human liver mesenchymal stem cells and its hybridoma cell line HLM1, which has the advantages of high specificity and good repeatability, can specifically recognize a surface antigen on human liver mesenchymal stem cells, provides a specific method for the study of human liver mesenchymal stem cells, and provides an efficient tool for the rapid sorting, identification and purification of human liver mesenchymal stem cells. Various detection technologies based on monoclonal antibodies of human liver mesenchymal stem cells can also be applied to the study of acute injury and regeneration of human liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The morphology of human liver mesenchymal stem cells obtained by four-step collagenase perfusion method and density gradient centrifugation combined with adherent culture method, among which, Figure 1 A in the figure is the morphology of cultured primary human liver mesenchymal stem cells (40X); Figure 1 B is a morphological picture of human liver mesenchymal stem cells at the fifth passage of culture (100X);

[0016] Figure 2 This is a growth curve of human liver mesenchymal stem cells, where P5 and P9 are the growth curves of the 5th and 9th generations of human liver mesenchymal stem cells, respectively;

[0017] Figure 3 Figure 1 is a flow cytometry analysis diagram, in which Figure 3 A in the figure is a flow cytometric analysis of human liver mesenchymal stem cells using CD29-FITC monoclonal antibody as a probe; Figure 3 B is a flow cytometric analysis of human liver mesenchymal stem cells using CD90-FITC monoclonal antibody as a probe; Figure 3 C in the figure is a flow cytometric analysis of human liver mesenchymal stem cells using CD45-PE monoclonal antibody as a probe; Figure 3 D in the figure is a flow cytometric analysis of human liver mesenchymal stem cells using CD34-FITC monoclonal antibody as a probe;

[0018] Figure 4 This is a diagram analyzing the multidirectional differentiation ability of human liver mesenchymal stem cells, among which, Figure 4 A in the figure is the morphology of the fifth generation of cultured human liver mesenchymal stem cells; Figure 4 B is an Oil Red O staining image of human liver mesenchymal stem cells directional differentiation into adipose tissue; Figure 4 C in the figure is an Alizarin red staining image of human liver mesenchymal stem cells differentiating into osteoblasts; Figure 4 D in the figure is a glycogen staining image of human liver mesenchymal stem cells differentiating into hepatocytes;

[0019] Figure 5This is the result of indirect immunofluorescence staining to detect human liver mesenchymal stem cells using HLM1 monoclonal antibody as primary antibody;

[0020] Figure 6 This is the result of immunohistochemical staining to detect human liver mesenchymal stem cells using HLM1 monoclonal antibody as the primary antibody. Figure 6 A in the figure is the immunohistochemical staining result of the negative control using mouse immunoglobulin as the primary antibody. Figure 6 Figure B shows the expression of the corresponding antigen of the monoclonal antibody HLM1 in donated adult liver tissue. Figure 6 C in the figure is the expression result of the corresponding antigen of monoclonal antibody HLM1 in adult liver inflammation and damaged liver;

[0021] Figure 7 This is the result of flow cytometry detection of human liver mesenchymal stem cells using HLM1 monoclonal antibody as primary antibody;

[0022] Figure 8 This is the result of immunoblotting to detect the expression of corresponding antigens in human liver mesenchymal stem cells using HLM1 monoclonal antibody as the primary antibody. DETAILED DESCRIPTION

[0023] 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.

[0024] The monoclonal antibody specifically recognizing human liver mesenchymal stem cells of the present invention can specifically bind to the membrane surface protein of human liver mesenchymal stem cells, and has no cross reaction with other cells such as rat bone marrow mesenchymal stem cells, mouse bone marrow mesenchymal stem cells, pig adipose mesenchymal stem cells, human liver cell lines huh751 and HepG2, human liver progenitor cells, and human liver stellate cell line.

[0025] In addition, the present invention also provides a mouse hybridoma cell line HLM1 that produces the above monoclonal antibody. The spleen cells of Balb / c mice immunized with human liver mesenchymal stem cells and mouse myeloma FO cells are obtained respectively, fused under the action of polyethylene glycol (PEG), 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 HLM1 is successfully constructed and preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: C202250. 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.

[0026] Example 1: Isolation, culture, identification, cryopreservation and recovery of human liver mesenchymal stem cells.

[0027] (1) Isolation and culture of human liver mesenchymal stem cells

[0028] Part of the donated liver tissue of an adult was obtained under sterile conditions. The surface of the liver tissue was rinsed with 500 ml of phosphate buffer precooled to 4°C. Part of the liver tissue was treated with a four-step in vitro perfusion method including split enzymes and collagenase to separate and obtain primary human liver cells. A liver cell suspension containing human liver mesenchymal stem cells was obtained by density gradient centrifugation. The freshly isolated primary liver cells were cultured in an incubator at 37°C and 5% (v / v) CO2 using DMEM culture medium (v / v) containing 10% fetal bovine serum. The culture medium was replaced every 3 days. Non-adherent cells and senescent and dead cells were removed by replacing the DMEM culture medium. Long spindle-shaped adherent cells were visible in about 2 weeks. When the cells reached 70% to 80% fusion, they showed a colony-like distribution and attached to the bottom of the culture dish in a vortex-like form. The morphology was as follows: Figure 1 As shown in A. 0.25% (w / v) trypsin was used for digestion, adherent cells were harvested, and continuous cell passages were performed and expanded for use. The morphology of human liver mesenchymal stem cells at the 5th generation after passage is shown in Figure 1 As shown in B.

[0029] (2) Identification of the growth characteristics of human liver mesenchymal stem cells:

[0030] The 5th and 9th generation HLMSCs were cultured and cultured at 1.0 × 10 4The cells were inoculated into 6-well plates at an initial density of 10 cells / well. Cell counting was started after 24 hours for a total of 9 days. Counting was performed every 24 hours. Cells from 3 wells were counted each time. The average value was obtained for cell proliferation kinetic analysis. The proliferation capacity of human liver mesenchymal stem cells of the 9th and 5th generations was still good. Figure 2 As shown, P5 and P9 are the growth curves of the 5th and 9th generations of human liver mesenchymal stem cells, respectively.

[0031] (3) Identification of the phenotype of human liver mesenchymal stem cells:

[0032] The fifth generation HLMSC cells were taken and the surface markers of human liver mesenchymal stem cells were analyzed by flow cytometry using CD29-FITC, CD34-FITC, CD90-FITC and CD45-PE monoclonal antibodies as probes. Figure 3 As shown in A~D, the horizontal axis is the fluorescence intensity, and the vertical axis is the number of cells. The surface molecules CD29 and CD90 of human liver mesenchymal stem cells are positively expressed, and the hematopoietic stem cell marker molecules CD34 and CD45 are negatively expressed. The RT-PCR method (Reverse Transcription-Polymerase Chain Reaction, reverse transcription PCR) was used to further detect the cell surface molecules of human liver mesenchymal stem cells. The results showed that the cells' CD29, CD71, CD73, and CD90 were all positively expressed.

[0033] (4) Identification of the multi-differentiation potential of human liver mesenchymal stem cells:

[0034] When the cells of the fifth generation 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 multipotential differentiation potential of HLMSCs was evaluated under adipogenic, osteogenic, and hepatogenic differentiation conditions, and undifferentiated 5th generation HLMSCs were used as the control group. Figure 4 A in the figure shows the morphology of the 5th generation of cultured human liver mesenchymal stem cells.

[0035] After 7 days of adipogenic induction, the cell volume increased and the morphology changed to an oval shape. With the continued induction of differentiation, the circular characteristics of the cell contour became more obvious. After 21 days of culture, Oil Red O staining confirmed that HLMSCs could differentiate into adipocytes. Figure 4 As shown in B.

[0036] After 3-4 days of osteogenic induction, the cells adhered to the wall and gradually grew larger, forming tighter intercellular connections. Calcification spots were observed after 7 days, and calcified nodule-like structures appeared in the cells around 14-21 days. After 21 days of induction, red-stained cells were observed to aggregate in specific areas after Alizarin red staining, indicating that HLMSCs have achieved osteogenic differentiation. Figure 4 As shown in C.

[0037] After 5-7 days of induction culture towards hepatocytes, the cells changed into flat epithelial-like cell morphology, while the control group still maintained a fibroblast-like spindle shape. After 21 days of culture, HE staining (hematoxylin-eosin staining) revealed extensive and regular polygonal cells with obvious nuclei and abundant cytoplasm. PAS staining (periodic acid-Schiff staining) showed that HLMSCs had good glycogen storage capacity after hepatocyte differentiation, indicating that they could differentiate into more functionally mature hepatocytes, such as Figure 4 As shown in D.

[0038] (5) Cryopreservation and thawing of human liver mesenchymal stem cells:

[0039] HLMSCs were stored in DMEM culture medium (v / v) containing 10% DMSO. After being frozen in liquid nitrogen for several months to one year, the MSC cell characteristics and functions were still maintained after resuscitation culture.

[0040] Example 2: Preparation and purification of anti-human liver mesenchymal stem cell monoclonal antibodies.

[0041] (1) Animal immunization:

[0042] HLMSCs of passage 5 were selected and the cell density was 1.0×10 6 Balb / 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.

[0043] (2) Preparation of mouse hybridoma cell lines:

[0044] Peritoneal macrophages and spleen cells of normal mice without immunization were collected as feeder cells, and spleen cells of mice immunized with HLMSC were obtained aseptically, mixed with myeloma cells FO at a ratio of 10:1, and cell fusion was performed in a 38°C water bath environment with a 50% (w / w) concentration of PEG as a medium. 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.

[0045] 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-grown human liver mesenchymal stem cells for slide fixation, take the culture supernatant of hybridoma cells as the primary antibody, incubate at 37°C for 1h, wash 3 times with PBS for 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.

[0046] (3) Preparation and purification of monoclonal antibodies:

[0047] Balb / c mice were pretreated with paraffin oil and injected intraperitoneally with well-growing hybridoma cells HLM1. Each mouse was injected with 5×10 6 Cells. The abdomen of the mice was obviously swollen after about 2 weeks. The mice were killed by eye bleeding and ascites was collected. The ascites and saline were mixed at a ratio of 1:1 (w / w), 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 HLM1 against human liver mesenchymal stem cells.

[0048] (4) Ascites titer and subtype identification of anti-human liver mesenchymal stem cell monoclonal antibody HLM1:

[0049] The purified monoclonal antibody HLM1 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 HLM1 antibody was 2.0×10 4 The hybridoma cell culture supernatant was obtained, and the subtype of the monoclonal antibody was determined to be IgG1 using the ELISA method (Enzyme-Linked Immunosorbent Assays).

[0050] Example 3: Specific identification of anti-human liver mesenchymal stem cell monoclonal antibodies.

[0051] (1) Tissue-specific detection of human liver:

[0052] Tissue materials: tendons, muscles, vascular walls, ligaments, nerves, liver, small intestine, gallbladder, skin, lungs, fat and bone marrow. The above tissue specimens are all paraffin specimens of tissues removed by surgery from different patients. Anti-human liver mesenchymal stem cell monoclonal antibody HLM1 was used as the primary antibody to detect the paraffin sections of each tissue by immunohistochemical staining. The results showed that scattered positive cells were visible in human liver tissue sections, and all other human tissues were negative, which shows that the monoclonal antibody has the characteristics of specific binding and recognition of human liver mesenchymal stem cells.

[0053] (2) Species-specific detection:

[0054] Cell materials: normal pig, rabbit, rat, mouse liver tissues and cultured rat hepatocytes, pig hepatocytes and pig adipose mesenchymal stem cells, etc. Human liver tissue and human liver mesenchymal stem cells were used as positive controls by immunohistochemical staining or cytochemical staining, and anti-human liver mesenchymal stem cell monoclonal antibody HLM1 was used as an antibody to detect paraffin sections of tissues and cultured cell slides of other species. The results showed that tissues and cells of other species were all negatively stained, which shows that monoclonal antibody HLM1 has a strong specificity for human liver mesenchymal stem cells.

[0055] Example 4: Application of monoclonal antibodies in detecting human liver mesenchymal stem cells by immunofluorescence assay.

[0056] In this example, the monoclonal antibody against human liver mesenchymal stem cells is used as the primary antibody, and the fluorescent-labeled goat or sheep anti-mouse immunoglobulin antibody is used as the secondary antibody to establish an indirect immunofluorescence staining method to detect human liver mesenchymal stem cells, and observe and analyze the positive expression of human liver tissue or human liver mesenchymal stem cells in culture under a fluorescence microscope. Specifically:

[0057] Well-growing human liver mesenchymal stem cells were selected for slide fixation, and the monoclonal antibody HLM1 was used as the primary antibody. The indirect immunofluorescence staining method (the specific method is the same as the preparation of the hybridoma cell line described in step (2) of Example 2) was used. The positive rate was calculated under a fluorescence microscope. The results are as follows: Figure 5 As shown, 100% of human liver mesenchymal stem cells were positive and all expressed on the cell membrane surface. The experiment showed that the monoclonal antibody HLM1 can be used as a surface marker of human liver mesenchymal stem 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 mesenchymal stem cells.

[0058] Example 5: Application of monoclonal antibody HLM1 in detecting human liver mesenchymal stem cells by immunohistochemistry.

[0059] In this example, a monoclonal antibody against human liver mesenchymal stem 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 mesenchymal stem cells, and to detect and analyze the positive expression of human liver tissue or human liver mesenchymal stem cells after culture. Specifically:

[0060] Well-grown human liver mesenchymal stem 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, after washing with distilled water, hematoxylin counterstaining for 2 minutes, and the distilled water washing method was the same as before. The results were observed under a microscope, and the results showed that the positive rate of human liver mesenchymal stem cells was 100%. The same method was used to detect human liver tissue sections of various liver diseases, and scattered positive cells were seen, such as Figure 6 As shown in B~C. Figure 6 A in the figure is the immunohistochemical staining result of the negative control with mouse immunoglobulin as the primary antibody, which shows that the monoclonal antibody has good specificity and sensitivity and can be effectively used in immunohistochemistry. The expression of corresponding antigens in human liver mesenchymal stem cells can be effectively observed under a microscope, which is helpful for the localization and quantitative analysis of human liver mesenchymal stem cells.

[0061] Example 6: Application of monoclonal antibodies in flow cytometry detection of human liver mesenchymal stem cells.

[0062] In this example, a monoclonal antibody against human liver mesenchymal stem 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 mesenchymal stem cells. The flow cytometer was used to detect and analyze the positive expression of human liver mesenchymal stem cells after culture. Specifically:

[0063] Well-growing human liver mesenchymal stem cells were selected, monoclonal antibody HLM1 was used as a probe, FITC-labeled anti-mouse immunoglobulin antibody was used as a secondary antibody, and flow cytometry detection results showed that the HLMSC positive rate was 95.4%. Figure 7 As shown, the horizontal axis is the fluorescence intensity and the vertical axis is the number of cells, indicating that the monoclonal antibody HLM1 can efficiently bind to the surface protein of human liver mesenchymal stem cells and is suitable for the quantitative analysis of human liver mesenchymal stem cells.

[0064] Example 7: Application of monoclonal antibodies in detecting human liver mesenchymal stem cells by western blot.

[0065] In this example, a monoclonal antibody against human liver mesenchymal stem 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 mesenchymal stem cells, and analyze the positive expression of human liver tissue or human liver mesenchymal stem cells after culture. Specifically:

[0066] The in vitro expanded human liver mesenchymal stem 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 electrotransfer. The monoclonal antibody HLM1 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 in a dark room. The results showed that a specific binding protein band appeared at a molecular weight of about 110KD (kilodaltons) of the protein MARK, 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 mesenchymal stem cells, and further extract the antigens and detect their expression and biological characteristics in liver mesenchymal stem cells.

[0067] 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 mesenchymal stem cells, characterized in that: It is secreted and produced by the hybridoma cell line HLM1 with the deposit number: CCTCC NO: C202250.

2. The monoclonal antibody for recognizing human liver mesenchymal stem cells according to claim 1, characterized in that: The monoclonal antibody is a mouse IgG1 monoclonal antibody.

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

5. A hybridoma cell line HLM1, characterized in that It was deposited in the China Center for Type Culture Collection on June 2, 2022, with the accession number: CCTCC NO: C202250.

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