Human hepatocellular carcinoma cell strain as well as construction method and application thereof

Abstract

The invention discloses a human hepatocellular carcinoma cell strain as well as a construction method and application thereof, and belongs to the technical field of biomedicine. The cell strain is named as a cell strain for malignant transformation of human normal hepatocytes caused by hexafluoropropylene dimer acid, and is preserved in China General Microbiological Culture Collection Center (CGMCC) on January 6, 2025, and the preservation number is CGMCC No.46315. The human hepatocellular carcinoma cell strain provided by the invention is helpful for identifying the carcinogenicity of the perfluorinated substance, which indicates that the low-dose long-term exposure of the hexafluoroepoxypropane dimer acid can cause the vicious transformation of hepatocytes, and provides a new cell model basis and a new research idea for the research of the carcinogenic mechanism of the perfluorinated or polyfluoroalkyl substance.

Description

Technical Field

[0001] The invention relates to a human hepatocellular carcinoma cell line and a construction method and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] Hexafluoropropylene oxide dimer acid (GenX) is often used as a substitute for long-chain per- and polyfluoroalkyl substances (PFASs) because it is believed to have a shorter carbon chain and exhibits lower bioaccumulation. It is widely used in various industrial production. However, studies have shown that GenX may be more harmful than the currently widely studied perfluorooctanoic acid (PFOA) because it has a higher toxicity score in the modeled serum and liver concentrations.

[0003] The liver is an important metabolic organ in the body, and it has a biotransformation effect on a variety of non-nutritional substances from inside and outside the body (such as poisons, drugs, and metabolites in the body). Current studies have shown that most PFOA substances circulate in the blood and accumulate in the liver tissue. Related cell experimental studies have shown that after exposure to GenX, cell proliferation increased significantly starting from a concentration of 100μM. In a mouse experiment, the liver pathological changes in the GenX group and the PFOA group were similar, both of which can lead to enlargement of hepatocytes, loss of cytoplasm, nuclear migration, inflammatory cell infiltration, and reduced glycogen storage.

[0004] Animal experimental toxicity studies: By studying the toxicity of GenX to pregnant mice and their developing embryo-placental units, it was found that the effects of low-dose and high-dose GenX exposure were similar, both of which could lead to increased maternal liver weight and liver histopathological changes. Adult male BALB / c mice were treated by gavage, and the results showed that GenX exposure could lead to increased relative liver weight in mice, bile acid metabolism disorders, and other phenomena. In another study, it was found that GenX could alter fetal development and antibody production in mice, and cause toxic reactions in the liver and kidneys of rodents. GenX exposure can also cause severe gastrointestinal disorders, aggravate microbial toxicity, liver toxicity, and metabolic disorders. In addition, studies have found that mice exposed to GenX showed an increase in the number of mitotic BALF macrophages and increased Ki67 immunostaining.

[0005] Cell experimental toxicity study: Existing studies on the dose-dependent effects of GenX on primary human hepatocytes (PHH) have shown that low doses of GenX can interfere with metabolic pathways, and high doses of GenX can induce fibroinflammatory changes in human hepatocytes. After HepG2 cells were exposed to GenX for 48 hours, the overall methylation level first decreased and then increased. In addition, after GenX acted on HepG2 cells for 12 hours, it was found that cell viability decreased, apoptosis increased significantly, and GenX increased the production of intracellular reactive oxygen species (ROS) and the expression of major apoptosis-related genes, indicating that GenX can mediate HepG2 cell apoptosis through ROS.

[0006] The results of previous animal experiments showed that GenX treatment of mice caused an increase in liver cancer-related biomarkers. This indicates that GenX exposure is also likely to induce cancer in normal human liver cells. However, there is still a lack of systematic in vitro cell experimental studies on the potential carcinogenicity of GenX to mice. Therefore, studies on the potential carcinogenicity of GenX exposure to normal human liver cells are of great significance for establishing the detection limit of GenX. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a human hepatocellular carcinoma cell line, which is helpful to identify the carcinogenicity of hexafluoroepoxypropane dimer acid and also provides a new idea and model basis for the study of its carcinogenic mechanism.

[0008] The technical solution of the present invention is:

[0009] The first object of the present invention is to provide a human hepatocellular carcinoma cell line, which is named hexafluoroepoxypropane dimer acid-induced malignant transformation of normal human liver cells, and was deposited in the General Microbiological Center of China Microbiological Culture Collection Committee on January 6, 2025, with a deposit number of CGMCC No. 46315. The address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] The second object of the present invention is to provide a method for constructing the above-mentioned human hepatocellular carcinoma cell line, comprising the following steps:

[0011] Prepare at least two normal human hepatocyte culture medium;

[0012] Using a culture medium containing hexafluoroepoxypropane dimer acid at a first concentration to continuously culture at least one normal human liver cell;

[0013] Using a culture medium containing hexafluoroepoxypropane dimer acid at a second concentration to continuously culture at least one other normal human liver cell, wherein the second concentration is greater than the first concentration;

[0014] Change the cell medium every 20 to 30 hours;

[0015] When the cell confluence reaches 75% to 85%, subculture and amplify;

[0016] The cell line of malignant transformation of normal human liver cells induced by hexafluoroepoxypropane dimer acid is obtained by subculturing to 20 to 40 generations.

[0017] Furthermore, the method for preparing the culture medium of hexafluoroepoxypropane dimer acid comprises the following steps:

[0018] The hexafluoroepoxypropane dimer acid stock solution was added to a dimethyl sulfoxide medium and shaken evenly to obtain a hexafluoroepoxypropane dimer acid mother solution;

[0019] The hexafluoroepoxypropane dimer acid stock solution is diluted by a culture medium gradient dilution method to a culture medium containing a first concentration of hexafluoroepoxypropane dimer acid and a culture medium containing a second concentration of hexafluoroepoxypropane dimer acid.

[0020] Furthermore, in the culture medium containing the first concentration of hexafluoroepoxypropane dimer acid and the culture medium containing the second concentration of hexafluoroepoxypropane dimer acid, the concentration of dimethyl sulfoxide in the culture medium is less than or equal to 0.1%.

[0021] Furthermore, the dimethyl sulfoxide medium is a dimethyl sulfoxide high-glucose complete medium.

[0022] Furthermore, the dimethyl sulfoxide culture medium includes 8% to 12% fetal bovine serum and 0.8% to 1.2% penicillin-streptomycin.

[0023] Furthermore, the first concentration is 25 μM, and the second concentration is 100 μM.

[0024] The third object of the present invention is to provide a human hepatocellular carcinoma cell line for use as a cell model for studying the carcinogenic mechanism of perfluoroalkyl or polyfluoroalkyl substances.

[0025] The fourth object of the present invention is to provide a human hepatocellular carcinoma cell line for use in screening or evaluating drugs for treating cancers caused by perfluoro or polyfluoroalkyl substances.

[0026] The fifth object of the present invention is to provide a human hepatocellular carcinoma cell line for use in detecting the secretion of MMP-2 / 9 or the cell migration ability or the soft agar colony formation ability of the cell or in indicating the malignant transformation of the cell.

[0027] The beneficial technical effects of the present invention are:

[0028] The present invention constructs a human hepatocellular carcinoma cell line, specifically a hexafluoroepoxypropane dimer acid (GenX)-induced malignant transformation cell line of normal human hepatocellular carcinoma cells. The human hepatocellular carcinoma cell line is helpful to identify the carcinogenicity of perfluorinated substances, and explains that low-dose long-term exposure to hexafluoroepoxypropane dimer acid (GenX) can cause malignant transformation of hepatocytes, providing a new cell model basis and new research ideas for the study of the carcinogenic mechanism of perfluorinated or polyfluoroalkyl substances.

[0029] Deposit of biological materials:

[0030] The hexafluoropropylene dimer acid-induced malignant transformation cell line of normal human liver cells was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 6, 2025, with the deposit number CGMCC No.46315. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The figure shows the changes in the morphology of normal hepatocytes after the cells were exposed to 25 μM and 100 μM PFOA and GenX for 20 and 40 generations (the black arrows indicate filamentous pseudopodia);

[0032] Figure 2 The effect of long-term exposure to GenX on MMPs, the main marker of liver cancer invasion and metastasis (compared with group 0, *P<0.05, **P<0.01, ***P<0.001);

[0033] Figure 3 Effects of long-term exposure to GenX on VEGF, a key marker of intratumoral angiogenesis, and KI67, a key marker of cell proliferation in liver cancer (compared with group 0, *P<0.05, **P<0.01, ***P<0.001);

[0034] Figure 4 Effects of long-term exposure to GenX on the proliferation of normal human hepatocytes (compared with group 0, *P<0.05, **P<0.01, ***P<0.001);

[0035] Figure 5 shows the changes in cell migration ability of cells exposed to 25μM and 100μM GenX for 20 and 40 generations; Figure 5-1 This is the observation picture of the scratch test microscope of the cells in the 20th generation exposure group and the control group; Figure 5-2 This is the observation picture of the scratch test microscope of the cells in the 40th generation exposure group and the control group; Figure 5-3 Yes Figure 5-1 and Figure 5-2 Quantitative analysis; (compared with group 0, *P<0.05, **P<0.01, ***P<0.001);

[0036] FIG6 shows the changes in the colony formation ability of cells exposed to 25 μM and 100 μM GenX for a long time for 40 generations; Figure 6-1 It is the effect of plate clone formation on cells of the 40th generation exposure group and the control group; Figure 6-2 This is the observation picture under the microscope of the soft agar cloning experiment of cells in the 40th generation exposure group and the control group. DETAILED DESCRIPTION

[0037] In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the specific implementation methods of the present invention are further described in detail below in conjunction with the drawings and examples. The following examples are used to illustrate the present invention but are not used to limit the scope of the present invention.

[0038] Example 1: Preparation of a culture medium containing a first concentration and a second concentration of hexafluoroepoxypropane dimer acid (GenX):

[0039] Hexafluoroepoxypropane dimer acid stock solution: GenX stock solution: P888213-5g, McLean, China.

[0040] Hexafluoroepoxypropane dimer acid stock solution (GenX stock solution) was purchased from MacLean Reagent Company, with a molecular weight of 330.05 and a density of 1.748±0.06 g / mL.

[0041] Take 303 μL of GenX stock solution and add it to 697 μL of dimethyl sulfoxide (DMSO) culture medium. Vortex and mix well to obtain a GenX solution with a concentration of 1.6 mol, which is stored at room temperature.

[0042] Furthermore, the mother solution of hexafluoroepoxypropane dimer acid is diluted by a culture medium gradient dilution method to a culture medium containing hexafluoroepoxypropane dimer acid at a first concentration and a culture medium containing hexafluoroepoxypropane dimer acid at a second concentration.

[0043] Furthermore, in the culture medium containing the first concentration of hexafluoroepoxypropane dimer acid and the culture medium containing the second concentration of hexafluoroepoxypropane dimer acid, the concentration of dimethyl sulfoxide in the culture medium is less than or equal to 0.1%.

[0044] Furthermore, the dimethyl sulfoxide medium is a dimethyl sulfoxide high-glucose complete medium.

[0045] Furthermore, the dimethyl sulfoxide culture medium includes 10% fetal bovine serum and 1% penicillin-streptomycin.

[0046] Determine the appropriate dose of poison:

[0047] Normal human liver cells HL-7702 are treated with culture media containing hexafluoroepoxypropane dimer acid at different concentrations for 24 hours. The culture media containing hexafluoroepoxypropane dimer acid at different concentrations include: a culture media without hexafluoroepoxypropane dimer acid (concentration is 0), a culture media containing hexafluoroepoxypropane dimer acid at a first concentration, and a culture media containing hexafluoroepoxypropane dimer acid at a second concentration.

[0048] Considering the toxicity of long-term exposure to GenX to cells, a low dose (a culture medium containing the first concentration of hexafluoroepoxypropane dimer acid) was selected for research, and the dose with the highest cell survival rate was used as the long-term exposure dose, and the long-term exposure dose of GenX was finally determined to be 25μM and 100μM. That is, the first concentration is 25μM and the second concentration is 100μM. In other words, when preparing the culture medium, the culture medium is gradiently diluted to a culture medium containing 25μM and 100μM concentrations of GenX.

[0049] Example 2: Method for constructing human hepatocellular carcinoma cell line:

[0050] The present invention provides a method for constructing the above-mentioned human hepatocellular carcinoma cell line, comprising the following steps:

[0051] Prepare at least two normal human hepatocyte culture medium;

[0052] Using a culture medium containing hexafluoroepoxypropane dimer acid at a first concentration to continuously culture at least one normal human liver cell;

[0053] Using a culture medium containing hexafluoroepoxypropane dimer acid at a second concentration to continuously culture at least one other normal human liver cell, wherein the second concentration is greater than the first concentration;

[0054] Change the cell medium every 20 to 30 hours;

[0055] When the cell confluence reaches 75% to 85%, subculture and amplify;

[0056] The cell line of malignant transformation of normal human liver cells induced by hexafluoroepoxypropane dimer acid is obtained by subculturing to 20 to 40 generations.

[0057] Specifically, two dishes of normal HL-7702 cells (passage 0 cells) were prepared, one dish was continuously cultured with DMEM complete medium containing 25 μM GenX, the cell medium was changed every 24 hours, and the cells were subcultured when the cell confluence reached 80%, and the subculture was continued to the 40th generation. The other dish was continuously cultured with DMEM complete medium containing 100 μM GenX, the cell medium was changed every 24 hours, and the cells were subcultured when the cell confluence reached 80%, and the subculture was continued to the 40th generation.

[0058] Comparative Example:

[0059] Two dishes of normal HL-7702 cells (passage 0 cells) were prepared. One dish was cultured with DMEM complete medium containing 25 μM PFOA for long-term continuous exposure. The cell medium was changed every 24 hours. When the cell confluence reached 80%, the cells were subcultured and amplified. The cells were subcultured to the 40th generation. The other dish was cultured with DMEM complete medium containing 100 μM PFOA for long-term continuous exposure. The cell medium was changed every 24 hours. When the cell confluence reached 80%, the cells were subcultured and amplified. The cells were subcultured to the 40th generation.

[0060] Control group:

[0061] Normal culture was performed with DMEM complete medium (GenX concentration was 0), and normal HL-7702 cells (passage 0 cells) were cultured in the same way. The cell medium was changed every 24 hours, and the cells were subcultured when the cell confluence reached 80%, and the cells were subcultured normally until the 40th generation as the subculture control group.

[0062] Every 20 generations was set as a monitoring point to detect the doubling time of the 20th and 40th generations of cells in the poisoned group, comparative group and control group.

[0063] Example 3: Whether cells undergo malignant transformation is determined by detecting changes in matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), cell doubling time, cell migration ability, and cell anchorage-independent growth.

[0064] Figure 1 The study revealed changes in the morphology of normal human liver cells after exposure to 25 μM and 100 μM PFOA and GenX for 20 and 40 generations. Figure 1 It can be seen that after exposing normal human liver cells to 25μM and 100μM PFOA and GenX for 20 and 40 generations, the cell morphology changed significantly. The cells in the control group were smooth cobblestone-like, while the liver cells exposed to PFOA and GenX changed to spindle-shaped morphology, and the liver cells showed filamentous pseudopodia, showing the characteristics of cancer cell migration.

[0065] Figure 2 The effects of long-term exposure to GenX on MMPs, the main marker of liver cancer invasion and metastasis, were revealed. By comparing the content of the target protein with GAPDH in the samples, the changes in protein expression between samples can be evaluated, thereby calibrating and standardizing the experimental results. Figure 2 From left to right in the figure are the mRNA expression level of MMPs, protein bands and protein band quantification graphs.

[0066] Depend on Figure 2It can be seen that after the cells were exposed to high concentrations of 100μM PFOA and GenX for 20 / 40 generations, the mRNA expression levels of MMP2 and MMP9 were significantly increased compared with the control group. When the low concentration of 25μM PFOA and GenX was cultured for 20 / 40 generations, the promotion of cell mRNA expression by GenX was more obvious than that by the PFOA treatment group. In addition, after long-term exposure to GenX and PFOA, the protein expression levels of MMP2 and MMP9 were significantly increased compared with the control group, and the protein expression levels of MMP2 and MMP9 increased with the increase in the number of culture generations.

[0067] Figure 3 The effect of long-term exposure to GenX on VEGF, a key marker of intratumoral angiogenesis, and KI67, a key marker of cell proliferation in liver cancer (compared with group 0, *P<0.05, **P<0.01, ***P<0.001). Figure 3 It can be seen that after the cells were exposed to 25μM and 100μM PFOA and GenX, the VEGF mRNA expression level was significantly higher than that of the control group. Moreover, when the first concentration (25μM) was cultured for 20 generations and the second concentration (100μM) was cultured for 40 generations, the VEGF mRNA expression level of the treatment group containing GenX was higher than that of the treatment group containing PFOA.

[0068] In addition, after the cells were exposed to 25μM and 100μM PFOA and GenX, the mRNA levels of Ki67 increased significantly at the 20th and 40th generations, and the effect of GenX was higher than that of PFOA when the cells were cultured at 25μM exposure concentration for 20 generations and 100μM exposure concentration for 40 generations.

[0069] Therefore, both GenX and PFOA exposure can lead to an increase in VEGF, a key marker of intratumoral angiogenesis in liver cancer, and Ki67, a key marker of cell proliferation, indicating that GenX can promote related production and cell proliferation.

[0070] Figure 4 Effects of long-term exposure to GenX on the proliferation of normal human hepatocytes. Figure 4 It can be seen that after HL-7702 cells were continuously passaged with 25μM PFOA for 20, 30, and 40 generations, the cell proliferation rates of the cells in the treatment group containing 25μM PFOA increased by 16.5%, 24.4%, and 37.4% respectively compared with the control group. After HL7702 cells were continuously passaged with 25μM GenX for 20, 30, and 40 generations, the cell proliferation rates of the cells in the treatment group containing 25μM GenX increased by 26%, 35.2%, and 49.2% respectively compared with the control group. The proliferation rates of each treatment group were significantly higher than those of the control group (P<0.05).

[0071] After HL-7702 cells were continuously passaged with 100μM PFOA for 20, 30, and 40 generations, the cell proliferation rates of the cells in the treatment group containing 100μM PFOA increased by 16.2%, 37.4%, and 46.7% respectively compared with the control group. After HL-7702 cells were continuously passaged with 100μM GenX for 20, 30, and 40 generations, the cell proliferation rates of the cells in the treatment group containing 100μM GenX increased by 28.5%, 35.2%, and 56.6% respectively compared with the control group. The proliferation rates of each treatment group were significantly higher than those of the control group (P<0.05).

[0072] In addition, by Figure 4 It can be seen that under the same treatment concentration and the same treatment time, the cell proliferation rate of the GenX treatment group is higher than that of the PFOA treatment group. As the number of passages increases, cell proliferation increases. After continuous passage treatment of HL7702 cells with 100μM PFOA and GenX, the cell proliferation rate of each group was significantly higher than that of the control group, but there was no significant difference in the cell proliferation rate compared with the 25μM treatment group. Therefore, the cell proliferation ability of cells was significantly increased after exposure to 25μM and 100μM PFOA and GenX, and the cell proliferation ability became stronger as the number of passages increased.

[0073] Figure 5 shows the changes in cell migration ability of cells exposed to 25μM and 100μM GenX for 20 and 40 generations. Figure 5-1 This is the observation picture of the scratch experiment under the microscope of the 20th generation exposure group and the control group cells. Figure 5-2 Figure 5-3 is the observation of the scratch test of the cells in the 40th generation exposure group and the control group under the microscope. Figure 5-3 is the quantitative graph of the migration ability of HL7702 cells after continuous passage of 20 and 40 generations by PFOA and GenX.

[0074] Depend on Figure 5-1 , Figure 5-2 and Figure 5-3It can be seen that 25μM PFOA increased the relative migration distance of HL-7702 cells by 14% and 30% after continuous passage for 20 and 40 generations, respectively. 25μM GenX increased the relative migration distance of HL-7702 cells by 29% and 34% after continuous passage for 20 and 40 generations, respectively. 100μM PFOA increased the relative migration distance of HL7702 cells by 24% and 37% after continuous passage for 20 and 40 generations, respectively, and 100μM GenX increased the relative migration distance of HL-7702 cells by 44% and 33% after continuous passage for 20 and 40 generations, respectively. The relative migration distance increase rate was statistically significant compared with the control group (P<0.05). This shows that long-term passage treatment with PFOA and GenX significantly enhanced the migration ability of HL-7702 cells. In addition, GenX has a stronger ability to migrate HL-7702 cells under the same concentration and the same treatment passage time. At the same passage time, high concentrations of PFOA and GenX have a stronger ability to affect the migration of HL7702 cells than low concentrations. It can be seen that when low and high concentrations of PFOA and GenX are poisoned and cultured to the 20th and 40th generations, the migration ability of cells is enhanced, and the 40th generation is more obvious.

[0075] FIG. 6 shows the changes in the colony-forming ability of cells exposed to 25 μM and 100 μM GenX for a long time for 40 generations.

[0076] in, Figure 6-1 The effect of PFOA and GenX long-term exposure on the plate colony formation of HL-7702 cells for 20 and 40 generations was tested. Figure 6-1 It can be seen that after long-term exposure to PFOA and GenX, the number of cell colony formation in the treatment group increased significantly compared with the control group, and the number of colony formation increased significantly with the increase of exposure concentration and exposure time. Under the same treatment concentration and the same exposure time, the number of colony formation in the GenX treatment group increased more than that in the PFOA treatment group (P<0.05). This shows that long-term exposure to GenX can promote the formation of HL-7702 cell colonies and has the risk of inducing cell carcinogenesis. In addition, GenX has a stronger ability to induce cancer than PFOA.

[0077] Figure 6-2 This is a picture of the soft agar clone experiment of cells in the 40th generation exposure group and the control group under a microscope. Figure 6-2It can be seen that the cells in the control group only have individual micro-colony shapes in soft agar, that is, the number of soft agar clones formed in the control group is small. After continuous subculture treatment with PFOA and GenX, the cells can form obvious clonal colonies in soft agar, that is, the number of soft agar clones formed increases. Moreover, the number of soft agar clones formed increases significantly with the increase of exposure concentration and exposure time. Under the same treatment concentration and the same exposure time, the number of soft agar clones formed in the GenX treatment group increased more than that in the PFOA treatment group. The results of this part show that long-term exposure to PFOA and GcnX can enhance the anchorage-independent growth ability of HL-7702 cells. GenX has the risk of inducing cell carcinogenesis, and its carcinogenic ability is stronger than PFOA.

[0078] The secretion of MMP-2 and MMP-9, cell doubling time, cell migration ability and soft agar clone formation ability mentioned above are all commonly used indicators for identifying in vitro cell malignant transformation. According to the above experimental results, it is shown that long-term exposure to 25μM hexafluoroepoxypropane dimer acid (GenX) and subculture to the 20th generation can induce malignant transformation of HL-7702 cells.

[0079] In summary, the present invention constructs a human hepatocellular carcinoma cell line, specifically a hexafluoroepoxypropane dimer acid (GenX)-induced malignant transformation cell line of normal human hepatocytes. The human hepatocellular carcinoma cell line is helpful to identify the carcinogenicity of perfluorinated substances, and illustrates that low-dose long-term exposure to hexafluoroepoxypropane dimer acid (GenX) can cause malignant transformation of hepatocytes, providing a new cell model basis and new research ideas for the study of the carcinogenic mechanism of perfluorinated or polyfluoroalkyl substances.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the technical principles of the present invention, and these improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A human hepatocellular carcinoma cell line, characterized in that The cell line was named hexafluoropropylene dimer acid-induced malignant transformation of normal human liver cells cell line, and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 6, 2025, with the deposit number CGMCC No.46315.

2. A method for constructing a human hepatocellular carcinoma cell line as claimed in claim 1, characterized in that: The steps include: Prepare at least two normal human hepatocyte culture medium; Using a culture medium containing hexafluoroepoxypropane dimer acid at a first concentration to continuously culture at least one normal human liver cell; Using a culture medium containing hexafluoroepoxypropane dimer acid at a second concentration to continuously culture at least one other normal human liver cell, wherein the second concentration is greater than the first concentration; Change the cell medium every 20 to 30 hours; When the cell confluence reaches 75% to 85%, subculture and amplify; The cell line of malignant transformation of normal human liver cells induced by hexafluoroepoxypropane dimer acid is obtained by subculturing to 20 to 40 generations.

3. The method for constructing a human hepatocellular carcinoma cell line according to claim 2, characterized in that: The preparation method of the culture medium of hexafluoroepoxypropane dimer acid comprises the following steps: The hexafluoroepoxypropane dimer acid stock solution was added to a dimethyl sulfoxide medium and shaken evenly to obtain a hexafluoroepoxypropane dimer acid mother solution; The hexafluoroepoxypropane dimer acid stock solution is diluted by a culture medium gradient dilution method to a culture medium containing a first concentration of hexafluoroepoxypropane dimer acid and a culture medium containing a second concentration of hexafluoroepoxypropane dimer acid.

4. The method for constructing a human hepatocellular carcinoma cell line according to claim 3, characterized in that: In the medium containing the first concentration of hexafluoroepoxypropane dimer acid and the medium containing the second concentration of hexafluoroepoxypropane dimer acid, the concentration of dimethyl sulfoxide in the medium is less than or equal to 0.1%.

5. The method for constructing a human hepatocellular carcinoma cell line according to claim 3, characterized in that: The dimethyl sulfoxide medium is a dimethyl sulfoxide high-glucose complete medium.

6. The method for constructing a human hepatocellular carcinoma cell line according to claim 3, characterized in that: The dimethyl sulfoxide culture medium includes 8% to 12% fetal bovine serum and 0.8% to 1.2% penicillin-streptomycin.

7. The method for constructing a human hepatocellular carcinoma cell line according to any one of claims 2 to 6, characterized in that: The first concentration is 25 μM, and the second concentration is 100 μM.

8. Use of the human hepatocellular carcinoma cell line as claimed in claim 1 as a cell model for studying the carcinogenic mechanism of perfluoroalkyl or polyfluoroalkyl substances.

9. Use of a human hepatocellular carcinoma cell line as claimed in claim 1 in screening or evaluating drugs for treating cancers caused by perfluoroalkyl or polyfluoroalkyl substances.

10. Use of a human hepatocellular carcinoma cell line as claimed in claim 1 in detecting the secretion of MMP-2 / 9 or the ability of cell migration or the ability of soft agar colony formation or in indicating the occurrence of malignant transformation of cells.

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