A human hepatocellular carcinoma cell line and its application

Abstract

The present invention provides a human hepatocellular carcinoma cell line and its application. The cell line is named human hepatocellular carcinoma cell line HCCP-ZJ, which was deposited in the China Center for Type Culture Collection on April 21, 2021, with a deposit number of CCTCC NO: C202188. The human hepatocellular carcinoma cell line of the present invention can be passaged in vitro under conventional cell culture conditions, has stable in vitro properties, and conforms to the biological characteristics of clinical tumors, has tumorigenicity, and can prepare a subcutaneous heterotopic transplant tumor model of liver cancer, a liver carcinoma in situ model, and a liver cancer lung metastasis model, all of which can be used for the study of the mechanism of liver cancer occurrence and development and the in vivo evaluation of drug effects. The hepatocellular carcinoma cell line forms liver in situ tumors and lung metastases quickly, which is conducive to the development of scientific research. In addition, the hepatocellular carcinoma cell line can be used for the in vitro evaluation of the effect of liver cancer drugs. The human hepatocellular carcinoma cell line of the present invention can be used to guide clinical medication, which is conducive to the discovery of new anti-liver cancer drugs, and has good scientific research value and economic benefits.

Description

Technical Field

[0001] The invention relates to the fields of biology and oncology and to a human hepatocellular carcinoma cell line and a construction method and application thereof. Background Art

[0002] Hepatocellular carcinoma is one of the top five malignant cancers with the highest incidence and the top four mortality rates in the world, placing a heavy burden on the global medical system. Due to the lack of typical symptoms in early liver cancer, most patients have already progressed and metastasized when they are first diagnosed, and cannot undergo radical surgery. Transcatheter arterial chemoembolization, percutaneous alcohol injection, local radiotherapy, and radiofrequency ablation are commonly used local palliative treatments for liver cancer. Liver cancer cells are less sensitive to traditional chemotherapy drugs. New multi-kinase inhibitors represented by sorafenib, lenvatinib, and regorafenib have certain benefits in prolonging the survival time of liver cancer patients. Overall, the prognosis of liver cancer patients is still very poor, with a five-year overall survival rate of less than 6%. There is an urgent need to seek more effective drugs through in-depth scientific research to further improve the quality of life of liver cancer patients.

[0003] Human liver cancer cell lines are commonly used tools in liver cancer research. Human liver cancer cell lines retain the biological behavior of liver cancer, and can be continuously passaged in vitro and the response of cells to various drug treatments can be monitored in real time through cell biology methods. At the same time, some liver cancer cell lines can form subcutaneous heterotopic transplants in experimental animals, and even fewer liver cancers can form orthotopic transplants in situ in the liver and spontaneously metastasize to the lungs, which can be used for systematic evaluation of liver cancer treatment.

[0004] However, primary liver cancer has great heterogeneity. Multiple factors such as hepatitis virus infection, alcohol abuse, liver steatosis, liver fibrosis, and drug toxicity have a combined impact on the development of the disease, resulting in liver cancer cell lines from different patients often showing significantly different tumor biological characteristics. This high degree of heterogeneity requires researchers to use multiple liver cancer cell lines simultaneously during the research process to ensure that the research results have a wider applicability. Moreover, there are currently few hepatocellular carcinoma cell lines that can simultaneously carry out in vitro passage, subcutaneous heterotopic transplants, liver orthotopic transplants, and lung metastasis models, and lung metastasis takes a long time (more than two months). It is urgent to establish new hepatocellular carcinoma cell lines that can quickly metastasize from the liver to the lungs to ensure that researchers can cover more types of hepatocellular carcinoma. Summary of the invention

[0005] The purpose of the present invention is to provide a human hepatocellular carcinoma cell line and a construction method and application thereof.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] A human hepatocellular carcinoma cell line, named human hepatocellular carcinoma cell line HCCP-ZJ, was deposited in the China Center for Type Culture Collection on April 21, 2021, with the deposit number CCTCC NO: C202188.

[0008] The present invention also provides progeny cells of the human hepatocellular carcinoma cell line described above.

[0009] The present invention also provides the use of the human hepatocellular carcinoma cell line as described above, including:

[0010] a. To study the occurrence, development, metastasis and treatment mechanism of liver cancer;

[0011] b. Preparing a tumor cell model or a tumor animal model;

[0012] c. In vitro screening and / or in vitro evaluation of prepared tumor therapeutic drugs; wherein, the method for screening tumor therapeutic drugs can be: by adding different tumor therapeutic drugs to the culture medium of the human hepatocellular carcinoma cell line, observing the changes in cell morphology, and obtaining preliminary effective candidate drugs. Then, the candidate drugs are administered to the cells, the half-inhibitory concentration (I50) of the screened effective drugs is calculated, and the drug with the lowest IC50 is selected to further act on the animal model, and the survival period, tumor size, metastasis, etc. of the animals in the non-administered group are compared to screen for potential therapeutic drugs;

[0013] d. Develop tumor drug targets, such as flow cytometry antibodies with specific markers, gene detection kits, etc.;

[0014] e. Preparation of tumor diagnostic products;

[0015] f. In vitro screening of tumor biotherapeutic drugs / reagents; the tumor biotherapeutic drugs / reagents are tumor vaccines;

[0016] g. Develop and detect tumor-related bioengineering products in vitro; the tumor-related bioengineering products can be liver cancer-specific molecular diagnostic PCR kits, fluorescent in situ hybridization kits, etc.

[0017] Furthermore, the tumor animal model includes an animal liver cancer subcutaneous heterotopic transplant tumor model, a liver carcinoma in situ model and a lung metastasis model. The liver cancer is hepatocellular carcinoma. A liver cancer subcutaneous heterotopic transplant tumor model is obtained by inoculating a certain number of human hepatocellular carcinoma cells into the subcutaneous tissue of immunodeficient mice. A liver carcinoma in situ model is obtained by inoculating a certain number of human hepatocellular carcinoma cells into the left leaf of the liver of immunodeficient mice. When the liver carcinoma in situ model progresses to the end stage of the disease (one month later), metastatic lesions will form in the lungs of mice, and a liver cancer lung metastasis model is obtained.

[0018] Furthermore, the animal is an immunodeficient mouse (such as a nude mouse).

[0019] The present invention also provides a method for constructing the human hepatocellular carcinoma cell line, comprising the following steps:

[0020] After obtaining fresh liver cancer tissue surgical specimens (0.5mm×0.5mm×0.5mm) in the laminar flow operating room, they were immediately stored in 0.5ml of 4℃ sterile William'E culture medium and transferred to the biosafety cabinet within 0.5 hours for the following aseptic operation. First, the liver cancer tissue was mechanically separated, and then digested with 1ml tissue digestion solution (William'E culture medium containing 2mg / ml type IV collagenase, 0.02mg / ml type I nuclease, 10% fetal bovine serum, 5ng / ml recombinant human hepatocyte growth factor, 0.5U / ml recombinant human insulin, 1.6μmol / L dexamethasone sodium phosphate, 100U / ml penicillin, 100μg / ml streptomycin sulfate, and 0.25μg / ml amphotericin B) at 37℃ for 3 hours under shaking conditions. After the digested cell suspension was filtered through a 70μm filter to obtain a single cell suspension, it was centrifuged at 1200rpm for 5 minutes, the supernatant was discarded, and the cell pellet was obtained. After washing the cells three times with pH 7.4 phosphate buffer, resuspend them in 2 ml complete culture medium (William'E culture medium containing 10% fetal bovine serum, 5 ng / ml recombinant human hepatocyte growth factor, 0.5 U / ml recombinant human insulin, 1.6 μmol / L dexamethasone sodium phosphate, 100 U / ml penicillin, 100 μg / ml streptomycin sulfate, 0.25 μg / ml amphotericin B) and plant them in a six-well plate coated with rat tail collagen, and place them in a 37°C cell culture incubator containing 5 vol% CO2 for static culture. After 24 hours, change the medium and wash away the non-adherent cells. After that, change the culture medium every 3 days until the cells grow to 80% conjugation, and pass them at a ratio of 1:5. After passage to the 30th generation, a cell line with uniform morphology and stable passage was obtained, and the cell line could be cultured in a DMEM high-glucose medium containing 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin sulfate.

[0021] The beneficial effects of the present invention are as follows: the human hepatocellular carcinoma cell line of the present invention can be passaged in vitro under conventional cell culture conditions, has stable in vitro properties, and conforms to the biological characteristics of clinical tumors. The human hepatocellular carcinoma cell line has tumorigenicity, and can be used to prepare a subcutaneous heterotopic transplant tumor model of liver cancer, a liver carcinoma in situ model, and a liver cancer lung metastasis model, all of which can be used for the study of the mechanism of liver cancer occurrence and development and the in vivo evaluation of drug effects. The hepatocellular carcinoma cell line forms liver in situ tumors and lung metastases quickly, which is conducive to the development of scientific research. In addition, the hepatocellular carcinoma cell line can be used for the in vitro evaluation of the effects of liver cancer drugs. In general, the human hepatocellular carcinoma cell line can be used to guide clinical drug use, is conducive to the discovery of new anti-liver cancer drugs, and has good scientific research value and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The cell morphology of HCCP-ZJ under light microscope.

[0023] Figure 2 Cell growth curves of HCCP-ZJ, LM3 (commercial liver cancer cell line), and Huh1 (commercial liver cancer cell line).

[0024] Figure 3 Cell viability-drug dose curves of sorafenib (left) and lenvatinib (right) for HCCP-ZJ, LM3 (commercial liver cancer cell line), and Huh1 (commercial liver cancer cell line).

[0025] Figure 4 The figures show the results of migration experiment (upper) and invasion experiment (lower) of HCCP-ZJ, LM3 (commercial liver cancer cell line), and Huh1 (commercial liver cancer cell line).

[0026] Figure 5 This is a diagram showing the results of a nude mouse subcutaneous tumor formation experiment of HCCP-ZJ-luc.

[0027] Figure 6 This is the subcutaneous tumor growth curve of HCCP-ZJ-luc.

[0028] Figure 7 This is a model of HCC-PZJ-luc liver tumor in situ with lung metastasis. (A) Fluorescence imaging of mice in a small animal in vivo imager, (B) Photo of liver tumor in situ obtained after dissection, (C) Fluorescence imaging of lung metastasis, (D) HE staining of lung metastasis.

[0029] Figure 8 This is the result of genome karyotype identification of HCCP-ZJ. DETAILED DESCRIPTION

[0030] The present invention is further described below with examples, but the present invention is not limited thereto. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions.

[0031] Example 1 Preparation of HCCP-ZJ cells.

[0032] Primary cell culture: Fresh liver cancer tissue surgical specimens (53-year-old Chinese male hepatocellular carcinoma patient, no hepatitis virus, no HIV, CT showed a tumor diameter of 4.5 cm, serum AFP concentration of 2036 mAU / ml) were obtained in the laminar flow operating room. 0.5 mm × 0.5 mm × 0.5 mm in size were stored in 0.5 ml of 4°C sterile William'E culture medium and transferred to the biosafety cabinet within 0.5 hours for the subsequent sterile operation. First, the liver cancer tissue was mechanically separated, and then digested with 1 ml of tissue digestion solution (William'E culture medium containing 2 mg / ml type IV collagenase, 0.02 mg / ml type I nuclease, 10% fetal bovine serum, 5 ng / ml recombinant human hepatocyte growth factor, 0.5 U / ml recombinant human insulin, 1.6 μmol / L dexamethasone sodium phosphate, 100 U / ml penicillin, 100 μg / ml streptomycin sulfate, and 0.25 μg / ml amphotericin B) at 37°C for 3 hours under shaking conditions. The digested cell suspension was filtered through a 70 μm filter to obtain a single cell suspension, and then centrifuged at 1200 rpm for 5 minutes, and the supernatant was discarded to obtain a cell pellet. After washing the cells three times with phosphate buffered saline (PBS) at pH 7.4, resuspend them in 2 ml complete culture medium (William'E culture medium containing 10% fetal bovine serum, 5 ng / ml recombinant human hepatocyte growth factor, 0.5 U / ml recombinant human insulin, 1.6 μmol / L dexamethasone sodium phosphate, 100 U / ml penicillin, 100 μg / ml streptomycin sulfate, and 0.25 μg / ml amphotericin B) and plant them in a six-well plate coated with rat tail collagen, and place them in a 37°C cell culture incubator containing 5 vol% and CO2 for static culture. After 24 hours, change the medium and wash away the non-adherent cells, and then change the above culture medium every 3 days.

[0033] Cell subculture: When the cells grow to 80% conjugation, cell subculture is performed in a biosafety cabinet. The specific steps are as follows: discard the old culture medium, wash the cells with sterile PBS at pH 7.4 and discard. Digest with 0.05wt% trypsin solution until the cells are suspended, add culture medium containing serum to terminate digestion, and collect all cells into a sterile centrifuge tube, centrifuge at 1000rpm and discard the supernatant. Resuspend the cells with fresh culture medium and subculture them in a new culture bottle at a ratio of 1:5. The first 30 generations use William'E culture medium containing 10vol% fetal bovine serum, 5ng / ml recombinant human hepatocyte growth factor, 0.5U / ml recombinant human insulin, 1.6μmol / L dexamethasone sodium phosphate, 100U / ml penicillin, 100μg / ml streptomycin sulfate, and 0.25μg / ml amphotericin B. After passage 30, the cells can be cultured in DMEM high-glucose medium containing 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin sulfate.

[0034] In the present invention, hepatocellular carcinoma presents an epithelial-like morphology, uniform cell morphology, contact inhibition, and stable cell growth rate. The cell was named human hepatocellular carcinoma cell line HCCP-ZJ, and was deposited in the China Center for Type Culture Collection on April 21, 2021, with a deposit number of CCTCC NO: C202188, and the deposit address is Wuhan University, Wuhan, China, with a postal code of 430072.

[0035] Example 2 Biological characteristics and applications of human liver cancer cells.

[0036] Morphological observation

[0037] The culture flask containing HCCP-ZJ cells was placed under an inverted microscope for observation and photography. Figure 1 As shown, the cells were polygonal in shape, showing the morphological characteristics of epithelial cells; and it was observed that the cells grew malignantly but still maintained contact inhibition.

[0038] Determination of cell doubling time

[0039] The cells were seeded in a 96-well plate at a density of 2000 cells / well, and the cell viability was measured by CCK-8 method at 0, 24, 48, 72, and 96 hours. Figure 2 ), the doubling time of HCCP-ZJ was calculated to be 27 hours. Under the same conditions, the doubling times of the commonly used liver cancer cell lines LM3 and Huh1 are 28 and 21 hours, respectively. It can be seen that the doubling time of HCCP-ZJ is between LM3 and Huh1.

[0040] Determination of IC50 of half-lethal dose of sorafenib and lenvatinib

[0041] The cells were seeded in a 96-well plate at a density of 2000 cells / well. After 24 hours, different concentrations of sorafenib or lenvatinib were added. After 48 hours of drug treatment, the cell viability was measured using the CCK-8 method, and the half-lethal dose of the drug was calculated based on the drug concentration and cell viability. Figure 3 As shown, the sorafenib IC50 of HCCP-ZJ is 16.66 μM, which is between the commercial liver cancer cell lines LM3 (22.45 μM) and Huh1 (11.91 μM); the lenvatinib IC50 of HCCP-ZJ is 47.06 μM, which is between LM3 (52.44 μM) and Huh1 (22.29 μM).

[0042] Evaluation of migration and invasion ability in vitro

[0043] To evaluate the migration ability of cells, a cell perforated chamber with a pore size of 8 μm is placed in a 24-well plate, and 100 μl of serum-free culture medium containing 100,000 cells is added to the upper chamber (the upper space of the chamber), and 600 μl of culture medium containing 20% ​​fetal bovine serum is added to the lower chamber (the outer space of the chamber). After 24 hours of static culture in a cell culture incubator, the chamber is taken out, and the cells on the upper side of the chamber bottom membrane are wiped off with a cotton swab. The cells on the lower side of the chamber bottom membrane are fixed with a 4% paraformaldehyde solution and stained with crystal violet, and the number of cells that migrated to the lower side of the bottom membrane is observed under an optical microscope. The method for evaluating invasion ability is similar to this, except that the invasion experiment requires the cell perforated chamber to be coated with 50 μl of 1:9 diluted matrix gel in advance, and the subsequent operation method is exactly the same as the migration experiment. Experimental results ( Figure 4 ) showed that the migration ability of HCCP-ZJ was stronger than that of commercialized LM3 and Huh1 liver cancer cell lines, but its invasion ability was poor, weaker than Huh1 and similar to LM3.

[0044] Evaluation of subcutaneous tumorigenicity in vivo

[0045] Subcutaneous tumor formation experiments were performed on male or female BALB / C nude mice aged 4-6 weeks. HCCP-ZJ cells were resuspended in PBS and the cell concentration was adjusted to 2×10 7 After mixing with an equal volume of matrix gel, the cell concentration was 1×10 7 0.1 ml of the cell / matrigel mixture was injected subcutaneously near the axilla of the left forelimb of the nude mouse. The injection site was observed every 3 days. After 15 days of inoculation, a tumor gradually formed and a protruding mass ( Figure 5 ). Use a vernier caliper to measure the long axis diameter and short axis diameter of the subcutaneous tumor (the two are perpendicular to each other), according to "tumor volume = long axis diameter × short axis diameter 2 / 2” to calculate the tumor volume. On day 36 after inoculation, the average tumor volume reached 1000mm 3 ( Figure 6 ).

[0046] Evaluation of liver in situ tumorigenicity and lung metastasis

[0047] Male nude mice aged 6-8 weeks were used for the liver orthotopic tumorigenesis experiment. HCCP-ZJ cells were pre-infected with a lentivirus that overexpressed luciferase to obtain HCCP-ZJ-luc cells that can be tracked and quantified using fluorescent signals. After the animals were anesthetized with chloral hydrate, the skin and peritoneum were cut about 1 cm along the ventral midline below the xiphoid process to expose the liver surface. The liver lobe was adhered with a cotton swab and gently dragged out of the body through the incision. A microsyringe with an insulin needle was used to inject 1×10 6 HCCP-ZJ-Luc cells expressing luciferase (resuspended in 10μl PBS containing 50% matrix gel), wait for 10 seconds after the injection, and withdraw the needle after the matrix gel solidifies into a jelly state. At the same time, gently press the injection site with a cotton swab to prevent leakage of the cell suspension or bleeding in the liver. After the bleeding on the surface of the liver stops, the liver lobe is stuffed back into the abdominal cavity and the peritoneum and skin are sutured. After the mouse recovers, it is put back into the cage. At weeks 2, 3, and 4, the nude mice were anesthetized, injected with luciferin substrate, and in situ liver tumors were imaged in vivo. After imaging was completed in week 4, the mice were killed by rapid cervical dislocation, the lungs were isolated, and the fluorescent signals in the lungs were imaged. Experimental results ( Figure 7 ) showed that HCCP-ZJ could rapidly form in situ tumors in the liver and metastasize to the lungs by the fourth week, forming multiple micrometastatic lesions.

[0048] Genome karyotype identification

[0049] HCCP-ZJ cells were treated with colchicine overnight, digested, collected by centrifugation, and washed once with PBS. After treatment with hypotonic KCl (0.075M) for 30 minutes, 1 / 10 volume of fixative was added and centrifuged at 1500rpm for 5 minutes. The supernatant was discarded and the last 1ml of liquid was retained. The cells were gently resuspended by blowing, and the fixative was slowly added while shaking until the tube was full. The washing and centrifugation process with the fixative was repeated twice. Overnight at 4 degrees, the supernatant was removed by centrifugation and the last 1ml of liquid was retained. A small amount of the resuspended liquid was quickly dropped from a high place on a pre-cooled slide at 0 degrees, and then quickly flared or air-dried. The slide was stained with Giemsa and observed under a microscope and compared with the standard karyotype. The short arm of the eighth pair of chromosomes of HCCP-ZJ is missing ( Figure 8 ).

[0050] Short tandem repeat (STR) identification

[0051] STR (Short Tandem Repeat) gene loci are composed of short tandem repeat sequences with a length of 3 to 7 base pairs. These repeat sequences are widely present in the human genome and can be used as highly polymorphic markers, which are called DNA fingerprints of cells. The identification process is as follows: extract the genomic DNA of HCCP-ZJ, amplify the STR sequence by polymerase chain reaction, separate the amplified products by capillary electrophoresis, and then identify the copy number of the STR sequence by fluorescence detection. Finally, compare it with the cell STR database to infer the name of the cell line or possible cross-contamination of the cell line. The experimental results (Table 1) show that the STR fingerprint of the HCCP-ZJ cell line is completely consistent with that of the patient's peripheral blood, and they are from the same source, that is, there is no cross-contamination during the correct culture of the cell source. And unlike all known cell lines, it is a brand new liver cancer cell line.

[0052] Table 1 STR identification results of HCCP-ZJ

[0053]

[0054] Table 2 Summary of SNP and INDEL results from exon sequencing of HCCP-ZJ cells

[0055]

[0056] Note: M means that only mutant type was detected in the cells; WT / M means that both wild type and mutant type were detected in the cells.

[0057] Table 2 shows the results of SNP and INDEL sequencing of HCCP-ZJ cell exon. The results in the table show that the cell line cells are positive for multiple gene mutations related to tumor malignancy, such as TP53, CTNNB1, ARID1A, NFE2L2, CDKN2A, CDKN1A, KEAP1, RB1, HGF, RAC2, etc. This suggests that the cells have certain value in studying the mechanism of disease treatment or pathogenesis and screening targeted drugs for the above mutations.

[0058] In summary, the present invention uses HCCP-ZJ cells cultured in DMED high-glucose culture medium containing 10% fetal bovine serum to enable long-term growth in vitro and stable passage. Observed under a microscope, HCCP-ZJ cells have a typical epithelial cell-like morphology. The results of the CCK8 experiment showed that the doubling time of HCCP-ZJ was 27 hours, the half-lethal dose (IC50) of sorafenib was 16.66μM, and the half-lethal dose (IC50) of lenvatinib was 47.06μM. Perforation experiments and invasion experiments showed that HCCP-ZJ exhibited strong migration and invasion abilities in vitro. A certain amount of HCCP-ZJ cells can form tumors in the subcutaneous and liver parts of nude mice and spontaneously metastasize to the lungs, indicating that the cells have tumorigenic and metastatic abilities. The results of exome sequencing analysis showed that HCCP-ZJ contained multiple gene mutations related to tumor malignancy, such as TP53, CTNNB1, ARID1A, NFE2L2, CDKN2A, CDKN1A, KEAP1, RB1, HGF, and RAC2.

Claims

1. A human hepatocellular carcinoma cell line, named human hepatocellular carcinoma cell line HCCP-ZJ, was deposited in the China Center for Type Culture Collection on April 21, 2021, with the deposit number CCTCC NO: C202188.

2. A progeny cell of the human hepatocellular carcinoma cell line according to claim 1.

3. A use of the human hepatocellular carcinoma cell line according to claim 1, characterized in that: Used to prepare liver cancer cell models or liver cancer animal models.

4. The use according to claim 3, characterized in that The liver cancer animal models include animal liver cancer subcutaneous heterotopic transplant tumor models, liver carcinoma in situ models and lung metastasis models.

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