Human intrahepatic bile duct cancer cell line and application thereof

Abstract

The invention discloses a human intrahepatic bile duct cancer cell line and application thereof. The human intrahepatic bile duct cancer cell line comprises human intrahepatic bile duct cancer cells JXQ-3D-6951R; the human intrahepatic bile duct cancer cell JXQ-3D-6951R is preserved in the China Center for Type Culture Collection on November 22, 2023, and the preservation number of the human intrahepatic bile duct cancer cell JXQ-3D-6951R is CCTCC (China Center for Type Culture Collection) NO: C2023370. The human intrahepatic bile duct cancer cell line provided by the invention can be massively amplified and subcultured in vitro, has clone formation ability, is sensitive to cis-platinum and gemcitabine, and is relatively insensitive to 5-FU. A human bile duct cancer cell bank is enriched, and a new research material is provided for exploration of occurrence, development, metastasis mechanism and drug resistance mechanism of primary bile duct cancer and research and development of new drugs.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a human intrahepatic bile duct cancer cell line and an application thereof. Background Art

[0002] Intrahepatic cholangiocarcinoma (ICC) is a malignant tumor that originates from the bile duct epithelium above the confluence of the left and right hepatic ducts. As the second most common liver malignancy, it accounts for approximately 25% of all liver cancers, and its morbidity and mortality rates are steadily increasing. With a high degree of malignancy, ICC patients have a limited 1-year and 5-year overall survival rates of only 30% and 18%, respectively. Long-term survival for ICC patients depends on surgical resection, including liver resection and portal lymph node dissection. However, most patients are diagnosed with locally advanced or metastatic disease, making resection a suitable option for only 20%-30%. Furthermore, the 5-year survival rate after surgical resection is only 11%-40%, and most patients will relapse after resection. The 70%-80% of ICC patients who cannot undergo surgical resection are left with systemic chemotherapy, which has very limited efficacy. Therefore, it is urgent to understand the pathogenesis and treatment strategies of cholangiocarcinoma.

[0003] Human primary tumor cells are derived from the tumor tissue of clinical patients themselves, without any transformation or modification. They can reflect the characteristics of tumor tissue and are important biological materials for tumor biology research. At the same time, they can study the biological functions, mechanisms and sensitivity to chemotherapy drugs of human primary tumor cells in a suitable culture environment, thereby reflecting the genotype and phenotypic characteristics of tumor tissue of clinical patients. However, there are few existing ICC cell models, and there are only about 79 ICC cell lines reported so far. According to the literature and cell databases such as ATCC, according to the population source classification, the reported ICC cell lines include: (1) China: LICCF, IHC-ST1, ICC-X1, ICC-X2, ICC-X3, HCCC-9810 and HKGZ-CC; (2) Europe and the United States: 82.3, CC-LP-1, CC-SW-1, LIV27, MT-CHC01 and MT-CHC01R1.5; (3) Japan: ETK-1, HChol- Y1, HuCC-T1-G10, HuCC-T1-G100, HuH-28, KMC-1, NCC-CC1, NCC-CC3-1, NCC-CC3-2, NCC-CC4-1, NCC-CC4-2, NCC-CC4-3, NCC-CC6-1, NCC-CC6-2, OZ, RBE, RBE-Luc2-tdT, SSP-25, HuCC-T1, and TKKK; (4) South Korea: SNU-1079; (5) Southeast Asia, Thailand: HubCCA-1, HuCC-A1, HuCC-A1Nu, KKK-D049, KKK-D068, KKK-D131, KKK-D138, KKU-023, KKU-21 3A, KKU-213B, KKU-213C, KKU-213L5, KKU-M055, KKU-M055 / 46, KKU-M055 / 8, KKU-M139, KKU-M139 / GEM, KK U-M213L0H, KKU-M213L5H, KKU-M214 / 246, KKU-M214 / 9, KKU-M214 / GEM, KKU-OCA17, and RMCCA-1; (6) Unspecified populations: ICC1, ICC2, ICC3, ICC4, ICC5, ICC6, ICC8, ICC9, ICC10, ICC10-6, ICC10-8, ICC12, ICC13-7, ICC15, and SG231. In addition, a study by Dong et al. also elucidated ICC cell lines derived from 6 patients (PMID: 29551704).

[0004] In summary, there are currently very few ICC model cells. Although the existing ICC cell lines are all derived from cholangiocarcinoma, due to the existence of tumor heterogeneity, patients with ICC lack obvious common gene mutation characteristics, which cannot fully cover the characteristics of this disease. In addition, due to the different genetic backgrounds, in order to better study the disease characteristics of ICC in Chinese people, it is necessary to establish more Chinese-specific ICC model cell lines. Therefore, it is necessary to establish more ICC cell lines, enrich the cholangiocarcinoma cell library, and target the respective gene mutation characteristics of these cells and detect the sensitivity characteristics of different therapeutic drugs to provide more basic cell experimental models in clinical treatment.

[0005] In summary, discovering new human cholangiocarcinoma cell lines is one of the urgent issues to be addressed in the field of intrahepatic cholangiocarcinoma. Summary of the Invention

[0006] To address the shortcomings of existing technologies and meet practical needs, the present invention provides a human intrahepatic cholangiocarcinoma cell line and its application. The aim is to establish a novel primary cholangiocarcinoma cell line derived from intrahepatic cholangiocarcinoma tissue. The cell line exhibits stable biogenetic traits, a well-defined cell generation history, and is sensitive to cisplatin and gemcitabine, three chemotherapy drugs, but relatively insensitive to 5-fluorouracil. The establishment of this cell line further enriches the human cholangiocarcinoma cell library, providing new research materials for exploring the occurrence, progression, metastasis, and drug resistance mechanisms of primary cholangiocarcinoma in the Chinese population, as well as for the development of new drugs.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides a human intrahepatic bile duct cancer cell line, which includes human intrahepatic bile duct cancer cell JXQ-3D-6951R; the human intrahepatic bile duct cancer cell JXQ-3D-6951R was deposited in the China Center for Type Culture Collection on November 22, 2023, with the preservation number CCTCC NO: C2023370.

[0009] In this study, a novel human intrahepatic cholangiocarcinoma cell line was developed. The cell line exhibits stable properties and morphology, is capable of large-scale expansion, and can be subcultured in vitro for long periods. The cell line is sensitive to cisplatin and gemcitabine, but relatively insensitive to 5-FU. This provides a new experimental model for studying the etiology, metastasis mechanisms, drug resistance, and drug screening of intrahepatic cholangiocarcinoma.

[0010] Preferably, the human intrahepatic cholangiocarcinoma cell line further includes a progeny cell line of the human intrahepatic cholangiocarcinoma cell line JXQ-3D-6951R.

[0011] In a second aspect, the present invention provides a cell model, wherein the cell model comprises the human intrahepatic bile duct carcinoma cell line described in the first aspect.

[0012] In a third aspect, the present invention provides a use of the human intrahepatic cholangiocarcinoma cell line described in the first aspect in preparing a cell model for studying the pathogenesis of intrahepatic cholangiocarcinoma.

[0013] The present invention creatively discovered a new human intrahepatic cholangiocarcinoma cell line, JXQ-3D-6951R. The cell line has sensitivity characteristics to different therapeutic drugs and is sensitive to the three chemotherapy drugs used in the present invention. It can be used as a good in vitro research model in the field of scientific research, such as for theoretical research on the etiology of intrahepatic cholangiocarcinoma, for screening more drugs for the treatment of intrahepatic cholangiocarcinoma, etc.

[0014] In a fourth aspect, the present invention provides use of the human intrahepatic cholangiocarcinoma cell line described in the first aspect in preparing an intrahepatic cholangiocarcinoma animal model.

[0015] Preferably, the animal model is used to investigate the development or metastasis of intrahepatic cholangiocarcinoma cells.

[0016] According to the research results of the present invention, the novel human intrahepatic cholangiocarcinoma cell line JXQ-3D-6951R prepared by the present invention has a certain migration ability. Therefore, the intrahepatic cholangiocarcinoma animal model prepared by the cell line JXQ-3D-6951R is used in the field of scientific research to provide new research materials for exploring the development or metastasis of intrahepatic cholangiocarcinoma cells.

[0017] Preferably, the animal model is used to explore drug resistance pathways in intrahepatic cholangiocarcinoma.

[0018] According to the research results of the present invention, the new human intrahepatic cholangiocarcinoma cell line JXQ-3D-6951R prepared by the present invention is sensitive to cisplatin and gemcitabine, and is relatively insensitive to 5-FU. The drug-resistant characteristics of the cell line are used in the field of scientific research to provide new research materials for exploring the drug resistance mechanism and resistance pathway of intrahepatic cholangiocarcinoma cells.

[0019] In a fifth aspect, the present invention provides a method for constructing an intrahepatic bile duct carcinoma animal model, the method comprising:

[0020] The human intrahepatic bile duct carcinoma cell line described in the first aspect is used to infect immunodeficient animals to form tumors.

[0021] Preferably, the immunodeficient animal includes an immunodeficient nude mouse, an immunodeficient rat or a humanized mouse.

[0022] In a sixth aspect, the present invention provides use of the human intrahepatic cholangiocarcinoma cell line described in the first aspect in preparing a model for screening drugs for treating intrahepatic cholangiocarcinoma.

[0023] In a seventh aspect, the present invention provides use of the human intrahepatic cholangiocarcinoma cell line described in the first aspect in screening drugs for preventing or treating intrahepatic cholangiocarcinoma.

[0024] Preferably, the screening comprises determining whether the candidate drug can be used to prevent or treat intrahepatic cholangiocarcinoma based on the effects of the candidate drug on the human intrahepatic cholangiocarcinoma cell line before and after use.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The newly established human intrahepatic cholangiocarcinoma cell line JXQ-3D-6951R has a stable biological morphology and shape, can be stably passaged for a long time, and has been identified as a new single cell line by STR. It has strong proliferative activity, can be expanded in large quantities and cultured in vitro, and has clone-forming ability.

[0027] 2. The JXQ-3D-6951R cell line established by the present invention is an intrahepatic cholangiocarcinoma cell line derived from intrahepatic cholangiocarcinoma tissue of a Chinese individual, and can expand and enrich the cell resource library of intrahepatic cholangiocarcinoma, especially intrahepatic cholangiocarcinoma of a Chinese individual.

[0028] 3. The JXQ-3D-6951R established in the present invention has obvious biological functions, with strong proliferation, migration and clone formation abilities. It is sensitive to cisplatin and gemcitabine among the three chemotherapy drugs used in the present invention, and is relatively insensitive to 5-FU. It can be used as a good in vitro research model, providing a strong cell material basis for the occurrence and development, metastasis mechanism, drug resistance mechanism and new drug development and screening of intrahepatic bile duct carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a morphological observation image of human intrahepatic cholangiocarcinoma cell JXQ-3D-6951R (magnification 40).

[0030] Figure 2 This is a cell proliferation curve of human intrahepatic cholangiocarcinoma cell JXQ-3D-6951R.

[0031] Figure 3 This is a photograph of human intrahepatic cholangiocarcinoma cell JXQ-3D-6951R cells scratched at 0 hours (magnification 40).

[0032] Figure 4 This is a photograph of human intrahepatic cholangiocarcinoma cell JXQ-3D-6951R cells after scratching for 6 hours (magnification 40).

[0033] Figure 5 This is a photograph of human intrahepatic cholangiocarcinoma cell JXQ-3D-6951R cells after scratching for 24 hours (magnification 40).

[0034] Figure 6This is a photograph of human intrahepatic cholangiocarcinoma cell JXQ-3D-6951R cells after scratching for 48 hours (magnification 40).

[0035] Figure 7 This is a photograph of the colony formation of human intrahepatic cholangiocarcinoma cell JXQ-3D-6951R.

[0036] Figure 8 This is the result of the cisplatin drug sensitivity test on human intrahepatic bile duct cancer cell JXQ-3D-6951R.

[0037] Figure 9 This is the result of the 5-FU drug sensitivity test on human intrahepatic bile duct cancer cell JXQ-3D-6951R.

[0038] Figure 10 This is the result of the gemcitabine drug sensitivity test on human intrahepatic bile duct cancer cell JXQ-3D-6951R. DETAILED DESCRIPTION

[0039] To further illustrate the technical means and effects of the present invention, the present invention is further described below with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0040] For experimental methods with specific conditions not explicitly stated in the examples of the present invention, conventional conditions and methods in the art may be used, and reasonable adjustments may be made to the conditions. For details, reference may be made to the instructions of the suppliers of all the experimental kits used or to the Molecular Cloning Laboratory Manual (3rd edition or later, Cold Spring Harbor Laboratory Press, New York), etc.

[0041] All instruments, consumables, and reagents not specifically described in the examples of the present invention are conventional commercially available products in the art and can be obtained through commercial channels. The specific experimental materials and their sources listed in the following examples are merely illustrative and are not intended to limit the present invention. Materials of the same or similar type, model, quality, properties, or function as the following tissues, cells, reagents, and instruments can be used to practice the present invention.

[0042] In a specific embodiment of the present invention, the human intrahepatic cholangiocarcinoma cell line JXQ-3D-6951R Homo sapiens was deposited in the China Center for Type Culture Collection on November 22, 2023, with the deposit number CCTCCNO: C2023370; the address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0043] Example 1

[0044] This example establishes a novel human intrahepatic cholangiocarcinoma cell line.

[0045] The cell line established in this example was derived from a 66-year-old Chinese male patient with intrahepatic bile duct carcinoma invading the diaphragm in the left lobe of the liver. Fresh intrahepatic bile duct carcinoma tissue was obtained surgically, washed multiple times with pre-cooled sterile phosphate-buffered saline (PBS), and after removing excess blood and connective tissue, the tissue was cut into 1 mm 3 The small pieces were washed three times with pre-cooled sterile PBS. The small pieces were then transferred to a 10 cm culture dish and incubated in DMEM / F12 complete medium, which consisted of 90 parts DMEM / F12 (Gibco™, C11330500BT), 10 parts fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, USA), 1 part non-essential amino acids (Gibco™, 11140076), and 1 part penicillin (100 U / mL) / streptomycin (100 μg / mL) (Gibco, 15140-122; Thermo Fisher Scientific). When the cell density reached approximately 30%, the supernatant and remaining tissue fragments were discarded, and the cells were digested with 0.25% trypsin-EDTA (Gibco™, 25200072; Thermo Fisher Scientific) and transferred to 24-well plates for further culture for 5-6 generations to obtain purified tumor cells. The cells were then expanded using the aforementioned DMEM / F12 complete medium and cultured in a 37°C, 5% CO2 incubator.

[0046] Example 2

[0047] This example analyzes the biological characteristics and applications of cell lines

[0048] (1) Cell morphology

[0049] The morphology of the tumor cells obtained in Example 1 was observed under an inverted microscope. Figure 1 As shown (magnification 40), the cells grew adherently, were evenly dispersed, had relatively uniform size, and had a short spindle-shaped morphology. After multiple passages, the cell morphology remained consistent.

[0050] (2) STR identification

[0051] Short tandem repeats (STRs), also known as microsatellite DNA, are a type of DNA sequence on chromosomes consisting of a core unit of several base pairs (2-6 base pairs) repeated in tandem. The number of repeats ranges from 10 to 60, with gene fragments under 400 base pairs. The number of repeats of each core unit varies between individuals, resulting in alleles of varying fragment lengths. Therefore, the number of repeats of an STR sequence is virtually unique across individuals, serving as a hallmark of their genetic identity and a primary method for identifying cell identity and origin in cell biology.

[0052] STR analysis was performed on the tumor cells obtained in Example 1 and their derived tumor tissues. The cells were digested and collected with trypsin. Genomic DNA was extracted using a DNA extraction kit and amplified using a 20-locus STR amplification protocol. STR loci and the sex gene Amelogenin were analyzed on an ABI 3730XL genetic analyzer. The loci tested included: D5S818, D13S317, D7S820, D16S539, VWA, TH01, AMEL, TPOX, CSF1PO, D12S391, FGA, D2S1338, D21S11, D18S51, D8S1179, D3S1358, D6S1043, PENTAE, D19S433, and PENTAD. The results of the STR analysis of the tumor cells obtained in Example 1 are shown in Table 1.

[0053] Table 1

[0054]

[0055]

[0056] Note: Alle represents the chromosome where the test site is located, X represents sex chromosome X, and Y represents sex chromosome Y. Two sites X and Y represent male origin.

[0057] STR sequence searches were performed against the databases of the German Collection of Microorganisms (DSMZ) and the American Type Culture Collection (ATCC), but no identical STR results were found. No matching loci were found in the databases, indicating that the cell line was novel. No polyisotopic loci were found, indicating that the cell line was a single cell line with no contamination from other cells. The tumor cell line was named human intrahepatic cholangiocarcinoma cell line JXQ-3D-6951R.

[0058] (3) Cell proliferation

[0059] Confluent cells were digested with 0.25% trypsin to prepare a cell suspension. 20 μL of the cell suspension was mixed with 20 μL of trypan blue (Life, T10282). 20 μL of the mixture was slowly pipetted into a cell counting plate (Countstar, 12000550). Live cells were counted using an automatic cell counter (Countstar, IC 1000). Three fields of view were selected for counting in each counting well.

[0060] like Figure 2As shown in the figure, the cell counting results at four time points, 24h, 48h, 96h and 168h, were summarized. The measured time was used as the horizontal axis and the number of viable cells was used as the vertical axis. The measured values ​​at the four time points were plotted into a proliferation curve. The curve equation was Y = 3834*exp(0.02135*X), where Y is the number of cells, X is the proliferation time, and R 2 =0.996. Statistical data showed that the population doubling time of JXQ-3D-6951R cells was 32.47 h.

[0061] (4) Cell scratching

[0062] Before the cells are plated, use a pen to draw horizontal lines evenly on the back of the 6-well cell culture plate (using a ruler), approximately every 0.5-1 cm, and 5 lines are drawn in each well. Add 2 mL of complete culture medium to each well of the 6-well cell culture plate, and then calculate the number of cells added to each well based on the cell count results. 5 Prepare the volume of cell suspension required for each cell and inoculate it into a 6-well plate. Observe the cell seeding density under a microscope. After shaking the cells evenly, place them in an incubator and culture overnight. The next day, observe the cell density under a microscope. When the cell confluence reaches 90% or above, use a 200μL sterile pipette tip to scratch the top of the 6-well plate perpendicular to the horizontal line with uniform force, without pausing. Discard the culture medium and slowly add 2mL of PBS to wash the cells once they adhere to the wall. Discard the PBS and repeat this wash twice to remove any scratched cells. Add 2mL of fresh complete culture medium to each well.

[0063] Using an inverted microscope (Olympus, U-RFL-T) at 4× objective magnification (magnification 40), three scratch fields were selected for photographing and saved, and this time point was recorded as 0 h ( Figure 3 The cells were cultured in the incubator for 6 h, 24 h, and 48 h, respectively. The scratch field at the time point of 0 h was found and photographed.

[0064] The results showed that 6 hours after the cell scratch, some cells could be observed to migrate within the field of view ( Figure 4 ); 24 hours after cell scratching, many cells migrated ( Figure 5 ); 48 hours after cell scratching, the wound was completely healed ( Figure 6 ). This demonstrates that human intrahepatic cholangiocarcinoma cell line JXQ-3D-6951R has good migration ability and a fast migration speed.

[0065] (5) Clone formation

[0066] Add 2 mL of complete culture medium to each well of a 6-well cell culture plate. Calculate the volume of cell suspension required to add 500, 1000, 2000, 3000, 4000, and 5000 cells to each well based on the results of the cell count. Inoculate the cells into the 6-well plate and shake to mix. Observe the cells under a microscope to see if they are evenly distributed and place them in an incubator for culture. Observe the cell adhesion state on the next day, change the medium, and continue to culture for about 10 days. Based on the size and dispersion of the cell clones and whether the clones are connected together, determine that the optimal inoculation number for cell clone formation is 1000 cells / well. After digesting the cells, count the cells and calculate the volume of cell suspension required to add 1000 cells to each well. Inoculate the cells into a 6-well plate and observe under a microscope to see if they are evenly distributed and place them in an incubator for culture.

[0067] The next day, the cells were observed for attachment under a microscope. After changing the medium, the cells were cultured for approximately 10 days before terminating the culture. The culture medium in the 6-well plate was discarded, and each well was washed once with 1 mL of PBS. The PBS was discarded, and each well was fixed for 10 min at room temperature with 500 μL of methanol. The methanol was discarded, and each well was washed once with 1 mL of PBS. The PBS was discarded, and each well was fixed for 10 min at room temperature with 500 μL of 0.1% crystal violet (Solarbio, C8470) solution. The crystal violet solution was discarded, and each well was washed two to three times with 1 mL of ddH2O. The ddH2O was discarded, and the cells were air-dried. The entire cell culture well was photographed with a camera, and the number of cell colonies formed was counted using an optical microscope.

[0068] The results of the JXQ-3D-6951R cell plate clone formation experiment are as follows Figure 7 As shown, a large number of cell clones with clear edges were formed in the plate, indicating that JXQ-3D-6951R cells have the ability to form tumors in vitro and meet the characteristics of tumor cells.

[0069] (6) Drug sensitivity test

[0070] According to the counting results, 1000, 2000, 4000, 6000, 8000 and 10000 cells / 100 μL complete culture medium were added to the wells of a 96-well plate, and three replicates were made for each cell concentration. The culture was continued for 96 h. The cell growth status was observed under a microscope every day within 96 h, and the cell concentration that could fully grow the 96-well plate in 96 h (6000 cells / 100 μL) was selected for drug sensitivity testing.

[0071] Based on the count results, add 6,000 cells / 100 μL complete medium to each well of a 96-well plate. Perform three replicates for each cell concentration. Observe the cells under a microscope to ensure they are evenly shaken. Then, culture them in an incubator overnight. Observe the cell adhesion status under a microscope the next day.

[0072] The test uses three compounds: cisplatin, 5-fluorouracil (5-FU) and gemcitabine. Seven concentration gradients are set for each compound, and three replicates are set for each concentration. The drug concentrations are configured according to the following drug concentrations: the concentration gradient of cisplatin is 20 μM, 4 μM, 0.8 μM, 0.16 μM, 0.032 μM, 0.0064 μM and 0 μM (control); the concentration gradient of 5-fluorouracil is 900 μM, 30 0μM, 100μM, 33μM, 11μM, 3.7μM and 0μM (control); the concentration gradient of gemcitabine was 100μM, 20μM, 4μM, 0.8μM, 0.16μM, 0.032μM and 0μM (control), and then the supernatant culture medium of the 96-well plate was discarded, and 100μL of complete culture medium containing the above-mentioned drugs was added and continued to culture for 72h, and then the cell supernatant was discarded, 100μL of CTG reagent was added to each well and placed in a mixer, and mixed at 600rpm in the dark for 10min; after the mixing was completed, the cell supernatant was transferred to a 96-well white plate that could be protected from light, and the luminescence value was measured using an enzyme marker to calculate the cell viability.

[0073] The IC50 values ​​of JXQ-3D-6951R cells for cisplatin, 5-FU and gemcitabine were 1.547 μM ( Figure 8 )、81.62μM( Figure 9 ) and 1.02 μM ( Figure 10 ). The results show that the cells are highly sensitive to cisplatin and gemcitabine. In the present invention, the lower the IC50 value of the drug, the more sensitive the cell is to the drug. Conversely, the higher the IC50, the lower the cell sensitivity to the drug. After treating the cells with 4μM cisplatin, cell viability dropped sharply, and its IC50 was only 1.547μM. Similarly, the IC50 of gemcitabine was only 1.02μM, which is in the lower range, that is, relatively sensitive. The results of drug resistance to 5-FU showed that its IC50 was as high as 81.62μM, that is, low sensitivity to 5-FU, that is, relative resistance.

[0074] The above results indicate that the human cholangiocarcinoma cell line JXQ-3D-6951R of the present invention was derived from a 66-year-old Chinese male patient with intrahepatic bile duct carcinoma invading the diaphragm in the left lobe of the liver. The cells were relatively uniform in size and morphologically short and spindle-shaped, and their morphology remained stable after multiple passages. STR analysis identified the cell line as a novel cell line; no polymorphic loci were found, indicating that the cell line is a single cell line free of contamination by other cells. Regarding proliferation, the population doubling time of JXQ-3D-6951R cells was 32.47 hours. A scratch wound assay revealed that JXQ-3D-6951R cells possessed strong migration ability. A plate-based colony formation assay revealed that after 10 days of culture, the cells formed loose colonies on the plate. Regarding drug sensitivity, the human cholangiocarcinoma cell line JXQ-3D-6951R prepared by the present invention was sensitive to cisplatin and gemcitabine, but relatively insensitive to 5-FU.

[0075] The human intrahepatic cholangiocarcinoma cell line JXQ-3D-6951R established by the present invention has stable biological genetic traits, a clear cell generation number, and is identified as a new single cell line by STR analysis. It has strong proliferative activity, can be expanded in large quantities and subcultured in vitro, has clone-forming ability, and is sensitive to cisplatin and gemcitabine, three chemotherapy drugs, and is relatively insensitive to 5-FU. The establishment of this cell line further enriches the human cholangiocarcinoma cell library and provides new research materials for exploring the occurrence, development, metastasis mechanism, and drug resistance mechanism of primary cholangiocarcinoma in the Chinese population, as well as for the development of new drugs.

[0076] The applicant states that the present invention is intended to illustrate the detailed methods of the present invention through the above-described embodiments, but the present invention is not limited to the above-described detailed methods, that is, it does not mean that the present invention must rely on the above-described detailed methods in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions for various raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

Claims

1. A human intrahepatic cholangiocarcinoma cell line, characterized in that: The human intrahepatic bile duct carcinoma cell line includes the human intrahepatic bile duct carcinoma cell JXQ-3D-6951R; the human intrahepatic bile duct carcinoma cell JXQ-3D-6951R was deposited in the China Center for Type Culture Collection on November 22, 2023, with the preservation number CCTCCNO: C2023370.

2. The human intrahepatic cholangiocarcinoma cell line according to claim 1, characterized in that The human intrahepatic bile duct carcinoma cell line also includes a progeny cell line of the human intrahepatic bile duct carcinoma cell JXQ-3D-6951R.

3. A cell model, characterized in that The cell model comprises the human intrahepatic cholangiocarcinoma cell line according to claim 1 or 2.

4. Use of the human intrahepatic cholangiocarcinoma cell line according to claim 1 or 2 in preparing a cell model for studying the pathogenesis of intrahepatic cholangiocarcinoma.

5. Use of the human intrahepatic cholangiocarcinoma cell line according to claim 1 or 2 in preparing an intrahepatic cholangiocarcinoma animal model; Preferably, the animal model is used to investigate the development or metastasis of intrahepatic bile duct cancer cells; Preferably, the animal model is used to explore drug resistance pathways in intrahepatic cholangiocarcinoma.

6. A method for constructing an animal model of intrahepatic bile duct cancer, characterized in that: The method comprises: The human intrahepatic bile duct carcinoma cell line according to claim 1 or 2 is used to infect immunodeficient animals to form tumors.

7. The method for constructing an animal model of intrahepatic cholangiocarcinoma according to claim 6, characterized in that: The immunodeficient animals include immunodeficient nude mice, immunodeficient rats or humanized mice.

8. Use of the human intrahepatic cholangiocarcinoma cell line according to claim 1 or 2 in preparing a model for screening drugs for treating intrahepatic cholangiocarcinoma.

9. Use of the human intrahepatic cholangiocarcinoma cell line according to claim 1 or 2 in screening drugs for preventing or treating intrahepatic cholangiocarcinoma.

10. The use according to claim 9, characterized in that The screening includes determining whether the candidate drug can be used to prevent or treat intrahepatic bile duct cancer based on the effects of the candidate drug on the human intrahepatic bile duct cancer cell line before and after use.

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