Human gallbladder cancer cell line and application thereof

Abstract

The invention discloses a human gallbladder cancer cell line and application thereof, and relates to the technical field of cells. The human gallbladder cancer cell line disclosed by the invention is named as a human gallbladder cancer cell line IIAIM-GBC021A6 Homo sapiens, the human gallbladder cancer cell line is preserved in the China Center for Type Culture Collection on February 14, 2025, and the preservation number is CCTCC (China Center for Type Culture Collection) NO: C202559. The human gallbladder cancer cell line IIAIM-GBC021A6Homo sapiens is stable in character, has strong proliferation ability, scratch healing ability, migration ability and in-vitro tumor formation ability, and has different sensitivities to different chemotherapeutic drugs such as cis-platinum, carboplatin, gemcitabine, oxaliplatin, 5-fluorouracil (5-Fu) and the like.

Description

Technical Field

[0001] The present invention relates to the field of cell technology, and more particularly to a human gallbladder cancer cell line and its applications. Background Art

[0002] Gallbladder carcinoma (GBC) is a malignant tumor originating from the mucosal epithelial cells of the gallbladder. It has a very high degree of malignancy. 80% of tumor patients have a survival period of no more than one year, the median survival period is only 3 months, and only 2%-5% of tumor patients have a 5-year survival period. Currently, for early-stage GBC, surgical resection is the main treatment, while for advanced GBC, systemic drug treatment is mainly used. Since the onset of GBC is relatively hidden, most patients have no obvious symptoms or only show symptoms of chronic cholecystitis at the early stage of onset, making it difficult to be diagnosed. Once patients present symptoms such as persistent abdominal pain, mass, jaundice, etc., they are mostly in the advanced stage and have lost the opportunity for surgical treatment. However, advanced gallbladder cancer cell lines are insensitive to commonly used clinical chemotherapy drugs, and the effect of targeted drug treatment is also very limited.

[0003] Tumor cell lines are important tools for tumor biology research, providing important experimental tools and research platforms for the study of tumor pathogenesis, drug development, personalized treatment, etc. To date, the number of GBC cell lines worldwide is very limited, and most GBC cell lines are derived from the same patient. From a genetic perspective, cell lines derived from the same tumor patient usually have the same genetic background and biological characteristics. Therefore, the existing gallbladder cancer cell lines cannot well reflect the pathological characteristics of the vast majority of gallbladder cancer patients, and there is an urgent need to establish more patient-derived gallbladder cancer cell lines to provide more experimental materials for the study of GBC pathogenesis and new drug development. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. For this purpose, the present invention provides a human gallbladder cancer cell line, which has stable characteristics, strong proliferation ability, scratch healing ability, migration ability, and in vitro tumorigenesis ability, and has different sensitivities to different chemotherapy drugs such as cisplatin, carboplatin, gemcitabine, oxaliplatin, 5-fluorouracil (5-Fu), etc.

[0005] The present invention also provides a method for constructing a gallbladder cancer animal model.

[0006] The present invention also provides a method for screening drugs for the treatment and / or prevention of gallbladder cancer.

[0007] The present invention also provides the applications of the above-mentioned gallbladder cancer cell line or animal model.

[0008] The human gallbladder cancer cell line according to the embodiment of the first aspect of the present invention is named as human gallbladder cancer cell line IIAIM-GBC021A6 Homo sapiens. The human gallbladder cancer cell line IIAIM-GBC021A6 Homo sapiens was deposited at the China Center for Type Culture Collection on February 14, 2025, with the deposit number of CCTCC NO: C202559.

[0009] The gallbladder cancer cell line according to the embodiment of the present invention has at least the following beneficial effects:

[0010] The human gallbladder cancer cell line IIAIM-GBC021A6 Homo sapiens in the embodiment has stable traits, can be stably passaged multiple times, and remains stable after being passaged in vitro to the 50th generation. It has strong proliferation ability, scratch healing ability, migration ability, and in vitro tumorigenesis ability, meeting the characteristics of tumor cells, with a tumorigenesis rate of 100%. It has different sensitivities to chemotherapeutic drugs such as cisplatin, carboplatin, gemcitabine, oxaliplatin, 5-Fu, etc., and is particularly sensitive to gemcitabine. The human gallbladder cancer cell line IIAIM-GBC021A6 Homo sapiens enriches the human gallbladder cancer cell line library, provides new experimental materials for preclinical studies on revealing the mechanisms of gallbladder cancer occurrence, development, and metastasis and the development of corresponding drugs, and has good application prospects in preparing human gallbladder cancer xenograft models, screening candidate drugs for treating human gallbladder cancer, etc.

[0011] A method for constructing a gallbladder cancer animal model according to the embodiment of the second aspect of the present invention includes the following steps:

[0012] Inoculate the human gallbladder cancer cell line described in the embodiment of the first aspect into a model animal.

[0013] According to some embodiments of the present invention, the inoculation method is selected from any one of orthotopic gallbladder injection, intravenous injection, intraperitoneal injection, subcutaneous injection, intradermal injection, and intramuscular injection.

[0014] According to some embodiments of the present invention, the model animal is an immunodeficient animal.

[0015] According to some embodiments of the present invention, the immunodeficient animal includes at least one of immunodeficient nude mice, immunodeficient rats, or humanized mice.

[0016] A method for screening a drug for treating and / or preventing gallbladder cancer according to the embodiment of the third aspect of the present invention includes the following steps:

[0017] Treat the human gallbladder cancer cell line described in the embodiment of the first aspect with the drug to be tested, detect the survival situation of the human gallbladder cancer cell line, and select the drug to be tested that meets the expectation as a candidate drug.

[0018] Use of the human gallbladder cancer cell line according to the first aspect of the embodiment of the fourth aspect of the present invention or the animal model obtained by the construction method according to the second aspect of the embodiment.

[0019] According to some embodiments of the present invention, the use is for studying the pathogenesis, development and / or metastasis mechanism of gallbladder cancer, or for preparing a product for studying the pathogenesis, development and / or metastasis mechanism of gallbladder cancer.

[0020] According to some embodiments of the present invention, the use is for screening or evaluating drugs for treating gallbladder cancer, or for preparing a product for screening or evaluating drugs for treating gallbladder cancer.

[0021] According to some embodiments of the present invention, the use is for developing treatment targets for gallbladder cancer or for preparing a product for developing treatment targets for gallbladder cancer.

[0022] According to some embodiments of the present invention, the use is for screening biomarkers for gallbladder cancer or for preparing a product for screening biomarkers for gallbladder cancer.

[0023] According to some embodiments of the present invention, the use is for studying the mechanism of gemcitabine sensitivity of gallbladder cancer or for preparing a product for studying the mechanism of gemcitabine sensitivity of gallbladder cancer.

[0024] According to some embodiments of the present invention, the use is for non-disease diagnosis and treatment purposes.

[0025] According to some embodiments of the present invention, the product is selected from drugs, reagents or kits.

[0026] Other features and advantages of the present invention will be described in the subsequent specification, and in part will be obvious from the specification, or will be understood by implementing the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1A It is a morphological observation diagram of the 10th passage of IIAIM-GBC021A6 cells;

[0028] Figure 1B It is a morphological observation diagram of the 20th passage of IIAIM-GBC021A6 cells;

[0029] Figure 1C It is a morphological observation diagram of the 30th passage of IIAIM-GBC021A6 cells;

[0030] Figure 2 It is a proliferation curve diagram of IIAIM-GBC021A6 cells;

[0031] Figure 3AIt is the result graph of IIAIM-GBC021A6 cell scratch at 0 h;

[0032] Figure 3B It is the result graph of IIAIM-GBC021A6 cell scratch at 24 h;

[0033] Figure 3C It is the result graph of IIAIM-GBC021A6 cell scratch at 48 h;

[0034] Figure 4 It is the colony formation result of IIAIM-GBC021A6 cells;

[0035] Figure 5 It is the cell migration photo of IIAIM-GBC021A6;

[0036] Figure 6 It is the result graph of the drug sensitivity test of IIAIM-GBC021A6 cells to cisplatin;

[0037] Figure 7 It is the result graph of the drug sensitivity test of IIAIM-GBC021A6 cells to gemcitabine;

[0038] Figure 8 It is the result graph of the drug sensitivity test of IIAIM-GBC021A6 cells to carboplatin;

[0039] Figure 9 It is the result graph of the drug sensitivity test of IIAIM-GBC021A6 cells to oxaliplatin;

[0040] Figure 10 It is the result graph of the drug sensitivity test of IIAIM-GBC021A6 cells to 5-Fu;

[0041] Figure 11 It is the tumor growth curve graph of IIAIM-GBC021A6 cells inoculated into nude mice;

[0042] Figure 12 It is the tumor tissue photo formed by inoculating IIAIM-GBC021A6 cells into nude mice. Specific implementation mode

[0043] The concept of the present invention and the technical effects generated will be clearly and completely described below in combination with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0044] In the examples where specific conditions are not indicated, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase.

[0045] Unless otherwise specified, "room temperature" in the present invention means (25 ± 5)°C.

[0046] Example 1

[0047] This example discloses a method for establishing the human gallbladder cancer cell line IIAIM-GBC021A6 Homo sapiens, and the steps are as follows:

[0048] Fresh gallbladder cancer tissue was obtained through surgery (from a 73-year-old female patient with gallbladder adenocarcinoma from China). The tissue was washed with pre-cooled sterile phosphate buffer (PBS) to remove excess blood and connective tissue. After that, the tissue was cut into tissue blocks of about 2*2*2 mm and washed 3 times with pre-cooled sterile PBS. The tissue blocks were placed in a 6-well plate, and DMEM / F12 complete medium (containing 20% FBS + 1% P / S) was added, and it was placed in an incubator at 37°C and 5% CO 2 overnight. The next day, the old culture medium was aspirated and discarded, and DMEM / F12 complete medium (containing 20% FBS + 1% P / S) was added. After that, the DMEM / F12 complete medium (containing 20% FBS + 1% P / S) was changed every 2 - 3 days, and the content of penicillin / streptomycin (P / S) was gradually reduced. When the cell density reached about 30%, the supernatant and tissue blocks were removed, and the cells were digested with 0.25% trypsin (containing EDTA) and transferred to a 6-well plate for continued culture for 4 - 5 generations, thereby obtaining pure tumor cells, and the growth rate of the cells changed from slow to fast and tended to be stable. The cells were named the human gallbladder cancer cell line IIAIM-GBC021A6 Homo sapiens (Homo sapiens, also known as: IIAIM-GBC021A6 cells).

[0049] At 37°C and 5% CO 2 In an incubator, the human gallbladder cancer cell line IIAIM-GBC021A6 Homo sapiens was passaged / amplified using DMEM / F12 complete medium (containing 10% FBS).

[0050] The human gallbladder cancer cell line IIAIM-GBC021A6 Homo sapiens was deposited at the China Center for Type Culture Collection (CCTCC) on February 14, 2025. The address of the depositary institution is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postcode 430072, and the deposit number is CCTCC NO: C202559.

[0051] Example 2

[0052] In this example, the biological characteristics of the human gallbladder cancer cell line IIAIM-GBC021A6 Homo sapiens were analyzed.

[0053] (1) Morphological observation:

[0054] The culture flasks containing IIAIM-GBC021A6 cells at different passages (P10, P20, and P30) were placed under an inverted microscope and observed under bright field.

[0055] The optical morphology pictures of IIAIM-GBC021A6 cells at P10, P20, and P30 passages are as Figure 1A - Figure 1C shown.

[0056] After multiple passages, the characteristics of IIAIM-GBC021A6 cells were stable. The cells at P10, P20, and P30 passages had the same morphology. The cells adhered to the wall and grew in patches, were evenly dispersed, had relatively consistent sizes, and were polygonal in shape. This indicates that the cell morphology was stable after multiple passages and the cells had the ability to be stably passaged.

[0057] (2) Analysis of short tandem repeat (STR) of genetic material and homology identification:

[0058] STR, also known as microsatellite DNA, refers to a class of DNA sequences formed by tandem repeats of several base pairs (2 - 6 base pairs) as the core unit on chromosomes (the number of repeats is more than 10 - 60 times, and the gene fragment is less than 400 base pairs); the number of repeats of each core unit shows individual differences, thus forming alleles with different fragment lengths. Therefore, the number of repeats of a set of STR sequences is almost unique in different individuals, which is the genetic identity characteristic of an individual and also the main method for cell biology to identify the identity and origin of cells.

[0059] DNA from IIAIM-GBC021A6 cells and their derived gallbladder cancer tissues was extracted using a genomic extraction kit (Axygen), amplified using a 21-STR amplification protocol, and the STR loci and sex gene Amelogenin were detected on an ABI3730XL genetic analyzer. Testing services were provided by Shanghai Fuda Testing Group. STR loci and Amelogenin points include: D5S818, D13S317, D7S820, D16S539, VWA, TH01, AMEL, TPOX, CSF1PO, D12S391, FGA, D2S1338, D21S11, D18S51, D8S1179, D3S1358, D6S1043, PENTAE, D19S433, PENTAD, and D1S1656.

[0060] The results of STR analysis of the genetic material of IIAIM-GBC021A6 cells and their source tissues are shown in Table 1.

[0061] Table 1

[0062]

[0063]

[0064] Note: Allele refers to a gene variant located at the same position on homologous chromosomes; AMEL is a sex identification site (XX indicates female).

[0065] The genotyping results of the STR loci and Amelogenin loci of IIAIM-GBC021A6 cells and their source tissues matched the STR data collected in the American Type Culture Collection (ATCC), the German Collection of Microorganisms (DSMZ, which collected STR data of 2,490 cell strains from ATCC, DSMZ, JCRB and RIKEN), and the ExPASy cell bank (ExPASy collected STR data of about 8,000 human cell strains from databases such as ATCC, DSMZ, Japan JCRB Cell Collection, European Certified Cell Culture Collection (ECACC) and RIKEN), and no identical STR test results were found. In other words, no loci matching those of IIAIM-GBC021A6 cells were found in the database. This indicates that IIAIM-GBC021A6 cells are a new cell line.

[0066] Furthermore, the analysis results showed that no polyisotopes were found, which indicated that IIAIM-GBC021A6 cells were single cells and no cross contamination with other cells occurred during the culture process.

[0067] The STR genotype matching degree of IIAIM-GBC021A6 cells and their tissues is 100%. According to the identification criteria of the ATCC Standards Committee (ANSI / ATCC ASN-0002-2011), a matching degree of ≧80% is considered that the samples are of the same origin. This indicates that the IIAIM-GBC021A6 cell line is derived from human gallbladder cancer tissue, well preserves the characteristics of gallbladder cancer patients, and has high clinical guiding value.

[0068] (3) Cell proliferation experiment:

[0069] Digest the cells with 0.25% trypsin (containing EDTA) and prepare a cell suspension. Take 10 μL of the cell suspension and count it with a cell counter. Prepare a cell suspension of 1000 cells / 100 μL, evenly inoculate it in a 96-well plate, and after standing for 2 h - 4 h, add trichloroacetic acid (TCA) with a final concentration of 10% to fix the first case of cells, denoted as 0, and place it in an incubator at 37 °C and 5% CO 2 and continuously culture. Fix one case every other day (24 h) for a total of 9 days (216 h). Remove the plate, discard the 10% TCA solution, and wash it once with ddH 2 O; discard ddH 2 O, dry it in an oven, add 100 μL of 0.4% sulforhodamine B (SRB) staining solution to each well, and fix it at 25 °C for 1 h; discard the 0.4% SRB staining solution, and wash it 3 times with ddH 2 O; discard ddH 2 O, dry it in an oven, add 100 μL of 10 mM tris to dissolve SRB, mix well, and then use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value at 540 nm. Detect the cell number at each time period by detecting the absorbance. Summarize the absorbance of the cells on days 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9, and calculate the growth multiple relative to day 0 at each time period. Take the measured time as the abscissa and the growth multiple as the ordinate, and plot the proliferation curve for the 9-day measurements.

[0070] The results are as Figure 2 shown.

[0071] As Figure 2 can be seen, the IIAIM-GBC021A6 cell line is in the doubling state on days 1, 2, 3, 4, 5, 6, 7, and 8, and the average doubling time of the cells is about 31 h. On the 9th day, it enters the plateau and stable phase, with strong proliferation ability.

[0072] (4) Cell scratch experiment:

[0073] First, use a marker pen to evenly draw horizontal lines on the back of a 6-well cell culture plate (with a ruler), about one line every 0.5 - 1 cm, and 5 lines pass through each well. Add 2 mL of complete culture medium to each well of the 6-well cell culture plate, and inoculate 3.5×105 One IIAIM-GBC021A6 cell. After the cells were shaken evenly, they were placed in an incubator for overnight culture. The next day, the cell density was observed under a microscope. When the cell confluence reached 90% or more, a 200 μL sterile pipette tip was used to make a scratch perpendicular to the horizontal line. The pipette tip should be perpendicular and not tilted, and the force should be consistent without pausing in the middle. Discard the culture medium, slowly add 2 mL of PBS to the adherent cells to wash the cells, discard the PBS, and repeat the washing 2 times in total to remove the scratched cells. Add 2 mL of fresh serum-free culture medium to each well. Use a microscope to select 3 scratch fields of view for photographing and saving, and record this time point as 0 h. Continue to culture the cells in the incubator. At 24 h and 48 h respectively, find the scratch field of view at the 0 h time point for photographing and saving.

[0074] The results are as Figure 3A - Figure 3C shown.

[0075] After 24 h of scratching of IIAIM-GBC021A6 cells, the scratch in the field of view became significantly narrower; after 48 h, the scratch was completely healed. This indicates that IIAIM-GBC021A6 cells have the ability of scratch healing and migration ability.

[0076] (5) Plate colony formation assay:

[0077] Add 2 mL of complete culture medium to each well of a 6-well cell culture plate. Inoculate 500, 1000, 2000, and 4000 IIAIM-GBC021A6 cells into each well respectively. After shaking and mixing evenly, place them in an incubator for culture. Observe the cell attachment status the next day. After changing the medium, continue to culture for about 10 days. Determine that the optimal seeding number for cell colony formation is 500 cells / well according to "the size of cell colonies, the dispersion situation, and whether the colonies are connected together".

[0078] Add 2 mL of DMEM / F12 complete medium (containing 10% FBS) to each well of a 6-well cell culture plate. Inoculate 500 IIAIM-GBC021A6 cells into each well, shake and mix evenly, and then place them in an incubator for culture. Observe the cell attachment status under a microscope the next day. After changing the medium, continue to culture for about 14 days, and then terminate the culture; discard the culture medium in the 6-well plate, add 2 mL of 10% TCA solution to each well, and fix at 4 °C for 2 h; discard the 10% TCA solution, wash once with ddH 2 O; discard ddH 2 O, dry in an oven, add 2 mL of 0.4% SRB staining solution to each well, and stain at 25 °C for 1 h; discard the 0.4% SRB staining solution, wash 3 times with ddH 2 O; discard ddH 2 O, dry in an oven. Use a camera to photograph the overall effect of the cell culture wells.

[0079] The results are asFigure 4 as shown

[0080] The results showed that IIAIM-GBC021A6 cells had the ability to form tumors in vitro and met the characteristics of tumor cells.

[0081] (6) Cell migration assay:

[0082] After adding 600 μL of DMEM / F12 complete medium (containing 10% FBS) to each well of a 24-well plate, place the chamber. Add 200 μL of serum-free medium containing 2 × 10 5 IIAIM-GBC021A6 cells into the chamber and culture for 24 h. Add 600 μL of 4% paraformaldehyde fixative to a new well, transfer the chamber to the well containing 600 μL of 4% paraformaldehyde fixative, and add 200 μL of 4% paraformaldehyde fixative to the chamber. Fix at room temperature for 2 h - 4 h. After fixation, wash the chamber 3 times with ddH 2 2O. Add 600 μL of crystal violet to a new well, place the washed chamber into it, add 100 μL of crystal violet to the chamber, and stain at room temperature for 30 min - 60 min. After staining, wash the chamber 3 times with ddH 2 2O. Place the washed chamber in a new well. After wiping the upper chamber with a cotton swab, observe the staining under a microscope and take pictures.

[0083] The results were as Figure 5 shown.

[0084] IIAIM-GBC021A6 cells had a certain migratory ability.

[0085] (7) Drug sensitivity test:

[0086] Add 100 μL of DMEM / F12 complete medium (containing 10% FBS) containing 6000 IIAIM-GBC021A6 cells to each well of a 96-well cell culture plate. After shaking well, place it in an incubator and culture overnight. Observe the cell attachment status under a microscope the next day. Discard the culture medium, and add 100 μL of DMEM / F12 complete medium (containing 10% FBS) containing cisplatin, carboplatin, 5-Fu, gemcitabine, or oxaliplatin at a set concentration to each well, and then culture for 72 h. Fix the cells with TCA at a final concentration of 10%. Discard the 10% TCA solution, and wash once with ddH 2 2O; discard ddH 2O, dried in an oven, 100 μL of 0.4% SRB staining solution per well, stained at 25 °C for 1 h; discard the 0.4% SRB staining solution, wash with 1% glacial acetic acid 3 times; discard the 1% glacial acetic acid, dry in the oven, add 100 μL of 10 mM tris to dissolve SRB, mix well and measure the absorbance at 540 nm using a microplate reader. Calculate the cell survival rate at each concentration of each compound. Using the compound concentration as the abscissa and the cell survival rate as the ordinate, make a non-linear regression and curve fitting graph to obtain the IC 50 value. Set 2 replicate wells for each concentration.

[0087] The set treatment concentrations of each compound are as follows:

[0088] Cisplatin: 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, 0.15625 μM, and 0 μM (control);

[0089] Gemcitabine: 20 μM, 0.8 μM, 0.032 μM, 0.0064 μM, 0.00128 μM, 0.000256 μM, 0.0000512 μM, and 0 μM (control);

[0090] Carboplatin: 640 μM, 320 μM, 160 μM, 80 μM, 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, and 0 μM (control);

[0091] Oxaliplatin: 200 μM, 40 μM, 8 μM, 1.6 μM, 0.32 μM, 0.064 μM, and 0 μM (control);

[0092] 5-Fu: 1500 μM, 300 μM, 60 μM, 12 μM, 2.4 μM, 0.48 μM, and 0 μM (control).

[0093] The results are as Figure 6 - Figure 10 shown.

[0094] The IC 50 values of IIAIM-GBC021A6 cells for cisplatin, gemcitabine, carboplatin, oxaliplatin, and 5-Fu are 2.93 μM, 0.008 μM, 66.04 μM, 2.39 μM, and 29.82 μM, respectively. IIAIM-GBC021 cells have different sensitivities to cisplatin, gemcitabine, carboplatin, oxaliplatin, and 5-Fu, and are particularly sensitive to gemcitabine.

[0095] (8) Tumorigenicity experiment: Detect the in vivo tumorigenicity of the cell line IIAIM-GBC021A6 by subcutaneous tumorigenesis experiment in nude mice

[0096] The IIAIM-GBC021A6 cells in good growth state were digested with 0.25% trypsin to prepare a cell suspension (cell concentration: 1×10 8 cells / mL). 100 μL of the cell suspension was inoculated into the unilateral axilla of 5-week-old SPF-grade female nude mice (purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd., experimental animal license number: SCXK (Jiangsu) 2023-0009) (3 mice were inoculated in total), and then observed. All experimental animals were housed in an SPF-grade breeding room. 14 days after inoculation, stable xenograft tumors the size of mung beans were observed on the nude mice. Subsequently, the growth of the tumors was recorded every 2-3 days. On the 42nd day, the mice were dissected, the tumors were removed, and the excess tissue was removed for observation and photography.

[0097] The results are shown in Figure 11 and Figure 12 .

[0098] The IIAIM-GBC021A6 cells could form tumors in nude mice, and the tumorigenesis rate was 100% (3 / 3), indicating tumorigenicity. This shows that the IIAIM-GBC021A6 cells can be used to construct an animal model for drug screening and efficacy evaluation.

[0099] In summary, the human gallbladder cancer cell line IIAIM-GBC021A6 Homo sapiens can be amplified in large numbers and can be passaged in vitro for a long time. It has different sensitivities to cisplatin, carboplatin, 5-Fu, gemcitabine, and oxaliplatin, enriching the human gallbladder cancer cell bank. It can be used to construct an animal model, providing a new experimental model for the etiology, metastasis mechanism, drug resistance mechanism, drug screening, and drug efficacy evaluation of gallbladder cancer, and providing new experimental materials for the preclinical study of revealing the occurrence and development mechanism of gallbladder cancer in the Chinese population and the development of corresponding drugs.

[0100] The embodiments of the present invention have been described in detail above in combination with the examples. However, the present invention is not limited to the above embodiments. Various changes can be made without departing from the spirit of the present invention within the scope of knowledge possessed by those of ordinary skill in the art.

Claims

1. A human gallbladder cancer cell line, characterized in that The human gallbladder cancer cell line IIAIM-GBC021A6 Homo sapiens was named and deposited in the China Center for Type Culture Collection on February 14, 2025, with the deposit number of CCTCC NO: C202559.

2. A method for constructing an animal model of gallbladder cancer, characterized in that: The following steps are involved: The human gallbladder cancer cell line according to claim 1 is inoculated into a model animal.

3. The construction method according to claim 2, characterized in that: The inoculation method is selected from any one of gallbladder in situ injection, intravenous injection, intraperitoneal injection, subcutaneous injection, intradermal injection and intramuscular injection.

4. The construction method according to claim 2, characterized in that: The model animals are immunodeficient animals.

5. A method for screening drugs for treating and / or preventing gallbladder cancer, characterized in that: The steps include: The human gallbladder cancer cell line according to claim 1 is treated with the drug to be tested, the survival of the human gallbladder cancer cell line is detected, and the drug to be tested that meets the expectations is selected as a candidate drug.

6. Use of the human gallbladder cancer cell line according to claim 1 or the animal model obtained by the construction method according to any one of claims 2 to 4 in studying the pathogenesis, development and / or metastasis mechanism of gallbladder cancer, or in preparing a product for studying the pathogenesis, development and / or metastasis mechanism of gallbladder cancer.

7. Use of the human gallbladder cancer cell line according to claim 1 or the animal model obtained by the construction method according to any one of claims 2 to 4 in screening or evaluating drugs for treating gallbladder cancer, or in preparing products for screening or evaluating drugs for treating gallbladder cancer.

8. Use of the human gallbladder cancer cell line according to claim 1 or the animal model obtained by the construction method according to any one of claims 2 to 4 in developing therapeutic targets for gallbladder cancer or preparing products for developing therapeutic targets for gallbladder cancer.

9. Use of the human gallbladder cancer cell line according to claim 1 or the animal model obtained by the construction method according to any one of claims 2 to 4 in screening gallbladder cancer biomarkers or preparing products for screening gallbladder cancer biomarkers.

10. Use of the human gallbladder cancer cell line according to claim 1 or the animal model obtained by the construction method according to any one of claims 2 to 4 in studying the mechanism of gallbladder cancer sensitivity to gemcitabine or in preparing a product for studying the mechanism of gallbladder cancer sensitivity to gemcitabine.

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