Human gallbladder cancer cell line and application thereof

Abstract

The invention discloses a human gallbladder cancer cell line and application thereof. The human gallbladder cancer cell line comprises a human gallbladder cancer cell JXQ-3D-4160R4; the human gallbladder cancer cell JXQ-3D-4160R4 is preserved in the China Center for Type Culture Collection on November 22, 2023, and the preservation number of the human gallbladder cancer cell JXQ-3D-4160R4 is CCTCC (China Center for Type Culture Collection) NO: C2023369. The human gallbladder 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 5-fluorouracil, but has lower sensitivity to gemcitabine, is a good in-vitro cell model, and can be used for preparing a human gallbladder cancer cell line. An important research basis is provided for the pathogenesis and drug resistance mechanism of the primary gallbladder cancer as well as research, development and screening of new drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a human gallbladder cancer cell line and application thereof. BACKGROUND

[0002] Gallbladder cancer (GBC) is the most common biliary tract malignancy, accounting for 80-95% of biliary tract tumors. Gallbladder cancer at the neck of the gallbladder is very similar to the hilar or common bile duct cancer, and the two are difficult to distinguish, but gallbladder cancer is more invasive. The incidence of gallbladder cancer continues to rise with age, and due to the lack of typical early symptoms, gallbladder cancer is usually diagnosed at an advanced stage, with a poor prognosis. The 5-year survival rate of patients with stage I cancer is about 50%, while that of patients with stage IV is only 3%, and the overall average survival rate is only about 6 months. Currently, surgical resection is still the main treatment for gallbladder cancer patients, only about 10% of patients can be timely resected, and almost about 50% of patients have lymph node metastasis. The recurrence rate within 5 years after surgical resection is about 64%, and patients who cannot be treated by surgery can only rely on radiotherapy and chemotherapy. Therefore, in-depth study of the genetic characteristics, development, metastasis and drug resistance mechanisms of gallbladder cancer is expected to improve the treatment effect of gallbladder cancer patients.

[0003] The exploration of the pathogenesis and drug resistance mechanism of human malignant tumors depends on the basic research of tumor cell characteristics. Clinical basic research explores the biological function of tumor cells and related signaling pathways, and then develops and screens compounds to inhibit the proliferation, metastasis and invasion ability of tumor cells; at the same time, it explores the sensitivity of tumor cells to chemotherapy drugs and the drug resistance mechanism, which helps the research and application of new treatment schemes. Compared with artificially modified and transformed tumor cell lines, the physiological state of primary cells derived from clinical patient tumor tissues is more similar to the characteristics of tumor cells in the patient's body, and thus is more conducive to the study of tumor drug sensitivity and the exploration of new treatment schemes. However, there are too few existing primary gallbladder cancer cell models. Comprehensive statistics of literature and related cell databases (Cellosaurus, etc.) show that there are only about 95 gallbladder cancer cell models. Due to the extensive existence of tumor heterogeneity, each case of gallbladder cancer has its own unique genetic mutation characteristics, so there is no obvious consistent genetic mutation characteristics between different gallbladder cancer patients, and it is difficult to comprehensively cover the characteristics of this disease. At present, about 59 gallbladder cancer cell strains are derived from 13 Chinese populations. Based on the differences in genetic background, it is still seriously insufficient to in-depth study the genetic mutation characteristics of gallbladder cancer in Chinese population with only this number. Some of the reported GBC cell lines have not been included in the STR authentication of the Cellosaurus database, and lack of genetic mutation characteristic map. GBC cell lines that have not passed STR authentication and lack of genetic mutation characteristic map are not suitable for GBC preclinical basic research.

[0004] Therefore, it is currently urgent to establish more gallbladder cancer cell lines derived from the Chinese population, to enrich the gallbladder cancer cell library, to comprehensively cover the genetic mutation characteristics of gallbladder cancer in the Chinese population and even the global population, to provide more comprehensive cell materials for preclinical research of gallbladder cancer, and to explore the sensitivity differences of different types of gallbladder cancer to drug treatment, and to provide more basic cell experimental models for clinical treatment.

[0005] In summary, mining new human gallbladder cancer cell lines is one of the problems to be solved in the field of gallbladder cancer. SUMMARY

[0006] In view of the deficiencies of the prior art and actual needs, the present application provides a human gallbladder cancer cell line and its application, aiming to establish a new type of gallbladder cancer primary cell line of gallbladder cancer tissue, which has strong proliferation, migration and colony formation ability, and is sensitive to three kinds of chemotherapy drugs cisplatin and 5-fluorouracil, but has lower sensitivity to gemcitabine, providing an important research basis for the pathogenesis, drug resistance mechanism, and new drug research and screening of primary gallbladder cancer.

[0007] To achieve the purpose of the present application, the following technical solutions are adopted:

[0008] In a first aspect, the present application provides a human gallbladder cancer cell line, which comprises human gallbladder cancer cells JXQ-3D-4160R4; the human gallbladder cancer cells JXQ-3D-4160R4 were deposited with the China Center for Type Culture Collection on November 22, 2023, and the deposit number is CCTCC NO: C2023369.

[0009] In the present application, a new type of human gallbladder cancer cell line is mined and prepared, which has stable cell line characteristics and morphology, can be amplified in large quantities, and can be cultured in vitro for a long time. The cell line is sensitive to three kinds of chemotherapy drugs cisplatin and 5-fluorouracil, but has lower sensitivity to gemcitabine, providing an important research basis for the pathogenesis, drug resistance mechanism, and new drug research and screening of primary gallbladder cancer.

[0010] Preferably, the human gallbladder cancer cell line further comprises a progeny cell line of human gallbladder cancer cells JXQ-3D-4160R4.

[0011] In a second aspect, the present application provides a cell model containing the gallbladder cancer cell line of the first aspect.

[0012] In a third aspect, the present application provides the use of the human gallbladder cancer cell line of the first aspect in the preparation of a cell model for studying the pathogenesis of gallbladder cancer.

[0013] The application creatively discovers a new human gallbladder cancer cell JXQ-3D-4160R4, which has sensitive characteristics to different therapeutic drugs, is sensitive to three kinds of chemotherapy drugs used in the application, and can be used as a good in-vitro research model for scientific research fields, such as gallbladder cancer etiology theory research, screening of more drugs for treating gallbladder cancer, and the like.

[0014] In a fourth aspect, the application provides application of the human gallbladder cancer cell line in the first aspect in preparation of a gallbladder cancer animal model.

[0015] Preferably, the animal model is used for exploring gallbladder cancer cell development or metastasis.

[0016] According to the research results of the application, the new human gallbladder cancer cell JXQ-3D-4160R4 prepared by the application has a certain migration ability, therefore, the gallbladder cancer animal model prepared by the cell strain JXQ-3D-4160R4 is used for scientific research fields, and provides new research materials for exploring gallbladder cancer cell development or metastasis.

[0017] Preferably, the animal model is used for exploring gallbladder cancer drug resistance pathways.

[0018] According to the research results of the application, the new human gallbladder cancer cell JXQ-3D-4160R4 prepared by the application is sensitive to drugs cisplatin and 5-fluorouracil, but has lower sensitivity to gemcitabine, and the drug resistance characteristics of the cell line are used for scientific research fields, and provide new research materials for exploring the drug resistance mechanism and drug resistance pathways of gallbladder cancer cells.

[0019] Preferably, the animal model is used for screening gallbladder cancer biomarkers.

[0020] In a fifth aspect, the application provides a method for constructing a gallbladder cancer animal model, the method comprising:

[0021] The human gallbladder cancer cell line in the first aspect is used to infect an immunodeficient animal to form a tumor.

[0022] Preferably, the immunodeficient animal comprises an immunodeficient nude mouse, an immunodeficient rat, or a humanized mouse.

[0023] In a sixth aspect, the application provides application of the human gallbladder cancer cell line in the first aspect in preparation of a model for screening drugs for treating gallbladder cancer.

[0024] In a seventh aspect, the application provides application of the human gallbladder cancer cell line in the first aspect in screening drugs for preventing or treating gallbladder cancer.

[0025] Preferably, the screening comprises determining whether a candidate drug can be used for preventing or treating gallbladder cancer based on the effect of the candidate drug on the human gallbladder cancer cell line before and after use.

[0026] Compared with the prior art, the present application has at least the following beneficial effects:

[0027] 1. The new gallbladder cancer cell line JXQ-3D-4160R4 established by the present application is an adherent cell with stable properties, long foot-like morphology, strong proliferation activity, and the ability to be amplified and subcultured in vitro.

[0028] 2. The JXQ-3D-4160R4 cell and its progeny cells established by the present application are a new single gallbladder cancer cell line identified by STR, which can further enrich the gallbladder cancer cell library, especially the gallbladder cancer cell library of Chinese population.

[0029] 3. The JXQ-3D-4160R4 established by the present application has obvious gallbladder cancer cell biological functions, is sensitive to three kinds of chemotherapeutic drugs cisplatin and 5-fluorouracil, but has lower sensitivity to gemcitabine, and is a good in vitro cell model, which provides an important research basis for the pathogenesis, drug resistance mechanism, and new drug research and screening of primary gallbladder cancer. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is a morphological observation diagram of human gallbladder cancer cells JXQ-3D-4160R4 (magnification is 40 times).

[0031] Figure 2 is a cell proliferation curve diagram of human gallbladder cancer cells JXQ-3D-4160R4.

[0032] Figure 3 is a photograph of human gallbladder cancer cells JXQ-3D-4160R4 cell scratch 0 hours (magnification is 40 times).

[0033] Figure 4 is a photograph of human gallbladder cancer cells JXQ-3D-4160R4 cell scratch 6 hours (magnification is 40 times).

[0034] Figure 5 is a photograph of human gallbladder cancer cells JXQ-3D-4160R4 cell scratch 24 hours (magnification is 40 times).

[0035] Figure 6 is a photograph of human gallbladder cancer cells JXQ-3D-4160R4 cell scratch 48 hours (magnification is 40 times).

[0036] Figure 7is the photograph of human gallbladder cancer cell JXQ-3D-4160R4 cell scratch 72 hours.

[0037] Figure 8 is the photograph of human gallbladder cancer cell JXQ-3D-4160R4 clone formation.

[0038] Figure 9 is the test result of human gallbladder cancer cell JXQ-3D-4160R4 sensitivity to cisplatin drug.

[0039] Figure 10 is the test result of human gallbladder cancer cell JXQ-3D-4160R4 sensitivity to 5-FU drug.

[0040] Figure 11 is the test result of human gallbladder cancer cell JXQ-3D-4160R4 sensitivity to gemcitabine drug. DETAILED DESCRIPTION

[0041] The technical solutions of the present application will be further described below in conjunction with the accompanying drawings and through specific embodiments. However, the following examples are only simple examples of the present application and do not represent or limit the protection scope of the present application, and the protection scope of the present application is subject to the claims.

[0042] The technical solutions adopted by the present application are common methods and conditions in the art, and certain reasonable adjustments can be made during specific operation, and specific reference can be made to "Molecular Cloning Laboratory Guide" (third edition or updated version, New York, Cold Spring Harbor Laboratory Press) and the like.

[0043] The reagents, consumables and instruments used in the present application can be obtained through commercial channels and are common commercially available products in the art. The specific experimental materials and their sources listed in the following examples are only exemplary and are not intended to limit the present application. Materials with the same or similar types, models, qualities, properties or functions of the following organizations, cells, reagents and instruments can be used to implement the present application.

[0044] In specific embodiments of the present application, human gallbladder cancer cell line JXQ-3D-4160R4 was deposited with the China Center for Type Culture Collection on November 22, 2023, and the deposit number is CCTCC NO: C2023369; the address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0045] Example 1

[0046] This example establishes a new human gallbladder cancer cell line.

[0047] The cell strain established by the application is derived from a 46-year-old Chinese female with gallbladder cancer, and the pathological result shows that it is of medium-low differentiation and is accompanied by liver and gallbladder duct lymph node metastasis. The gallbladder cancer tissue obtained by surgery is washed with sterile PBS for multiple times to remove excess blood and connective tissue, and then transferred to a clean bench for multiple washing with sterile PBS. Subsequently, the tissue block is cut into 1 mm 3 pieces using a sterile blade and transferred to a 10 cm culture dish, and DMEM / F12 complete culture medium is added, and the culture is incubated in a 37°C, 5% CO2 incubator. The culture medium is composed of 88% DMEM / F12 (Gibco TM , C11330500BT), 10% fetal bovine serum (FBS) (Gibco TM , 12657-029), 1% non-essential amino acids (Gibco TM , 11140076), and 1% penicillin (100 U / mL) / streptomycin (100 μg / mL) (Gibco TM , 15140-122). When the cell density reaches about 30%, the supernatant and tissue block are discarded, and the adherent tumor cells are digested and inoculated into a 24-well plate for continued culture using trypsin. After about 5-6 generations, purified tumor cells are obtained, and the above-mentioned complete culture medium is used for further expansion of the culture.

[0048] Example 2

[0049] This example analyzes the biological characteristics and application of the cell line

[0050] (1) Cell morphology

[0051] The morphology of the tumor cells obtained in Example 1 was observed under an inverted microscope (Olympus, U-RFL-T), as shown in FIG. 1 (magnification 40x). It can be seen that the cells grow adherently, are evenly distributed, have consistent size, and have a long foot-like shape. After multiple passages, the cell morphology remains consistent. Figure 1

[0052] (2) STR identification

[0053] Short tandem repeat (STR) is also known as microsatellite DNA, which refers to a type of DNA sequence formed by the tandem repetition of several base pairs (2-6 base pairs) as the core unit (the number of repetitions is 10-60 times, and the gene fragment is below 400 bp). The number of repetitions of each core unit varies among individuals, resulting in alleles with different fragment lengths. Therefore, the number of repetitions of a STR sequence is almost unique in different individuals, which is a genetic identity characteristic of an individual and is also a main method for identifying the identity and source of cells in cell biology.

[0054] ​The tumor cells obtained in Example 1 and the tumor tissue from which they were derived were subjected to STR identification, trypsinized and collected, and the cell genomic DNA was extracted using a DNA extraction kit, amplified using a 20-site STR amplification protocol, and the STR sites and gender gene Amelogenin were detected on an ABI3730XL genetic analyzer. The sites detected 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.

[0055] Table 1

[0056]

[0057] Note: Alle represents the chromosome on which the detection site is located, X is the X sex chromosome, and two sites of X represent female origin.

[0058] The Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) database and the American Type Culture Collection (ATCC) database were searched for STR sequences, and no identical STR detection results were found. No matching sites were found in the database, indicating that the cell line is a new cell line; no multiple alleles were found, indicating that the cell strain is a single cell without other cell contamination. The tumor cell was named human gallbladder cancer cell JXQ-3D-4160R4.

[0059] (3) Cell proliferation

[0060] The long cells were digested with 0.25% trypsin and prepared into a cell suspension, 20 μL of the cell suspension and 20 μL of trypan blue (Life, T10282) were mixed and 20 μL of the mixed suspension was added to a cell counting plate (Countstar, 12000550), and the viable cells were counted in three fields of view in an automatic cell counter (Countsta, 12000550). 8000 cells per well (3 replicates) were inoculated into 4 96-well plates, and after 24 h, 48 h, 72 h and 96 h of culture, the number of cells was detected using the CCK8 reagent. 10 μL of CCK8 reagent (Bi Yun Tian, C0037) was added to each cell well, and then the incubator was continued for 3 h. The absorbance value at 450 nm was measured using a microplate reader, and a cell proliferation curve was plotted, as shown in Figure 2The cell proliferation curve shown, the curve equation is Y = 9.217 * exp (0.01281 * X), wherein Y is the number of cells, X is the proliferation time, R2=0.9895. Statistical data show that the population doubling time of JXQ-3D-4160R4 is 54.1 hours.

[0061] (4) Cell scratch

[0062] Before the cell plate, draw 5 straight lines on the back of the 6-hole plate with a pen. Add 2 mL of complete culture solution in the hole plate, calculate the required cell volume per hole, inoculate 5x10 5 cells per hole, shake after inoculation, observe the inoculation density under a microscope, and then put it in the incubator overnight. When the cells are confluent to more than 90%, use a 200 μL sterile gun head to scratch in a clean bench, the gun head is perpendicular to the hole plate and the line, and the force is uniform. Discard the supernatant and wash away the scratched cells with 2 mL of PBS per hole, and add 2 mL of fresh complete culture medium per hole.

[0063] Use an inverted microscope to select 3 scratch fields at 4x objective magnification, take pictures and save, and determine the time point as 0h( Figure 3 ). Then find the scratch field at the 0h time point and take pictures and save at 6h, 24h, 48h and 72h.

[0064] The results show that the cells migrate after 6h( Figure 4 ), a small amount of cell migration occurs after 24h( Figure 5 ), a large number of cells migrate after 48h( Figure 6 ), and the wound is mostly healed at 72h( Figure 7 ). It is proved that human gallbladder cancer cells JXQ-3D-4160R4 have good migration ability and fast migration speed.

[0065] (5) Cloning

[0066] In 6-well plates, each well was inoculated with 500, 1000, 2000, 3000, 4000 and 5000 cells respectively, 2 mL of complete fresh culture solution was added, and it was placed in the incubator for culture. The next day, the cell adhesion was observed and the liquid was changed for continuous culture for about 10 days. According to the size, dispersion and contact between the clones, the optimal clone inoculation number was determined to be 3000 cells / well. Then crystal violet was used for clone staining: the culture medium supernatant was discarded, 1 mL of PBS was added for cleaning once and then discarded, 500 μL of 4% methanol was added for fixation for 10 min, then the methanol was discarded, 1 mL of PBS was used for cleaning once, 100 μL of 0.1% crystal violet was added for incubation for 10 min, the crystal violet was discarded, 1 mL of double distilled water was added for cleaning twice, the double distilled water was discarded, and it was dried at room temperature. Then a camera was used to take pictures, and an optical microscope was used to take pictures of the cell number of the clone colony.

[0067] As Figure 8 As shown in the results of the plate clone formation experiment of human gallbladder cancer cells JXQ-3D-4160R4, a large number of cell clones with clear edges were formed in the plate, and the results showed that JXQ-3D-4160R4 cells had the ability to form tumors in vitro, which met the characteristics of tumor cells.

[0068] (6) Drug sensitivity test

[0069] The digested cells were counted and inoculated into 96-well plates at 1000, 2000, 4000, 6000, 8000 and 10000 cells / 100 μL of complete culture medium for continuous culture for 96 h, and three replicate wells were prepared for each cell concentration; the cell growth state was observed under a microscope every day, and the cell concentration that could just grow full at 96 h (8000 cells / 100 μL) was selected for drug sensitivity test;

[0070] The cells were inoculated into the wells of the 96-well plate according to the counting results at 8000 cells / 100 μL of complete culture medium, and were placed in the incubator for overnight culture. The next day, the cell adhesion was observed, and then a certain concentration of drug was added.

[0071] The experiment uses three compounds cisplatin, 5-fluorouracil (5-Fu) and gemcitabine, and six concentration gradients are set for each drug, and each has three duplicate holes, that is, cisplatin: 100 μM, 20 μM, 4 μM, 0.8 μM, 0.16 μM and 0 μM (control); 5-fluorouracil: 200 μM, 40 μM, 8 μM, 1.6 μM, 0.32 μM and 0 μM (control); gemcitabine: 200 μM, 40 μM, 8 μM, 1.6 μM, 0.32 μM and 0 μM (control). The supernatant culture medium of the 96-well plate with the cells is discarded, 100 μL of the complete culture medium containing the above-mentioned drugs is added, and then cultured for 72 h. Subsequently, 10 μL of CCK8 is added to the hole, and then transferred to a 37℃ incubator or oven for incubation for 3 h. After incubation, the absorbance at 450 nm is measured by using an enzyme label instrument, and the cell viability is calculated.

[0072] The IC50 of JXQ-3D-4160R4 cells to cisplatin, 5-Fu and gemcitabine is 5.096 μM ( Figure 9 ), 7.046 μM ( Figure 10 ) and 12.6 μM ( Figure 11 ), respectively. It can be known from the results that JXQ-3D-4160R4 cells have high sensitivity to cisplatin and 5-Fu. In the present application, the lower the IC50 value of the drug is, the more sensitive the cell is to the drug, and vice versa. The cell viability decreases sharply after being treated with 4 μM of cisplatin, and the IC50 is only 5.096 μM. Similarly, the IC50 of 5-Fu is only 7.046 μM, which belongs to a lower range, that is, relatively sensitive. It can be known from the results that after the addition of gemcitabine, the cell viability does not change much, that is, the sensitivity to gemcitabine is relatively low. Figure 11

[0073] It can be known from the above results that the human gallbladder cancer cell line of the present application is derived from a 46-year-old Chinese female gallbladder cancer patient, the cell morphology is consistent, the dispersion is uniform, the size is consistent, the morphology is long foot-shaped, and the cell morphology is stable after multiple passages. The cell line is identified as a new cell line by STR; no multiple alleles are found, indicating that the cell strain is a single cell without other cell contamination. In terms of proliferation, the population doubling time of JXQ-3D-4160R4 cells is 54.1 hours. It can be observed from the scratch test that JXQ-3D-4160R4 cells have strong migration ability. It can be observed from the plate colony formation experiment that loose colonies can be formed on the plate after 10 days of culture. In terms of drug sensitivity, the human gallbladder cancer cell JXQ-3D-4160R4 prepared in the present application is sensitive to cisplatin and 5-FU, and less sensitive to gemcitabine.

[0074] ​The JXQ-3D-4160R4 established by the application has obvious gallbladder cancer cell biological functions, stable biological genetic traits, clear cell generations, is a new single cell strain identified by STR, has strong proliferation activity, can be amplified and cultured in vitro, has a clone formation ability, is sensitive to three kinds of chemotherapy drugs cisplatin and 5-fluorouracil, but has lower sensitivity to gemcitabine, is a good in vitro cell model, and provides an important research basis for pathogenesis, drug resistance mechanism, and new drug research and screening of primary gallbladder cancer.

[0075] The applicant declares that the above is only a specific embodiment of the application, but the protection scope of the application is not limited thereto, and those skilled in the art should understand that any changes or replacements within the technical scope disclosed by the application, which can be easily thought of by any person skilled in the art, falls within the protection scope and disclosure scope of the application.

Claims

1. A human gallbladder carcinoma cell line, characterized in that, The human gallbladder cancer cell line comprises human gallbladder cancer cell JXQ-3D-4160R4; the human gallbladder cancer cell JXQ-3D-4160R4 was preserved in the China Center for Type Culture Collection on November 22, 2023, and the preservation number is CCTCC NO: C2023369.

2. The human gallbladder carcinoma cell line according to claim 1, characterized in that, The human gallbladder cancer cell line also comprises a subcell line of human gallbladder cancer cell JXQ-3D-4160R4.

3. A cellular model, characterized in that, The cell model contains the gallbladder cancer cell line of claim 1 or 2.

4. Use of the human gallbladder cancer cell line of claim 1 or 2 in the preparation of a cell model for the study of the pathogenesis of gallbladder cancer.

5. Use of the human gallbladder cancer cell line of claim 1 or 2 in the preparation of an animal model of gallbladder cancer. Preferably, the animal model is used to explore the development or metastasis of gallbladder cancer cells. Preferably, the animal model is used to explore the drug resistance pathway of gallbladder cancer. Preferably, the animal model is used to screen biomarkers of gallbladder cancer.

6. A method of constructing an animal model of gallbladder cancer, characterized by, The method comprises: Using the human gallbladder cancer cell line of claim 1 or 2 to infect an immunodeficient animal to form a tumor.

7. The method of constructing an animal model of gallbladder cancer according to claim 6, wherein The immunodeficient animal comprises an immunodeficient nude mouse, an immunodeficient rat, or a humanized mouse.

8. Use of the human gallbladder cancer cell line of claim 1 or 2 in the preparation of a model for screening drugs for treating gallbladder cancer.

9. Use of the human gallbladder cancer cell line of claim 1 or 2 in the screening of drugs for preventing or treating gallbladder cancer.

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

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