Human gallbladder cancer cell line and application thereof
By establishing the new human gallbladder cancer cell lines JXQ-3D-8529R1 and JXQ-3D-8529R2, the problem of insufficient existing models was solved, stable and diverse experimental models were provided for gallbladder cancer research and drug development, and the gallbladder cancer cell library was enriched.
Patent Information
- Application Number
- CN202410512751.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-04-26
- Publication Date
- 2025-10-28
AI Technical Summary
Existing research on gallbladder cancer cell models is limited, especially the insufficient number of cell lines of Chinese origin, and existing models fail to fully cover the high heterogeneity and gene mutation characteristics of gallbladder cancer, limiting the diversity of gallbladder cancer research and the effectiveness of drug development.
New human gallbladder cancer cell lines JXQ-3D-8529R1 and JXQ-3D-8529R2 were established. They have stable biological genetic traits, can be passaged for a long time in vitro, have strong migration ability, and have different sensitivities to drugs such as cisplatin, 5-fluorouracil and gemcitabine, enriching the gallbladder cancer cell library.
It provides a new experimental model that can be used to study the etiology, metastasis mechanism and drug screening of gallbladder cancer, enriches the gallbladder cancer cell resource library of the Chinese population, and provides diversity and effectiveness for preclinical research on gallbladder cancer.
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Figure CN120843431A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology and relates to human gallbladder cancer cell lines and their applications. Background Technology
[0002] Gallbladder carcinoma (GBC) originates from the epithelial cells of the gallbladder mucosa and is a malignant tumor of the biliary tract. Chronic cholecystitis with gallstones, dietary factors, chronic gallbladder infection, and environmental exposure to certain chemicals are considered major risk factors for the development of gallbladder carcinoma. Gallbladder carcinoma has unique malignant biological characteristics: high early local invasiveness, widespread hepatic lymph node metastasis, low surgical resection rate with high postoperative recurrence rate, low sensitivity to radiotherapy and chemotherapy, and poor prognosis. Therefore, in-depth research into the specific biological behavior, metastasis and recurrence mechanisms, and potential interventions of gallbladder carcinoma is of particular importance.
[0003] In recent years, primary tumor cell lines derived from patients have proven to be a powerful tool with great potential in drug screening, drug resistance studies, tumor microenvironment analysis, and the study of gallbladder cancer pathogenesis and metastasis. However, existing gallbladder cancer cell models are limited, including EH-GB1 / GB2, GBC-SD, GBC-SD / ATO, GBC-SD / HL, GBC-SD / M, GBC-SD / M3, and GBC-SD-Luc2-tdT. For a highly heterogeneous disease like gallbladder cancer, the currently reported gallbladder cancer cell lines are far from fully encompassing the characteristics of this disease. There is a need to establish more gallbladder cancer cell lines specific to the Chinese population to enrich the gallbladder cancer tumor cell research library and provide preclinical models for gallbladder cancer mechanism research and the development of new therapeutic drugs.
[0004] In summary, the number of existing gallbladder cancer cell lines derived from Chinese patients is limited, consisting of only 13 cases. To more fully investigate the characteristics of gallbladder cancer in the Chinese population, it is necessary to establish more gallbladder cancer cell lines derived from Chinese patients. Secondly, some existing cell lines lack gene mutation profiles, further limiting the number of cell lines available for research. Thirdly, due to tumor heterogeneity, each gallbladder cancer cell line possesses unique gene mutation characteristics, and the existing gallbladder cancer cell lines are far from sufficient to cover the genetic features of this disease. Therefore, to ensure the diversity of research models, it is urgent to establish novel gallbladder cancer cell lines to enrich the existing cell line library and provide preclinical models for gallbladder cancer mechanism research and the development of new therapeutic drugs. Summary of the Invention
[0005] In response to the shortcomings of existing technologies and practical needs, this invention provides a human gallbladder cancer cell line and its applications, establishes a novel human gallbladder cancer cell line, enriches the human gallbladder cancer cell database, and provides new experimental materials for revealing the mechanisms of occurrence, development or metastasis of primary gallbladder cancer in the Chinese population and for the development of anti-gallbladder cancer drugs.
[0006] To achieve this objective, the present invention adopts the following technical solution:
[0007] In a first aspect, the present invention provides a human gallbladder cancer cell line, which includes human gallbladder cancer cell line JXQ-3D-8529R1 or human gallbladder cancer cell line JXQ-3D-8529R2; the human gallbladder cancer cell line JXQ-3D-8529R1 was deposited at the China Center for Type Culture Collection on November 22, 2023, with accession number CCTCC NO:C2023372; the human gallbladder cancer cell line JXQ-3D-8529R2 was deposited at the China Center for Type Culture Collection on November 22, 2023, with accession number CCTCC NO:C2023371.
[0008] In this invention, two novel human gallbladder cancer cell lines were discovered and established. These lines exhibit stable biological and genetic traits, clear generation numbers, and the ability to be massively expanded. They can be passaged in vitro for extended periods, possess strong migration capabilities, and can form loose clones on plates. They also exhibit different sensitivities to cisplatin, 5-fluorouracil, gemcitabine, and other drugs. These novel experimental models provide new insights into the etiology, metastasis mechanisms, drug resistance mechanisms, and drug screening of gallbladder cancer.
[0009] Preferably, the human gallbladder cancer cell line further includes a progeny cell line of human gallbladder cancer cell line JXQ-3D-8529R1 or a progeny cell line of human gallbladder cancer cell line JXQ-3D-8529R2.
[0010] Secondly, the present invention provides the application of the human gallbladder cancer cell line described in the first aspect in the preparation of cell models for studying the pathogenesis of gallbladder cancer.
[0011] Thirdly, the present invention provides a cell model containing the human gallbladder cancer cell line described in the first aspect.
[0012] Fourthly, the present invention provides the application of the human gallbladder cancer cell line described in the first aspect in the preparation of animal models of gallbladder cancer.
[0013] Fifthly, the present invention provides a method for constructing an animal model of gallbladder cancer, the method comprising:
[0014] The human gallbladder cancer cell line described in the first aspect was used to infect immunodeficient animals to induce tumor formation.
[0015] Preferably, the immunodeficient animal includes immunodeficient nude mice, immunodeficient rats, or humanized mice.
[0016] In a sixth aspect, the present invention provides the application of the human gallbladder cancer cell line described in the first aspect in the preparation of a model for screening drugs for the treatment of gallbladder cancer.
[0017] In a seventh aspect, the present invention provides the use of the human gallbladder cancer cell line described in the first aspect in screening drugs for the prevention or treatment of gallbladder cancer.
[0018] Preferably, the screening includes determining whether the 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.
[0019] Compared with the prior art, the present invention has the following beneficial effects:
[0020] This invention establishes novel human gallbladder cancer cell lines JXQ-3D-8529R1 and JXQ-3D-8529R2. These human gallbladder cancer cell lines are stable, capable of multiple stable passages, and exhibit high cell proliferation activity, strong cell migration and colony formation ability. They also show varying sensitivities to drugs such as cisplatin, 5-fluorouracil, and gemcitabine. These cell lines can enrich the gallbladder cancer resource bank, especially for gallbladder cancer cells from the Chinese population, and serve as effective in vitro cell models for research, providing new experimental models for the etiology, metastasis mechanisms, drug resistance mechanisms, and drug screening of gallbladder cancer. Attached Figure Description
[0021] Figure 1 Morphological observation of JXQ-3D-8529R1 cells;
[0022] Figure 2 Morphological observation of JXQ-3D-8529R2 cells;
[0023] Figure 3 The growth curve of JXQ-3D-8529R1 cells;
[0024] Figure 4 The growth curve of JXQ-3D-8529R2 cells;
[0025] Figure 5 Scratch patterns for JXQ-3D-8529R1 and JXQ-3D-8529R2 cells;
[0026] Figure 6 Image showing the colony formation of JXQ-3D-8529R1 cells;
[0027] Figure 7 Image showing the colony formation of JXQ-3D-8529R2 cells;
[0028] Figure 8 The graph shows the cisplatin sensitivity test results for JXQ-3D-8529R1.
[0029] Figure 9 The graph shows the sensitivity test results for JXQ-3D-8529R2 cisplatin.
[0030] Figure 10 The graph shows the results of the JXQ-3D-8529R15-fluorouracil sensitivity test.
[0031] Figure 11 The graph shows the results of the JXQ-3D-8529R25-fluorouracil sensitivity test.
[0032] Figure 12 The graph shows the results of the gemcitabine sensitivity test for JXQ-3D-8529R1.
[0033] Figure 13 The graph shows the results of the gemcitabine sensitivity test for JXQ-3D-8529R2. Detailed Implementation
[0034] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.
[0035] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.
[0036] In a specific embodiment of the present invention, human gallbladder cancer cells JXQ-3D-8529R1 were deposited at the China Center for Type Culture Collection on November 22, 2023, with accession number CCTCC NO:C2023372; address: No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, postcode 430072.
[0037] Human gallbladder cancer cell line JXQ-3D-8529R2 was deposited at the China Center for Type Culture Collection (CCTCC) on November 22, 2023, with accession number CCTCC NO: C2023371; address: No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China (Wuhan University).
[0038] Example 1
[0039] This embodiment describes the construction of a human gallbladder cancer cell line.
[0040] (1) Primary culture
[0041] The cell lines JXQ-3D-8529R1 and JXQ-3D-8529R2 of this invention were derived from surgically removed tumor tissue of a 42-year-old Chinese male patient with gallbladder cancer and retroperitoneal lymph node metastasis. The isolated fresh tissue was rinsed in PBS buffer to remove blood and necrotic tissue. The tissue was then separated into approximately 1 mm pieces using ophthalmic forceps, ophthalmic scissors, and other surgical instruments. 3 The small pieces were spread evenly on the bottom of a 10cm petri dish, and 10mL of DMEM / F12 complete medium containing 88% DMEM / F12 (Gibco) was added. TM It contains 10% FBS (Gibco, 12657-029) and 1% non-essential amino acids (Gibco). TM ,11140076) and 1% penicillin (100U / mL) / streptomycin (100μg / mL) (Gibco) TM (15140-122), culture dishes were placed in a 37℃, 5% CO2 incubator. Observe and change the culture medium in a timely manner. After single-cell clones have grown, use trypsin-EDTA (0.25%, Gibco) TM Digest the cells (25200072), transfer them to 24-well plates for culture, and obtain tumor cells after 6 generations.
[0042] (2) Transmission
[0043] When the cells reach 70-80% confluence, proceed with cell passage culture. In a clean bench, aspirate the culture medium from the culture dish. Wash the cells twice with a small amount of PBS to remove residual culture medium and dead cells. Add 1 mL of 0.25% trypsin and incubate in a CO2 incubator for 5 min to digest the cells. When microscopic observation shows that most cells exhibit cytoplasmic retraction, increased intercellular spaces, and enhanced refractive index, add 4 mL of complete culture medium to terminate the digestion. Repeatedly pipette the adherent cells and transfer the cell suspension to a centrifuge tube. Centrifuge at 800×g for 5 min at 25°C. Discard the supernatant, resuspend the cells in complete culture medium, and seed into new culture dishes at the desired concentration.
[0044] (3) Cryopreservation and thawing
[0045] Cryopreservation: The digestion and centrifugation methods for collecting cells are as described in (2) above. The centrifuged cell pellet is resuspended in cryopreservation buffer (containing 80% DMEM / F12, 10% FBS and 10% DMSO) and gently pipetted to disperse it evenly. 1 mL of cell suspension is transferred to each cryovial and sealed. The cryovials are stored in a gradient cryopreservation box at -80°C for at least 12 hours, and then transferred to a liquid nitrogen tank the next day.
[0046] Resuscitation: After removing the cryovials from liquid nitrogen, quickly place them in 37°C warm water. Once thawed, transfer the cell suspension to a centrifuge tube and centrifuge at 800×g for 5 min at 25°C, discarding the supernatant. Add 5 mL of complete culture medium to the cell pellet, resuspend the cells by pipetting, transfer to a culture dish, and incubate at 37°C in a 5% CO2 incubator.
[0047] Example 2
[0048] This embodiment analyzes the biological characteristics and application tests of gallbladder cancer cell lines.
[0049] (1) Morphological observation
[0050] The morphology of JXQ-3D-8529R1 and JXQ-3D-8529R2 cells was observed under an inverted microscope, such as... Figure 1 and Figure 2 As shown, the two cell lines have similar morphologies, both exhibiting adherent growth and irregular polygonal shapes.
[0051] (2) Passage stability
[0052] After multiple passages, the cell morphology remained consistent.
[0053] (3) STR identification
[0054] Freshly cultured cells and tumor tissue samples from patients were collected. Genomic DNA was extracted from the cells using the QIAamp DNA Mini Kit (Qiagen, 51304). A 20-STR amplification protocol was employed, and the amplified products were sequenced on an ABI 3730XL genetic analyzer. Analysis included the number of base repeats at the following 20 STR sites: D5S818, D13S317, D7S820, D16S539, VWA, TH01, AMEL, TPOX, CSF1PO, D12S391, FGA, D2S1338, D21S11, D18S51, D8S1179, D3S1358, D6S1043, PENTAE, D19S433, and PENTAD.
[0055] The STR results of JXQ-3D-8529R1 and JXQ-3D-8529R2 cells and primary tumor tissue samples are shown in Table 1. X represents sex chromosome X and Y represents sex chromosome Y. The STR results of the two cell lines are highly consistent with those of the tumor tissue. Detailed STR matching analysis with cell banks such as DSMZ, ATCC, and JCRB was performed, but no results were found. This indicates that JXQ-3D-8529R1 and JXQ-3D-8529R2 have unique STR characteristics and are novel cell lines.
[0056] Table 1
[0057] STR sites JXQ-3D-8529R1 JXQ-3D-8529R2 organize database D5S818 11,12 11,12 11,12 / D13S317 11 11 8,11 / D7S820 11 11 11 / D16S539 9,11 9,11 9,11 / VWA 14,16 14,16 14,16 / TH01 7 7 7,9 / AMEL X X X,Y / TPOX 9,11 9,11 9,11 / CSF1PO 11 11 11 / D12S391 17,19 17,19 17,19 / FGA 19,22 19,22 19,22 / D2S1338 18 18 18,20 / D21S11 27,28,29 27,28,29 27,28 / D18S51 15 15 13,15 / D8S1179 11,12 11,12 11,12 / D3S1358 15 15 15,17 / D6S1043 13 13 11,13 / PENTAE 5,12 5,12 5,12 / D19S433 13,14 13,14 13,14 / PENTAD 11,12 11,12 11,12 /
[0058] (4) Cell proliferation
[0059] JXQ-3D-8529R1 and JXQ-3D-8529R2 cells in good growth condition were trypsinized to prepare single-cell suspensions and counted. Accurate seeding was performed in 96-well plates at 8000 cells / well, with three plates per cell line and three replicates per plate. 100 μL was seeded per well and incubated at 37℃ in a 5% CO2 incubator. One plate was removed at 24h, 48h, and 72h, and the absorbance of each well was measured at 450nm according to the CCK-8 assay kit (Beyotime, C0037). Cell proliferation curves were plotted using GraphPad software, and the doubling time of JXQ-3D-8529R1 cells was calculated to be 48.9h. Figure 3 The doubling time of JXQ-3D-8529R2 cells was 63.2 h. Figure 4 ).
[0060] (5) Cell scratch test
[0061] In a 6-well plate, each well is 5×10 5 Cells were seeded, shaken well, and incubated overnight. The next day, cell adhesion was observed. When cell confluence reached 90% or higher, a 200 μL sterile pipette tip was used to vertically scratch the bottom of the dish. The culture medium was discarded, and the cells were washed twice with 2 mL of PBS to remove floating cells. 2 mL of complete culture medium was added to each well. The scratched area was photographed under an inverted microscope at 40X magnification and recorded as 0 h. Cells were cultured again, and photographs were taken at 6, 24, and 48 h to record cell migration distance. The scratch experiment results showed ( Figure 5 After 48 hours of culture, the scratches on the JXQ-3D-8529R1 and JXQ-3D-8529R2 cells were basically repaired, and their migration ability was strong.
[0062] (6) Plate cloning experiment
[0063] 3000 cells were seeded into each well of a 6-well plate, with 3 replicates per cell line. After mixing, the cells were incubated overnight. The cell adhesion was observed the next day, and the culture medium was replaced with fresh medium and cultured for approximately 10 days. The culture medium was discarded, and the cells were washed with 1 mL of PBS. The PBS was discarded, and the cells were fixed with 500 μL of 4% methanol solution at 25°C for 10 min. The methanol solution was discarded, and the cells were washed with 1 mL of PBS. The PBS solution was discarded, and the cells were fixed with 500 μL of 0.1% crystal violet solution (Solarbio, C8470) at 25°C for 10 min. The crystal violet solution was discarded, and the cells were washed twice with 1 mL of ddH2O. The ddH2O was discarded, the cells were air-dried, and photographed. The plate colony results of JXQ-3D-8529R1 cells are shown below. Figure 6 The plate cloning results of JXQ-3D-8529R2 cells are as follows: Figure 7 .
[0064] (7) Drug susceptibility testing
[0065] Accurately seed 8000 cells / well in 96-well plates, 100 μL per well, shake well, and incubate overnight. Observe cell adhesion on the second day. Discard the culture medium and replace each well with 100 μL of cisplatin culture medium at concentrations of 100, 20, 4, 0.8, 0.16, and 0.032 μM; 5-fluorouracil culture medium at concentrations of 200, 40, 8, 1.6, and 0.32 μM; and gemcitabine culture medium at concentrations of 20, 4, 0.8, 0.16, 0.032, and 0.0064 μM, respectively. Three replicates were set for each drug concentration, and a drug-free control and a blank control were also set up. After incubation for another 72 h, the absorbance of each well was measured at 450 nm according to the CCK-8 kit instructions.
[0066] The IC50 of cisplatin was calculated using GraphPad software, and the results showed that the IC50 of JXQ-3D-8529R1 was 13.58 μM. Figure 8 The IC50 of JXQ-3D-8529R2 is 13.66 μM. Figure 9 ).
[0067] The IC50 of 5-fluorouracil was calculated using GraphPad software, and the results showed that the IC50 of JXQ-3D-8529R1 was 2.92 μM. Figure 10 The IC50 of JXQ-3D-8529R2 is 3.41 μM. Figure 11 ).
[0068] The IC50 of gemcitabine was calculated using GraphPad software, and the results showed that the IC50 of JXQ-3D-8529R1 was 0.017 μM. Figure 12 The IC50 of JXQ-3D-8529R2 is 0.031 μM. Figure 13 ).
[0069] In summary, this invention provides two novel human gallbladder cancer cell lines, JXQ-3D-8529R1 and JXQ-3D-8529R2. These lines can be massively expanded and passaged in vitro for extended periods. The doubling times for JXQ-3D-8529R1 and JXQ-3D-8529R2 cells are 48.9 h and 63.2 h, respectively. They exhibit strong migration ability, forming loose colonies on agar plates, and show different sensitivities to cisplatin, 5-fluorouracil, and gemcitabine. This invention enriches the human gallbladder cancer cell database and provides new experimental materials for preclinical research into the mechanisms of gallbladder cancer development and progression in the Chinese population and for the development of corresponding drugs.
[0070] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.
Claims
1. A human gallbladder cancer cell line, characterized in that, The human gallbladder cancer cell line includes human gallbladder cancer cell line JXQ-3D-8529R1 or human gallbladder cancer cell line JXQ-3D-8529R2; The human gallbladder cancer cell line JXQ-3D-8529R1 was deposited at the China Center for Type Culture Collection on November 22, 2023, with accession number CCTCC NO:C2023372. The human gallbladder cancer cell line JXQ-3D-8529R2 was deposited at the China Center for Type Culture Collection on November 22, 2023, with accession number CCTCC NO:C2023371.
2. The human gallbladder cancer cell line according to claim 1, characterized in that, The human gallbladder cancer cell line also includes the progeny cell line of human gallbladder cancer cell line JXQ-3D-8529R1 or the progeny cell line of human gallbladder cancer cell line JXQ-3D-8529R2.
3. The application of the human gallbladder cancer cell line according to claim 1 or 2 in the preparation of cell models for studying the pathogenesis of gallbladder cancer.
4. A cell model, characterized in that, The cell model contains the human gallbladder cancer cell line as described in claim 1 or 2.
5. The use of the human gallbladder cancer cell line according to claim 1 or 2 in the preparation of animal models of gallbladder cancer.
6. A method for constructing an animal model of gallbladder cancer, characterized in that, The method includes: Infect immunodeficient animals with the human gallbladder cancer cell line described in claim 1 or 2 to induce tumor formation.
7. The method for constructing an animal model of gallbladder cancer according to claim 6, characterized in that, The immunodeficient animals include immunodeficient nude mice, immunodeficient rats, or humanized mice.
8. The use of the human gallbladder cancer cell line according to claim 1 or 2 in the preparation of a model for screening drugs for the treatment of gallbladder cancer.
9. The use of the human gallbladder cancer cell line according to claim 1 or 2 in screening drugs for the prevention or treatment of gallbladder cancer.
10. The application according to claim 9, characterized in that, The screening includes determining whether a candidate drug can be used to prevent or treat gallbladder cancer based on its effects on the human gallbladder cancer cell line before and after use.