CN112662628A - Gemcitabine-resistant gallbladder cancer cell line and application thereof - Google Patents
Gemcitabine-resistant gallbladder cancer cell line and application thereof Download PDFInfo
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- CN112662628A CN112662628A CN202110044979.2A CN202110044979A CN112662628A CN 112662628 A CN112662628 A CN 112662628A CN 202110044979 A CN202110044979 A CN 202110044979A CN 112662628 A CN112662628 A CN 112662628A
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- 201000010175 gallbladder cancer Diseases 0.000 title claims abstract description 63
- 208000022072 Gallbladder Neoplasms Diseases 0.000 title claims abstract description 62
- SDUQYLNIPVEERB-QPPQHZFASA-N gemcitabine Chemical compound O=C1N=C(N)C=CN1[C@H]1C(F)(F)[C@H](O)[C@@H](CO)O1 SDUQYLNIPVEERB-QPPQHZFASA-N 0.000 title claims abstract description 58
- 229960005277 gemcitabine Drugs 0.000 title claims abstract description 57
- 206010059866 Drug resistance Diseases 0.000 claims abstract description 39
- 239000003814 drug Substances 0.000 claims abstract description 29
- 229940079593 drug Drugs 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 13
- 238000004321 preservation Methods 0.000 claims description 5
- 239000003560 cancer drug Substances 0.000 claims description 3
- 230000007246 mechanism Effects 0.000 claims description 3
- 238000010171 animal model Methods 0.000 claims description 2
- 238000000338 in vitro Methods 0.000 claims description 2
- 238000001727 in vivo Methods 0.000 claims description 2
- 238000012216 screening Methods 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 claims description 2
- 238000011835 investigation Methods 0.000 abstract description 3
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- 229940044683 chemotherapy drug Drugs 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract description 2
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- 239000000243 solution Substances 0.000 description 5
- 230000014509 gene expression Effects 0.000 description 4
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 3
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- 230000003698 anagen phase Effects 0.000 description 3
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- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
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- 239000001963 growth medium Substances 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
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- 101150114688 1.7 gene Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
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- 101100014539 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) get-1 gene Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
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- 201000011510 cancer Diseases 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
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- 238000011354 first-line chemotherapy Methods 0.000 description 1
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- 201000007487 gallbladder carcinoma Diseases 0.000 description 1
- 238000011223 gene expression profiling Methods 0.000 description 1
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Abstract
The invention relates to a gemcitabine-resistant gallbladder cancer cell line and application thereof. The method is characterized in that wild-type gallbladder cancer NOZ cells are treated by gemcitabine which is a chemotherapeutic drug and is repeatedly and gradually increased in dosage from low dosage, the drug resistance of NOZ cells is gradually enhanced along with the increase of the dosing times, and a human gallbladder cancer gemcitabine drug-resistant cell strain NOZ-R with stronger drug resistance to gemcitabine is further screened, wherein IC50 is 163.50 mu M, and the drug resistance coefficient is 5.89. The result of drug resistance stability investigation shows that the cell strain has strong drug resistance stability, and still maintains strong drug resistance after being stored for 6 months and more than 20 generations. The invention provides a powerful research material for exploring the drug resistance mechanism of the gallbladder cancer cells to gemcitabine.
Description
Technical Field
The invention relates to the field of drug-resistant cell strains, in particular to a gemcitabine-resistant gallbladder cancer cell strain and application thereof.
Background
Gallbladder cancer is high in malignancy degree and difficult to diagnose in early stage, and is found to belong to middle and late stages and lose operation chance. Chemotherapy is particularly important at this time, and the first-line chemotherapy regimen based on gemcitabine is currently poorly effective, with a 5-year survival rate of only 5%. This requires that the drug resistance mechanism of gallbladder cancer cells to gemcitabine should be deeply explored to find effective molecular targets to improve the efficacy of gemcitabine.
Patent document CN108315303A, published japanese 2018.07.24, uses human gallbladder cancer cell lines GBC-SD and SGC-996 as parent cells, adopts gemcitabine to simulate chemotherapy process, and adopts large dose impact combined with stepwise dose increasing method to respectively establish acquired gallbladder cancer drug-resistant cell lines GBC-SD/GEM and SGC-996/GEM, and the drug resistance index is above 10.
However, NOZ gallbladder cancer cell line resistant to gemcitabine is not seen at present. Furthermore, the self-constructed drug-resistant cell strain is known to have the problems of unstable cell drug resistance and easy mutation. Therefore, it is necessary to establish NOZ-resistant gallbladder cancer cell line with strong drug resistance and stable passage.
Disclosure of Invention
The invention aims to provide an NOZ gallbladder cancer cell line with strong gemcitabine resistance and good drug resistance stability aiming at the defects in the prior art.
The invention further aims to provide application of the NOZ gallbladder cancer cell line.
In order to achieve the first purpose, the invention adopts the technical scheme that:
a gemcitabine-resistant gallbladder cancer cell strain with a preservation number of CCTCC NO of C2020230.
In a preferred embodiment of the invention, the gemcitabine-resistant gallbladder cancer cell line is established based on a wild-type NOZ gallbladder cancer cell line.
In another preferred embodiment of the present invention, the gemcitabine-resistant gallbladder cancer cell line is established by a gradual increase of drug concentration method.
In order to achieve the second object, the invention adopts the technical scheme that:
the gemcitabine-resistant gallbladder cancer cell line for use as described above, wherein the use is selected from any one of the following:
a) studying the mechanism of gallbladder cancer drug resistance;
b) searching a target point for reversing the drug resistance of the gallbladder cancer;
c) screening a medicine for treating gallbladder cancer;
d) constructing a gemcitabine-resistant gallbladder cancer animal model;
d) the study analyzed the correlation of drug sensitivity and drug resistance in vitro and in vivo.
The invention has the advantages that:
the method is characterized in that the wild-type gallbladder cancer NOZ cell strain is treated by the chemotherapeutic drug gemcitabine which is repeatedly and gradually increased from low dose, the drug resistance of NOZ cells is gradually enhanced along with the increase of the dosing times, the human gallbladder cancer gemcitabine drug-resistant cell strain NOZ-R with stronger drug resistance to gemcitabine is further screened, the IC50 is 163.50 mu M, and the drug resistance coefficient is 5.89. The result of drug resistance stability investigation shows that the cell strain has strong drug resistance stability, and still maintains strong drug resistance after being stored for 6 months and more than 20 generations. The cell strain provided by the invention is helpful for researching the drug resistance mechanism of the gallbladder cancer cell to gemcitabine so as to find a therapeutic target point for reversing drug resistance.
Drawings
FIG. 1: cell morphology of human gallbladder cancer gemcitabine resistant cell line NOZ-R.
FIG. 2 is a drawing: drug resistance curves of a wild-type gallbladder cancer NOZ cell strain and a human gallbladder cancer gemcitabine drug-resistant cell strain NOZ-R.
FIG. 3: the gene expression difference of a wild-type gallbladder cancer NOZ cell line and a human gallbladder cancer gemcitabine drug-resistant cell line NOZ-R.
Detailed Description
The following detailed description of the present invention will be made with reference to the accompanying drawings.
Example 1
1. Materials and methods
1.1 cells
The wild-type gallbladder cancer NOZ cell line was purchased from Health Science Research Resources Bank (Osaka, Japan).
1.2 drugs
Gemcitabine, available from MCE corporation (cat # HY-17026).
1.3 Induction of drug-resistant cell lines
A concentration gradient increasing method is adopted to establish a human gallbladder cancer gemcitabine drug-resistant cell strain NOZ-R. NOZ of logarithmic phase is taken, DMEM culture solution containing gemcitabine is added, after continuous action for 24 hours from low concentration (5 mu mol/L), sensitive cells start apoptosis, the culture solution is discarded, PBS buffer solution is used for washing for 3 times, fresh culture solution is replaced, drug-resistant cells are continuously cultured in the culture solution (without drugs) until logarithmic phase is entered, the gemcitabine concentration is gradually increased, repeated induction is carried out, solution replacement is timely carried out for passage and cell preservation until cells which can stably grow and pass in the culture medium with the gemcitabine concentration of 20 mu mol/L are obtained by culture. The obtained gemcitabine drug-resistant cell strain NOZ-R for 12 months is finally deposited in China center for type culture Collection (Wuhan university, China 430072) 12 months and 16 days 2020, and has the preservation number of CCTCC NO: C2020230 and the name of the culture (classified name): gemcitabine resistant cell line NOZ-R for human gallbladder cancer.
1.4 morphological Observation of cells
The cell morphology of the cell NOZ and the human gallbladder cancer gemcitabine-resistant cell line NOZ-R was observed under an inverted microscope in a culture dish during cell culture.
1.5 drug sensitivity assays
NOZ cells and NOZ-R cells of human gallbladder cancer gemcitabine drug-resistant cell strain in logarithmic growth phase are selected and respectively inoculated with 5 multiplied by 10 in a 96-well plate3After 24 hours of culture, gemcitabine culture medium was added at 0, 0.5, 2, 8, 32, 128, 512, 2048. mu. mol/L to a 96-well plate, and three duplicate wells were set for each concentration, and a drug-free control and a blank control were set. Culturing for 48h under the same condition. The test is repeated for three times, the absorbance value of each well is measured by a CCK8 method, the growth inhibition rate is calculated according to a formula, and the half inhibition concentration IC50 of the drug and the drug Resistance Index (RI) of the drug-resistant cell IC 50/parent cell IC50 are calculated by adopting graphpad software.
1.6 drug resistance stability test
And (3) placing the established drug-resistant cell strain in a freezer at the temperature of-80 ℃ for freezing storage, recovering after 6 months, and detecting the drug resistance again. The established drug-resistant cell lines were further passaged, and drug resistance was again examined after 20 passages.
1.7 Gene expression profiling
Total RNA was extracted from the cells NOZ in the logarithmic growth phase, and the total RNA was sampled 3 times to obtain 3 groups: wt _1, Wt _2, Wt _ 3; total RNA of gemcitabine-resistant cell line NOZ-R in logarithmic growth phase of human gallbladder cancer was extracted, and the total RNA was sampled 3 times to set 3 groups: get _1, Get _2, Get _3, RNA sequence assay, comparing the gene expression difference between NOZ cells (Wt group) and human gallbladder cancer gemcitabine-resistant cell line NOZ-R cells (Get group).
2. Results
2.1 results of cell morphology
Observing by an inverted microscope to find that the wild type gallbladder cancer NOZ cells are epithelial cells, grow adherent, have multiple antennae, clear cytoplasm and larger cell nucleus; in addition to the above characteristics, the gemcitabine-resistant cell line NOZ-R is enlarged and the cell nucleus is enlarged (FIG. 1).
2.2 results of cell drug resistance examination
CCK8 detects IC50 of a wild-type gallbladder cancer NOZ cell strain and a human gallbladder cancer gemcitabine resistant cell strain NOZ-R, and the results show that IC50 of the wild-type gallbladder cancer NOZ cell strain is 27.75 mu M, IC50 of the human gallbladder cancer gemcitabine resistant cell strain NOZ-R is 163.50 mu M, and the drug resistance coefficient is 5.89 (figure 2), and the success establishment of the human gallbladder cancer gemcitabine resistant cell strain NOZ-R with stronger drug resistance to gemcitabine is verified, and the drug resistance is remarkably improved.
2.3 results of investigation of stability of cell drug resistance
After the cells are recovered after being stored for 6 months or after being passed for 20 generations, the IC50 of gemcitabine is detected by using a CCK8 method, the result is shown in Table 1, and the calculated drug resistance coefficients after being stored for 6 months and after being passed for 20 generations are respectively 6.02 and 5.82, which shows that the drug resistance of the gemcitabine drug-resistant cell strain NOZ-R of the human gallbladder cancer does not obviously fluctuate and has good stability.
TABLE 1 preservation of Gemcitabine resistant cell line NOZ-R for human gallbladder carcinoma for 6 months, passage 20 generations of IC50
2.4 cellular Gene expression differential detection results
RNA sequence detection shows that the gene expression of a wild-type gallbladder cancer NOZ cell strain and a human gallbladder cancer gemcitabine drug-resistant cell strain NOZ-R is obviously different (figure 3), which indicates that the internal mechanism of the human gallbladder cancer gemcitabine drug-resistant cell strain NOZ-R is also changed.
Example 2
In the research process, 12 gemcitabine-resistant NOZ gallbladder cancer cell strains are constructed by a concentration gradient increasing method, wherein the drug resistance and stability of the gemcitabine-resistant cell strain NOZ-R of human gallbladder cancer are the strongest, and the IC50 of other 11 drug-resistant cell strains are shown in Table 2.
TABLE 2.11 IC50(μ M) of drug-resistant cell lines
Note: -, indicates no detection.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.
Claims (4)
1. A gemcitabine-resistant gallbladder cancer cell line is characterized in that the preservation number is CCTCC NO: C2020230.
2. The gemcitabine-resistant gallbladder cancer cell line of claim 1, wherein the gemcitabine-resistant gallbladder cancer cell line is established based on a wild-type NOZ gallbladder cancer cell line.
3. The gemcitabine-resistant gallbladder cancer cell line of claim 1, wherein the gemcitabine-resistant gallbladder cancer cell line is established by a gradual drug concentration increase method.
4. The gemcitabine-resistant gallbladder cancer cell line as claimed in claim 1, wherein the use is selected from any one of the following:
a) studying the mechanism of gallbladder cancer drug resistance;
b) searching a target point for reversing the drug resistance of the gallbladder cancer;
c) screening a medicine for treating gallbladder cancer;
d) constructing a gemcitabine-resistant gallbladder cancer animal model;
d) the study analyzed the correlation of drug sensitivity and drug resistance in vitro and in vivo.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114181906A (en) * | 2021-12-10 | 2022-03-15 | 深圳市第二人民医院(深圳市转化医学研究院) | Human bladder cancer gemcitabine drug-resistant cell strain and application thereof |
| CN114621927A (en) * | 2021-12-20 | 2022-06-14 | 北京大学 | Construction method for stably expressing pemetrexed-resistant human lung adenocarcinoma cell strain |
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| KR20100099442A (en) * | 2009-03-03 | 2010-09-13 | 서울대학교산학협력단 | Gemcitabine-resistant human bladder cancer cell line |
| CN102329775A (en) * | 2010-07-13 | 2012-01-25 | 上海睿智化学研究有限公司 | Gemcitabine-tolerant human pancreatic cancer cell line and application thereof |
| CN103937744A (en) * | 2014-04-10 | 2014-07-23 | 上海赛安生物医药科技有限公司 | Drug-resistant cell strain of human bile duct cancer and application of drug-resistant cell strain |
| CN108315303A (en) * | 2018-01-30 | 2018-07-24 | 复旦大学附属中山医院 | A method of preparing Human gallbladder carcinoma gemcitabine medicine-resistant cell line |
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2021
- 2021-01-13 CN CN202110044979.2A patent/CN112662628B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20100099442A (en) * | 2009-03-03 | 2010-09-13 | 서울대학교산학협력단 | Gemcitabine-resistant human bladder cancer cell line |
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| CN108315303A (en) * | 2018-01-30 | 2018-07-24 | 复旦大学附属中山医院 | A method of preparing Human gallbladder carcinoma gemcitabine medicine-resistant cell line |
Non-Patent Citations (1)
| Title |
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| 刘辉;罗荣城;孙婷婷;杨小民;李成浩;蔡艳军;: "人鼻咽癌吉西他滨耐药细胞系的建立及其生物学特性", 解放军医学杂志, vol. 32, no. 6, pages 555 - 557 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114181906A (en) * | 2021-12-10 | 2022-03-15 | 深圳市第二人民医院(深圳市转化医学研究院) | Human bladder cancer gemcitabine drug-resistant cell strain and application thereof |
| CN114181906B (en) * | 2021-12-10 | 2023-10-13 | 深圳市第二人民医院(深圳市转化医学研究院) | Gemcitabine resistant cell line for human bladder cancer and application thereof |
| CN114621927A (en) * | 2021-12-20 | 2022-06-14 | 北京大学 | Construction method for stably expressing pemetrexed-resistant human lung adenocarcinoma cell strain |
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