CN112662628B - Gall bladder cancer cell strain resistant to gemcitabine and application thereof - Google Patents
Gall bladder cancer cell strain resistant to gemcitabine and application thereof Download PDFInfo
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- CN112662628B CN112662628B CN202110044979.2A CN202110044979A CN112662628B CN 112662628 B CN112662628 B CN 112662628B CN 202110044979 A CN202110044979 A CN 202110044979A CN 112662628 B CN112662628 B CN 112662628B
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- 201000010175 gallbladder cancer Diseases 0.000 title claims abstract description 60
- 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
- 208000022072 Gallbladder Neoplasms Diseases 0.000 title claims abstract description 52
- 206010059866 Drug resistance Diseases 0.000 claims abstract description 36
- 239000003814 drug Substances 0.000 claims abstract description 26
- 229940079593 drug Drugs 0.000 claims abstract description 25
- 201000007487 gallbladder carcinoma Diseases 0.000 claims description 8
- 230000007246 mechanism Effects 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 238000010171 animal model Methods 0.000 claims description 2
- 239000003560 cancer drug Substances 0.000 claims description 2
- 238000012216 screening Methods 0.000 claims description 2
- 238000011160 research Methods 0.000 abstract description 7
- 230000008261 resistance mechanism Effects 0.000 abstract description 3
- 239000002246 antineoplastic agent Substances 0.000 abstract description 2
- 229940044683 chemotherapy drug Drugs 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 78
- 238000000034 method Methods 0.000 description 10
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- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 101100014539 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) get-1 gene Proteins 0.000 description 1
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Abstract
The invention relates to a gall bladder cancer cell strain resistant to gemcitabine and application thereof. According to the invention, the gemcitabine treatment of the chemotherapeutic drug which is repeatedly carried out on the wild type gall bladder cancer NOZ cells and gradually increases from low dose is carried out, the drug resistance of NOZ cells is gradually increased along with the increase of dosing times, and the human gall bladder cancer gemcitabine drug-resistant cell strain NOZ-R with stronger drug resistance to gemcitabine is further screened, wherein the IC50 is 163.50 mu M, and the drug resistance coefficient is 5.89. The research result of drug resistance stability shows that the cell strain has strong drug resistance stability, and still maintains stronger drug resistance after being stored for 6 months and passaged for more than 20 generations. The invention provides powerful research materials for exploring the drug resistance mechanism of gall bladder cancer cells to gemcitabine.
Description
Technical Field
The invention relates to the field of drug-resistant cell lines, in particular to a gall bladder cancer cell line resistant to gemcitabine and application thereof.
Background
The malignant degree of the gall bladder cancer is high, the early diagnosis is difficult, and the symptoms of the gall bladder cancer are mainly middle and late stages and the surgical opportunity is lost. At this time, chemotherapy is particularly important, and the current first-line chemotherapy regimen based on gemcitabine has poor efficacy and survival rate of only 5% in 5 years. This requires that we have extensively explored the mechanism of drug resistance of gallbladder cancer cells to gemcitabine in order to find effective molecular targets to improve the therapeutic efficacy of gemcitabine.
Patent document CN108315303A, publication date 2018.07.24, using human gallbladder cancer cell lines GBC-SD and SGC-996 as parent cells, adopting gemcitabine to simulate a chemotherapy process, adopting a large-dose impact combined stepwise increasing dose method, respectively establishing acquired gallbladder cancer drug resistant cell lines GBC-SD/GEM and SGC-996/GEM, wherein the drug resistance index is above 10.
However, there is currently no strain of NOZ gallbladder cancer cells that is resistant to gemcitabine. Furthermore, it is known that the difficulty of self-established drug-resistant cell lines is the problem of unstable cell resistance and easy variation. Therefore, it is necessary to establish NOZ drug-resistant gallbladder cancer cell lines that are highly resistant and stably passable.
Disclosure of Invention
The invention aims at overcoming the defects in the prior art and providing a NOZ gall bladder cancer cell strain with strong drug resistance to gemcitabine and good drug resistance stability.
It is a further object of the present invention to provide the use of the NOZ gallbladder cancer cell strain.
In order to achieve the first object, the invention adopts the following technical scheme:
a gall bladder cancer cell strain resistant to gemcitabine has a preservation number of CCTCC NO: C2020230.
As a preferred embodiment of the present invention, the gemcitabine-resistant gallbladder carcinoma cell line is established based on a wild-type NOZ gallbladder carcinoma cell line.
As another preferred embodiment of the present invention, the gemcitabine-resistant gallbladder cancer cell line is established by a drug concentration taper method.
In order to achieve the second purpose, the invention adopts the following technical scheme:
use of a gemcitabine resistant gallbladder cancer cell line as described above, selected from any one of the following:
a) Research on the drug resistance mechanism of gallbladder cancer;
b) Searching a target point for reversing drug resistance of the gallbladder cancer;
c) Screening medicines for treating gallbladder cancer;
d) Constructing a gemcitabine resistant gallbladder cancer animal model;
d) The research analyzes the relevance of drug sensitivity and drug resistance in vitro and in vivo.
The invention has the advantages that:
according to the invention, the gemcitabine treatment of the chemotherapeutic drug which is repeatedly carried out on a wild type gall bladder cancer NOZ cell strain and gradually increases from a low dose is carried out, the drug resistance of NOZ cells is gradually increased along with the increase of the dosing times, and the human gall bladder cancer gemcitabine drug-resistant cell strain NOZ-R with stronger drug resistance to gemcitabine is further screened, wherein the IC50 is 163.50 mu M, and the drug resistance coefficient is 5.89. The research result of drug resistance stability shows that the cell strain has strong drug resistance stability, and still maintains stronger drug resistance after being stored for 6 months and passaged for more than 20 generations. The cell strain provided by the invention is helpful for researching the drug resistance mechanism of gallbladder cancer cells to gemcitabine so as to find a therapeutic target for reversing drug resistance.
Drawings
FIG. 1: cell morphology of human gallbladder carcinoma gemcitabine resistant cell line NOZ-R.
Fig. 2: drug resistance profile of wild-type gall bladder cancer NOZ cell line and human gall bladder cancer gemcitabine resistant cell line NOZ-R.
Fig. 3: gene expression differences between wild type and human gallbladder carcinoma NOZ and gemcitabine resistant cell lines NOZ-R.
Detailed Description
The following detailed description of the invention provides specific embodiments with reference to the accompanying drawings.
Example 1
1. Materials and methods
1.1 cells
Wild type gallbladder carcinoma NOZ cell line was purchased from Health Science Research Resources Bank (Osaka, japan).
1.2 medicaments
Gemcitabine, available from MCE company (cat# HY-17026).
1.3 Induction of drug resistant cell lines
By adopting a concentration gradient increasing method, a gemcitabine resistant cell strain NOZ-R of the human gallbladder cancer is established. And NOZ in the logarithmic phase is taken, DMEM culture solution containing gemcitabine is added, after continuous action is carried out for 24 hours from low concentration (5 mu mol/L), sensitive cells start to apoptosis, the culture solution is discarded, PBS buffer solution is used for washing for 3 times, fresh culture solution is replaced, drug-resistant cells continue to be cultured in the culture solution (without drugs) until the logarithmic phase is entered, the concentration of gemcitabine is gradually increased, repeated induction is carried out, and the cells are replaced and passaged in time until the cells which can stably grow and passaged in the culture medium with the concentration of 20 mu mol/L of gemcitabine are obtained through culture. For 12 months, finally obtaining the gemcitabine resistant cell strain NOZ-R of the human gallbladder cancer, and preserving the gemcitabine resistant cell strain in China center for type culture collection (university of Chinese Wuhan, 430072) on 12 months and 16 days in 2020, wherein the preservation number is CCTCC NO: C2020230, and the culture name (classification and naming): a gemcitabine resistant cell line NOZ-R for human gallbladder cancer.
1.4 morphological observations of cells
During cell culture, the cell morphology of cells NOZ and human gallbladder carcinoma gemcitabine-resistant cell line NOZ-R was observed on a petri dish under an inverted microscope.
1.5 drug sensitivity detection
Selecting NOZ in logarithmic growth phase and gemcitabine resistant cell strain NOZ-R cells of human gallbladder cancer, inoculating 5×10 cells in 96-well plate 3 After 24 hours of incubation, three duplicate wells were set for each concentration, with no drug control and no drug control, using gemcitabine culture at addition concentrations of 0,0.5,2,8, 32, 128, 512, 2048 μmol/L in 96-well plates. Culturing for 48h under the same condition. The experiment was repeated three times, absorbance values of each well were measured by CCK8 method, growth inhibition rate was calculated according to the formula, half inhibition concentration IC50 of the drug was calculated using graphpad software, and drug Resistance Index (RI) =drug resistant cell IC 50/parent cell IC50.
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, recovering after 6 months, and detecting drug resistance again. And in addition, the established drug-resistant cell strain is subjected to passage, and drug resistance is detected again after passage for 20 generations.
1.7 Gene expression profiling
Total RNA of logarithmic growth phase cells NOZ was extracted, sampled 3 times respectively, and set to 3 groups: wt_1, wt_2, wt_3; total RNA of the human gallbladder cancer gemcitabine resistant cell line NOZ-R in the logarithmic growth phase was extracted, and sampled 3 times, respectively, to set 3 groups: get_1, get_2, get_3, send RNA sequence assays, compare the difference in gene expression of NOZ cells (Wt group) and human gallbladder cancer gemcitabine resistant cell line NOZ-R cells (Get group).
2. Results
2.1 morphological observations of cells
The observation by an inverted microscope shows that the wild gall bladder cancer NOZ cells are in an epithelial cell shape, grow in an adherent manner, have multiple tentacles, have clear cytoplasm and have larger cell nuclei; in addition to the above characteristics, the human gallbladder cancer gemcitabine resistant cell line NOZ-R had enlarged cells and nuclei (FIG. 1).
2.2 results of investigation of cell resistance to drugs
The IC50 of the wild type gall bladder cancer NOZ cell strain and the human gall bladder cancer gemcitabine drug resistant cell strain NOZ-R are detected by CCK8, and the result shows that the IC50 of the wild type gall bladder cancer NOZ cell strain is 27.75 mu M, the IC50 of the human gall bladder cancer gemcitabine drug resistant cell strain NOZ-R is 163.50 mu M, and the drug resistance coefficient is 5.89 (figure 2), so that the establishment of the human gall bladder cancer gemcitabine drug resistant cell strain NOZ-R with relatively strong drug resistance to gemcitabine is successful, and the drug resistance is remarkably improved.
2.3 results of investigation on cell drug resistance stability
After 6 months of cell resuscitation or 20 passages, the IC50 of gemcitabine is detected by using a CCK8 method, the results are shown in Table 1, and the calculated drug resistance coefficients after 6 months of storage and 20 passages are respectively 6.02 and 5.82, which indicate that the drug resistance of the gemcitabine resistant cell line NOZ-R of the human gallbladder cancer does not obviously fluctuate, and the stability is good.
TABLE 1 IC50 for gemcitabine resistant cell line NOZ-R for 6 months and passage 20 passages of human gallbladder carcinoma
2.4 results of differential Gene expression detection in cells
RNA sequence detection showed a significant difference in gene expression between wild-type gall bladder cancer NOZ cell line and human gall bladder cancer gemcitabine resistant cell line NOZ-R (FIG. 3), indicating that the intrinsic mechanism of human gall bladder cancer gemcitabine resistant cell line NOZ-R was also altered.
Example 2
In the research process, a concentration gradient increasing method is adopted to construct 12 gemcitabine-resistant NOZ gall bladder cancer cell strains altogether, wherein the drug resistance and the stability of the human gall bladder cancer gemcitabine-resistant cell strain NOZ-R are the strongest, and the IC50 of the other 11 drug-resistant cell strains are shown in Table 2.
TABLE 2 IC50 (μM) for 11 drug-resistant cell lines
Note that: -, no detection.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and additions may be made to those skilled in the art without departing from the method of the present invention, which modifications and additions are also to be considered as within the scope of the present invention.
Claims (2)
1. A gemcitabine resistant cell line NOZ-R for human gallbladder cancer is characterized in that the preservation number is CCTCC NO: C2020230.
2. Use of the human gallbladder carcinoma gemcitabine resistant cell line NOZ-R of claim 1, wherein the use is selected from any one of the following:
a) Study the mechanism of gallbladder cancer drug resistance to gemcitabine;
b) Searching a target point of reversing the drug resistance of the gall bladder cancer to the gemcitabine;
c) Screening for a drug for treating gemcitabine-resistant gallbladder cancer;
d) Constructing a gemcitabine resistant gallbladder cancer animal model.
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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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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 |
| 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 |
Non-Patent Citations (1)
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| 人鼻咽癌吉西他滨耐药细胞系的建立及其生物学特性;刘辉;罗荣城;孙婷婷;杨小民;李成浩;蔡艳军;;解放军医学杂志;32(6);第555-557页，参见全文 * |
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