A method for preparing gemcitabine drug-resistant cell line of human gallbladder cancer

A gemcitabine and drug-resistant cell technology, applied in the field of bioengineering, can solve problems such as drug resistance, lack of drug-resistant cell lines, lack of systematic research, etc., and achieve the effect of simple and easy method and short establishment time.

Active Publication Date: 2021-03-30
ZHONGSHAN HOSPITAL FUDAN UNIV +1
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Problems solved by technology

Combining gemcitabine-based chemotherapy with platinum-based chemotherapy has become the first choice for the treatment of advanced gallbladder cancer, but it is unavoidable that patients with gallbladder cancer will develop drug resistance to gemcitabine
[0004] At present, the research on the mechanism of gemcitabine resistance in gallbladder cancer mainly focuses on the expression of drug-resistance-related proteins and drug-resistant genes. Cancer drug resistance research is also very little, the main reason is the lack of gallbladder cancer gemcitabine drug-resistant cell lines

Method used

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  • A method for preparing gemcitabine drug-resistant cell line of human gallbladder cancer
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  • A method for preparing gemcitabine drug-resistant cell line of human gallbladder cancer

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Embodiment 1

[0020] Example 1: Induction of drug-resistant cell lines

[0021] The drug resistance of gallbladder cancer cells was induced by high-dose pulse combined with step-up dose increase. Human gallbladder cancer cells GBC-SD (BNF-2472) and SGC-996 (JN-C0997) in the logarithmic growth phase (both purchased from Shanghai Rongbai Biotechnology Co., Ltd.) were inoculated in a medium containing 20% ​​newborn fetal bovine serum. in DMEM medium at 37°C, 5% CO 2 Culture in a saturated humidity incubator, add gemcitabine to the DMEM medium containing 20% ​​newborn fetal bovine serum for induction, the initial concentration is 0.2 μmol / L, when the cells return to normal growth, the concentration of gemcitabine is increased by 0.1 μmol / L, When the cells can grow normally and be passaged in DMEM medium containing 0.5 μmol / L gemcitabine and containing 20% ​​newborn fetal bovine serum, shock culture with DMEM medium containing 1.0 μmol / L gemcitabine and containing 20% ​​newborn fetal bovine ser...

Embodiment 2

[0022] Embodiment 2: Morphological observation

[0023] The cell morphology of GBC-SD and SGC-996 cells before and after gemcitabine induction was observed with an inverted phase-contrast microscope, and photographed and recorded. Such as figure 1 As shown, the size and shape of GBC-SD cells are relatively regular, with clear outlines and tight intercellular structures, while GBC-SD / GEM cells are irregular in shape, with fuzzy outlines, and many long pseudopodia. Quasi-circular, without obvious edges and corners; such as figure 2 As shown, the SGC-996 cells are regular polygons and closely arranged, while the SGC-996 / GEM cells look messy, with large intercellular spaces and multiple pseudopodia. In general, the morphology of GBC-SD and SGC-996 cells changed significantly after drug resistance was induced by gemcitabine.

Embodiment 3

[0024] Embodiment 3: the mensuration of growth curve

[0025] GBC-SD, SGC-996 and their corresponding drug-resistant cells in the logarithmic growth phase were digested with trypsin and inoculated in a 96-well culture plate, and the cell density was adjusted to 4×10 4 cells / mL, with 5 replicate wells for each group, placed at 37°C, 5% CO 2 After culturing in the incubator for 24 hours, the absorbance value A of each group at 450nm was measured every day by the CCK-8 method for 5 consecutive days, and the growth curve was drawn with the number of culture days as the abscissa and the absorbance A as the ordinate.

[0026] Such as image 3 As shown, GBC-SD cells grew slowly on day 1-2, entered logarithmic growth phase on day 3, slowed down on day 4, and entered plateau phase. The growth trend of the drug-resistant cell line GBC-SD / GEM was similar to that of GBC-SD was basically the same, and the overall growth was slightly slower; compared with the GBC-SD drug-resistant group, ...

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Abstract

The invention discloses a method for preparing a human gallbladder carcinoma gemcitabine drug resistant cell line. The method comprises the following steps: inoculating human gallbladder carcinoma cells in a logarithmic phase into a DMEM culture solution containing neonatal bovine serum, putting the culture solution into a saturated humidity incubator for culturing, adding gemcitabine into a culture solution to perform induction, ensuring that the concentration is increased when cells recover normal growth, performing impact culture with 1.0 micro mol / L gemcitabine when normal growth and passage of the cells can be achieved in the concentration condition of 0.5 micro mol / L, replacing the culture medium with a culture medium with the gemcitabine concentration being 0.5 micro mol / L, performing culture for 5 to 10 days, and performing impact again when stable growth and passage of the cells are achieved till the cells can grow in 10.0 micro mol / L gemcitabine, so as to obtain the human gallbladder carcinoma gemcitabine drug resistant cell line in which stable growth and passage of the cell can be achieved in a culture system containing 10 micro mol / L gemcitabine. The method is simple and practical, and short in setting time; the resistant drug index can reach greater than 10.

Description

technical field [0001] The invention belongs to the field of bioengineering and relates to a cell line, in particular to a method for preparing a gemcitabine drug-resistant cell line of human gallbladder cancer. Background technique [0002] Gallbladder cancer is the most common malignant tumor of the bile duct system. In recent years, its incidence rate has been on the rise, and its mortality rate has remained high. The main reason is that patients with gallbladder cancer have no specific symptoms in the early stage, so most patients are already at an advanced stage when they see a doctor. In addition, although surgical resection is currently the main treatment, 80% of patients have metastases at the time of diagnosis, the surgical resection rate is less than 30%, the postoperative recurrence rate is high, and the prognosis is extremely poor. Cancer patients are especially important. [0003] Gemcitabine (Gemcitabine, GEM) is a nucleoside anti-metabolite drug that can comp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/09
CPCC12N5/0693C12N2501/06
Inventor 锁涛刘厚宝王刚
Owner ZHONGSHAN HOSPITAL FUDAN UNIV
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