Method for preparing human gallbladder cancer cell line resistant to oxaliplatin

A technology of oxaliplatin and drug-resistant cells, applied in the field of bioengineering, can solve problems such as lack, and achieve the effect of short establishment time and simple and easy method.

Active Publication Date: 2018-08-10
ZHONGSHAN HOSPITAL FUDAN UNIV +1
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  • Abstract
  • Description
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Problems solved by technology

[0004] Although chemotherapy is one of the main methods for the treatment of advanced gallbladder cancer, multidrug resistance (mμltidrμgresistance, MDR), which often appears in chemotherapy and is difficult to avoid and solve, is the main reason for chemotherapy failure. Therefore, research on the drug resistance mechanism of gallbladder cancer and Overcoming or reversing the multidrug resistance of gallbladder cancer is of great significance for improving the curative effect, but there is still a lack of gallbladder cancer cell lines resistant to oxaliplatin

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  • Method for preparing human gallbladder cancer cell line resistant to oxaliplatin
  • Method for preparing human gallbladder cancer cell line resistant to oxaliplatin
  • Method for preparing human gallbladder cancer cell line resistant to oxaliplatin

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Embodiment 1

[0017] Example 1: Induction of drug-resistant cell lines

[0018] Induced by high-dose shock combined with gradually increasing dose method, human gallbladder cancer SGC-996 cells in the logarithmic growth phase (JN-C0997, purchased from Shanghai Rongbai Biotechnology Co., Ltd.) were inoculated in DMEM containing 20% ​​newborn fetal bovine serum. In the culture medium (the DMEM culture medium containing 20% ​​newborn fetal calf serum is the base culture medium of the following culture medium), place at 37°C, 5% CO 2 cultured in a saturated humidity incubator. Oxaliplatin was added to the DMEM medium containing 20% ​​newborn fetal bovine serum for induction, the initial concentration was 0.2 μmol / L, and when the cells returned to normal growth, the concentration was increased by 0.1 μmol / L. For normal growth and subculture at a concentration of 1.0 μmol / L OXA and 20% newborn fetal bovine serum, impact culture for 12 hours, and immediately replace the culture medium with a conc...

Embodiment 2

[0019] Embodiment 2: Morphological observation

[0020] The morphology of SGC-996 before and after oxaliplatin induction was observed with an inverted phase-contrast microscope, and photographed and recorded. Such as figure 1 As shown, SGC-996 is polygon-like, and some cells are short spindle-shaped with sharp edges and corners, while the morphology of SGC-996 / OXA has undergone obvious changes, some cells are obviously elongated, long spindle-shaped, and some cells have multiple Pseudopodia, in addition, the cell volume also slightly increased.

Embodiment 3

[0021] Embodiment 3: the mensuration of growth curve

[0022] Take SGC-996 in the logarithmic growth phase and its drug-resistant cell SGC-996 / OXA, digest with trypsin and resuspend the cells with DMEM complete culture medium, adjust the cell density to 5×10 4 cells / mL, inoculated in 96-well culture plate, with 4 replicate wells for each group, placed at 37°C, 5% CO 2 After culturing overnight in the incubator, the absorbance value A of each group at 492nm was measured by MTT method every day 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.

[0023] Such as figure 2 As shown, the growth trend of SGC-996 and its drug-resistant cell SGC-996 / OXA is basically the same, the growth is slow in the first three days, the growth is accelerated in the next two days, and it enters the exponential growth phase. In general, the growth of SGC-996 before drug resistance was faster, but the growth of...

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Abstract

The invention provides a method for preparing a human gallbladder cancer cell line resistant to oxaliplatin. The method comprises the following steps that human gallbladder cancer cells in the exponential phase are inoculated in a DMEM culture solution containing newborn fetal calf serum for culturing, oxaliplatin is added into the culture solution for inducing, wherein after the cells recover thenormal growth, the concentration increases progressively, when the cells can normally grow and perform passage at the concentration of 0.5 [mu] mol/L, oxaliplatin is adopted for carrying out impact culture, a culture medium with the concentration of oxaliplatin being 0.5 [mu] mol/L is used for culturing, after the cells grow and perform passage stably, impact is carried out again, then the concentration increases progressively, oxaliplatin is used for carrying out impact inducing till the cells can grow in oxaliplatin of 10.0 [mu] mol/L, and finally, the human gallbladder cancer cell line resistant to oxaliplatin which can stably grow and perform passage in the culture system containing 10.0 [mu] mol/L of oxaliplatin is obtained. The method for establishing the human gallbladder cancer cell line resistant to oxaliplatin is simple, convenient and feasible, the establishing time is short, and the drug resistance index is close to 20.

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 human gallbladder cancer oxaliplatin-resistant cell line. 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. Although surgical resection is currently the main treatment, 80% of patients have metastases at the time of diagnosis, the rate of surgical resection is less than 30%, the postoperative recurrence rate is high, and the prognosis is extremely poor. Particularly important. [0003] Oxaliplatin ( Oxaliplatin , OXA) is the third-generation platinum drug, which mainly exerts anti-tumor effect by int...

Claims

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Application Information

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Patent Type & Authority Applications(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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