Abstract:

Objective The drug⁃resistant cell lines were constructed by oxaliplatin（OXA）concentration gradient method to explore the drug resistance mechanism of OXA in the treatment of gastric cancer and provide experimental basis for reversing drug resistance strategy. Methods The OXA concentration gradient was used to culture human gastric cancer cells MGC⁃803 to replicate the OXA resistant cell line model（MGC⁃803/OXA）. Use an inverted microscope to observe the morphological changes of the cells and detect the cell resistance index RI. Flow cytometry was used to detect cell cycle distribution and apoptosis. RT⁃qPCR was used to detect the expression of MDR⁃1，MRP，and ERCC1 mRNAs related to drug resistance. Western Blot detects the expression of drug resis⁃ tance⁃related gene proteins MDR⁃1，MRP，ERCC1 and autophagy⁃related proteins P62，Beclin⁃1 and LC3. At the same time，the number and structure of autophagosomes in the cells were observed with a fluoroscopy electron microscope. Results OXA concentration gradient culture successfully constructed drug⁃resistant cell line MGC⁃803/ OXA. The half inhibitory concentration（IC50）is 62.33 μmol/L，and the RI is 7.01（RI > 5 meets the requirements of resistant strains）. The cell proliferation of the MGC⁃803/OXA cell line slows down，the G2/M phase of the cell cycle is prolonged，and the cell apoptosis rate is reduced. The expression of resistance⁃related genes and proteins such as MDR ⁃1，MRP，ERCC1 is higher than that of MGC ⁃803. It shows that the GC drug ⁃ resistant cell model MGC⁃803/OXA has been successfully constructed. Compared with MGC⁃803，P62 expression in MGC⁃803/OXA decreased，Beclin⁃1 expression and LC3⁃Ⅱ/Ⅰ ratio increased. And there is a typical vacuole bilayer membrane⁃like structure of autophagosomes containing cytoplasmic components ，and the number has increased significantly. Conclusions The OXA concentration gradient method can successfully construct drug⁃resistant cell lines，which are characterized by slower proliferation，prolonged G2/M phase，reduced apoptosis，and enhanced autophagy levels.

Key words:

oxaliplatin, gastric cancer, resistance, autophagy, apoptosis