Mechanism Study Of Seleno-Chitosan On Apoptotic Effect In Human Gastric Cancer MKN45 Cells

In this study,human gastric cancer MKN45 cells were taken as the research object,and large-scale roller culture was established to enlarge the traditional vial culture.And then,the effect of the proliferation inhibition and apoptotic mechanism of MKN45 cells induced by Seleno-Chitosan were investigated.The results of single factor and orthogonal test indicated the inoculum is the most important factor to affect the cell yield,and the optimal conditions for MKN45 cells in roller bottle is inoculation amount 5×107 cells,medium amount 200 mL culture time 72 h,adherent rotation rate 20 r/h.The MTT assay showed that Seleno-Chitosan significantly suppressed the proliferation of MKN45 cells in a time and concentration dependent manner.The cell growth inhibitory rate of MKN45 could reach to 80%after induced by Seleno-Chitosan(200 ?g/mL)for 72 h;inverted microscope,Scanning Electron Microscopy and Hoechst33342/PI staining observed that the cells appeared typical apoptotic characteristics such as chromatin condensation,which further proved the induction of Seleno-Chitosan morphologically;AnnexinV-FITC/PI staining showed that MKN45 cell gradually changed from early apoptosis to late apoptosis after induced by Seleno-Chitosan for different time;in addition,studies on cell cycle showed that the cell cycle was arrested in S phase.Then,further explored exploration on apoptotic signaling pathway of MKN45 cells induced by Seleno-Chitosan was conducted.The results of flow cytometry showed that the level of reactive oxygen species significantly increased and mitochondrial membrane potential decreased significantly.Last,the apoptosis was further verified from the apoptotic protein level by SDS-PAGE and Western blotting.We found that the abnormal expression of Bcl-2 and Bax of MKN45 cells affected by Seleno-Chitosan can cause the opening of mitochondria permeability transition pore and collapse the mitochondrial membrane potential,then,cytochrome C was released and Caspase were further activated,and cell apoptosis finally.In summary,chitosan can inhibit the proliferation of MKN45 cells and induce apoptosis through mitochondrial pathway.Therefore,it is a promising anticancer substance.