Mechanism Study Of Seleno-chitosan On Apoptotic Effect In MGC-803 Cells Under Roller Bottle Culture

The large-scale roller bottle culture system of the human gastric cancer cell line MGC-803 was established, enlarging the traditional vial culture. Then the Seleno-Chitosan and MGC-803 cells were co-cultured, the inhibition and apoptosis induction mechanism of Seleno-Chitosan on MGC-803 cell in roller bottle was explored.First of all, by taking account of the cell state, cell viability and so on, MGC-803 cells were cultured in roller bottle by a one-time adding medium. The optimal initial speed was 10r/h, the culture time was 5h by single factor experiment. Then the inoculation, adherent rotation rate, incubation time and medium amount were evaluated by the orthogonal experiment with four factors and three levels. The results indicated the inoculum was the most important factor affecting the cell yield, and the optimal conditions for MGC-803 cells in roller bottle were inoculation amount 4 ×107 cells, adherent rotation rate 20r/h,medium amount 150mL, culture time 72h.After the establishment of MGC-803 roller bottle cultivation method, the apoptosis effect of Seleno-Chitosan on MGC-803 cell in roller bottle was explored. The MTT assays showed that Seleno-Chitosan significantly suppressed the proliferation of MGC-803 cells with a time and concentration dependent manner. The cell growth inhibitory rate of MGC-803 could reach 69% after induced by Seleno-Chitosan (100?g/mL) for 48h; The cell morphology had an obviously change; Hoechst 33258 staining showed that the cells appeared typical apoptotic characteristics such as chromatin condensation; Annexin V-FITC/PI staining showed that cell apoptosis gradually changed from early to late apoptosis after induced by Seleno-Chitosan for different time; cell cycle studies showed that the cell cycle was arrested in G2/M phase.Then, the apoptosis signaling pathway of MGC-803 cells induced by Seleno-Chitosan was further explored. By flow cytometry, SDS-PAGE, Western blotting, the results found that Seleno-Chitosan can affect the expression of Bcl-2, Bax in MGC-803 cells, and the mitochondria permeability transition pore was opened, the mitochondrial membrane potential was decreased, cytochrome C was released, Caspase-9 and Caspase-3 were further activated, cell apoptosis. Thus, Seleno-Chitosan induced MGC-803 cell apoptosis through mitochondrial pathway.