The Study Of HepG2 Cultured In Roller Bottle And Apoptosis Induced By Seleno-chitosan In HepG2

In this paper the hepatocellular carcinoma cell line HepG2 was regarded as the research object. Basing on the vial culture, to expand the scale of HepG2 in cultivation, the method of HepG2 roller bottle cultivation was explored, and then the effect and mechanism of apoptosis in HepG2 cells induced by seleno-chitosan was studied.In this study, the speed of the rotation was explored in the first place, the initial adherent speed and the range of speed after adsorbed of HepG2 when cultured in roller bottle was determined through single factor experiments. Secondly, the cell inoculation quantity, the dosage of medium, rotate speed and incubation time were treated as independent variables to carried out the orthogonal test of four factors and three levels.Through the analysis, it is concluded that the amount of cells and the cultured time had a significant effect on the cell yield. Combining single factor tests with orthogonal test, the final optimum scheme of HepG2 that cultured in roller bottle was determined: the early adherent speed was 10 r/h, the speed after adsorbed was 20 r/h, the inoculation of HepG2 was 4 ×107 per bottle, the cultured time was 48 h and the dosage of the medium was 150 mL.Basing on the method of HepG2 roller bottle cultivation, the effect of seleno-chitosan in inducing apoptosis of HepG2 when cultured in roller bottle was studied. The optimal concentration of seleno-chitosan in inhibiting the proliferation of HepG2 was determined by MTT test, and the apoptosis related phenomena that caused by seleno-chitosan on HepG2 was observed through using scanning electron microscopy, experiments of rhodamine123 staining, Annexin V-FITC/PI double staining. Moreover, the apoptosis rates after seleno-chitosan incubated for 24 h, 36 h, 48 h were 12.33 %, 63.63 %, 81.00 %, and the cell cycle was blocked in phase S?G2 and G2?M .14 differentially expressed protein spots were founded by using proteomics technology to contrased protein profiles of seleno-chitosan unincubated group and incubated group, of which 10 were down regulated and 4 were up-regulated. 9 of the most significant protein spots were identified by using MALDI-TOF/TOF-MS, and four differential proteins were identified, which were cytochrome C oxidase, calmodulin, tubulin and myosin, through the study of its function combining with the decreased expression, it can be inferred that the mechanism of seleno-chitosan in inducing apoptosis of HepG2 cells were the mitochondrial pathway and inhibited the cell cycle.