Scale Culture Of K562 Cells And Apoptosis Mechanism Induced By Selenic Acid Polysaccharide

In this study, the basic culture conditions of chronic myelogenous leukemia (CML) K562 cells were researched by bio-reactor. Simultaneously, the apoptosis of K562 cells induced by selenic acid cartilage polysaccharide were also studied. The cell morphological characteristics were observed by HE and Hoechst 33342/PI. The cell cycle arrest and mitochondrial membrane potential (△Ψm) were assessed by Flow Cytometry (FCM). The expression of apoptosis-related protein and gene were analyzed by immunofluorescence and Real-time PCR.Firstly, the basic culture conditions of bio-reactor including initial cell density, pH and stirring speed were researched. The results showed that the initial cell density 2.5×105/mL, the pH 7.4 and the stirring speed 70 r/min were the best conditions of K562 cells scale culture. The largest density could reach 1.57×106 cells/mL under the condition.Secondly, the proliferation inhibition and apoptosis induction of selenic acid cartilage polysaccharide to K562 cells were assessed. MTT showed that the proliferation of K562 cells was inhibited by selenic acid cartilage polysaccharide with dose-dependent and time-dependent manner and the inhibition rate could reach 80% after K562 cells were exposed to 100μg/mL selenic acid cartilage polysaccharide for 48h. Typical apoptotic characteristics of cell volume reduction and chromatin shrinkage were observed by inverted microscope, HE and Hoechst 33342/PI. After treatment of selenic acid cartilage polysaccharide, FCM analysis indicated that selenic acid cartilage polysaccharide could increase the number of cells in S phase from 40.75% to 62.93%, and reduce the △Ψm by 18%. It was revealed that Cyclin A, CDK2 and Bcl-2 were decreased significantly but p21 and caspase-9 were increased significantly by immunofluorescence and ELISA. RT-PCR showed that the genes of these proteins had the same trends. All the results proved that selenic acid cartilage polysaccharide could inhibit K562 cells proliferation and induce K562 cells to apoptosis by influencing cell cycle and the expression of apoptosis-related protein.Above all, this study laid the foundation for the produce of vaccines and antibodies by scale cell culture.