Pilot Study On The Induction And Preparation Of Apoptptic Bodies Of A549 Cells By Chitosan Selenate

In the study,the human non-small lung cancer A549 cells were used as the research object to establish a cell roller bottle culture model,and the optimal conditions for large-scale culture of A549 cells were explored.After determining the best process,A549 cells were co-cultured with Chitosan Selenate(CS)to investigate the pilot study on the induction and preparation of apoptotic bodies of A549 cells by CS.First,SeO42-was introduced into chitosan by a redox reaction to synthesize CS.Infrared Spectroscopy(IR)verified that CS exhibited a characteristic absorption peak of Se=O at a wavelength of 892 cm-1.High-performance gel permeation chromatography(HPGPC)showed that the molecular weight of CS was about 41.879 KDa,indicating selenium was successfully combined with chitosan.Secondly,the single factor experiment was carried out to screen out the appropriate factors and levels with the rotation speed,cell inoculation amount and culture time as the impact factors and cell yield as the evaluation indexes.The significance and interaction of each factor were analyzed by using the Box-Behnken response surface method,and the A549 cells roller bottle culture process was determined as follows:the inoculation dose was 4.06x107 cells/bottle,the rotation speed was 13.98 r/h and the culture time was 48.92 h.Under these conditions,cell yield was predicted to be 8.15×107 cells/bottle,and the actual number of cells was 7.93×107 cells/bottle.Compared with the theoretical prediction,the relative error was small.After determining the culture conditions of A549 cells in roller bottles,the induction mechanism of CS on apoptotic bodies of A549 cells was further investigated.MTT assay showed that CS inhibited the proliferation of A549 cells in a time-and concentration-dependent manner.When the concentration of CS was 250?g/mL and the time of action was 24 h,the survival rate of A549 cells was only 18.3%.Both Hoechst 33258 and Hoechst 33342/PI staining showed that CS could induce A549 cells to exhibit some typical features of apoptosis,such as cell contraction,formation of apoptotic bodies,chromatin condensation,cell membrane blebbing,etc.;The results of Annexin V-FITC/PI double staining showed that the apoptosis rate of A549 cells increased from 1.25%to 80.36%after the action of CS at different concentrations,indicating that the cells gradually transformed from early apoptosis to late apoptosis.Cell cycle assay showed that CS disrupted the continuity of cell cycle and triggered S and G2/M phase arrest in a dose-dependent manner.Subsequently,Sodium dodecylsulphate polyacrylamide gel electrophoresis(SDS-PAGE)and Western Blotting(WB)analysis showed that CS could induce A549 cells apoptosis through Fas/FasL-mediated signaling pathway,such as increasing the ratio of Bax/Bcl-2 and activating apoptotic related proteins in A549 cells.Finally,the purified apoptotic bodies of A549 cells were obtained by differential centrifugation.In summary,the mechanism of CS inducing apoptosis of lung cancer A549 cells to obtain apoptotic bodies was to inhibit the proliferation of cancer cells and induce apoptosis through Fas/FasL apoptosis pathway,which lays a foundation for the production of anti-tumor vaccines,antibodies and other biological products by large-scale cell culture.