In Vitro Studies About Mechanism Of Bone Marrow Derived Neural Stem Cells Death And Resisting Oxidative Damage

Research of neural stem cells(NSCs) has been one of the hot points in the basic investigation of neuromedicine.The transplantation or regeneration of NSCs in brain supply new expectation for the treatment of central nervous system(CNS) disease caused by injury or retrogression.Culture in vitro is the most basic and indispensable period in the study and clinical application of NSCs.The status of cells in vitro influences directly the effect of transplantation in vivo.Though the transplation of NSCs has been used in some clinical trial,there are still a series of complicated and important problems to avoid the deterioration and death of NSCs,to maintain its survival,development,differentiation and play function.The bone marrow stromal cells-derived neural stem cells(BMSC-D-NSCs) are more close to the clinical application than the NSCs derived from embryo or adult brain,but it also might be undergoing death because they need to be induced and controlled in vitro.So,it has important clinical significance to research the death mechanisms of BMSC-D-NSCs cultured in vitro and explore the methods of maintaining and improving its survival.BMSCs could be isolated,cultured,and promoted to differentiate to NSCs.We research the mechanisms of BMSCs-d-NSCs cultured in vitro undergoing degeneration and death via observing and detecting the apoptosis and necrosis of cells at a series of time period,and observe the effect of resisting oxidative damage by antioxygen EUK-134.Part 1.Degeneration and death of bone marrow stromal cells-derived neural stem cells cultured in vitroObjective To observe the degeneration and death of bone marrow strom cells-derived neural stem cells(BMSCs-d-NSCs) cultured in vitro,and study its mechanism and rule.Methods BMSCs were isolated using density gradient centrifugation,cultured,and promoted to differentiate to NSCs using the special medium.The degeneration and death of cells were researched by the methods of AO staining,Rhodamine 123 staining, TUNEL,Annexin V-EGFP/PI double staining.Apoptotic and necrotic cells were observed by fluorescent microscope or phase-contrast microscope.The ratio of apoptotic and necrotic cells was detected by flow cytometry.Results Apoptosis and necrosis were observed in the cells cultured in vitro after 1w, 2w,3w,4w,8w,respectively,and the ratio of apoptotic cells was more than the ratio of necrotic cells.The death rate of cells cultured after 4w was more than the death rate within 3w.Conclusion The mechanism of BMSCs-d-NSCs cultured in vitro undergoing degeneration and death are apoptosis and necrosis,especially the former.This study provides foundation for researching cytoprotection.Part 2.Resisting oxidative damage effect of EUK-134 to bone marrow stromal cells-derived neural stem cells cultured in vitro.Objective To observe and research the resisting oxidative damage effect of antioxygen EUK-134 to bone marrow strom cells-derived neural stem cells(BMSCs-d-NSCs) cultured in vitro.Methods BMSCs were isolated using density gradient centrifugation,cultured,and promoted to differentiate to NSCs using the special medium.EUK-134 of different concentration of 0.5mM,1mM,2mM were added in medium respectively for serial subcultivation.The growth of the cells could be observed by phase-contrast microscope. The ratio of apoptotic and necrotic cells could be detected by flow cytometry aider Annexin V-EGFP/PI double staining.Results The cells cultured in the medium with EUK-134 reflected more activity and proliferation.The ratio of apoptotic and necrotic cells in them were significantly less than the cells cultured in medium without EUK-134 after 2w and 4w,respectively.Conclusion EUK-134 produced a significant reduction in apoptosis and necrosis of BMSCs-d-NSCs cultured in vitro.Specifically,1.0mM or 2.0mM EUK-134 added in the medium displyed more conspicuous effect of antioxygen,it could improve the activity and proliferation of cells cultured in vitro,and reduce the occurrence of apoptosis and necrosis.SUMMARYWe dedicated to research the death mechanisms of BMSC-D-NSCs cultured in vitro and explore the methods of maintaining and improveing its survival.BMSCs were isolated and cultured in vitro,and promoted to differentiate to NSCs using the special medium.The degeneration and death of cells were observed by the methods of AO staining,Rhodamine 123 staining,TUNEL,Annexin V-EGFP/PI double staining.Apoptotic and necrotic cells were observed by fluorescent microscope or phase-contrast microscope.The ratio of apoptotic and necrotic cells were detected by flow cytometry.We confirmed that the mechanisms of BMSCs-d-NSCs cultured in vitro undergoing degeneration and death were apoptosis and necrosis,especially the former.EUK-134 produced a significant reduction in apoptosis and necrosis of BMSCs-d-NSCs cultured in vitro,specifically,more conspicuous effects of antioxygen were displayed at 1.0mM or 2.0mM EUK-134 added in the medium. EUK-134 could improve the activity and proliferation of cells cultured in vitro,and reduce the occurrence of apoptosis and necrosis.The strategy of using antioxygen EUK-134 might possess application perspective widely in the study of protecting NSCs cultured in vitro or transplanted in vivo.