Experimental Study On Apoptosis By TNF-α In Neural Stem Cells Of Neonatal SD Rat

In the study, we establish isolation, cultivation and identificationsystem of neonatal SD rat NSC in vitro and observe the outcome ofapoptosis by TNF-αin neonatal-rat cerebral neural stem cells. It providesthe novel promising potential targets and experimental basis for clinicalresearch.The study includes two parts:PartⅠIsolation, Cultivation and Identification of neonatal SDrats neural stem cells in vitro.Objective To isolate, cultivate and identify the neural stem cellsfrom neonatal SD rats(1-3 days) and observe the character on the growth,proliferation of NSC.Methods cells were isolated from the central nervous tissue ofneonatal rats by mechanical dissociation. The obtained cells in suspensionwere cultured by non-serum clonal technology. The expression of Nestinantigen were quantitive investigated by immunofluorescencecytochemistry staining to calculate the NSC proportion of the primaryand passaged neurospheres. Meanwhile, the study on cryo-preservationand revitalization of NSC has been made.Results the NSC of primary and passaged cell suspensions hadpotential of self-proliferation. They were able to form large numbers of cell groups named neurospheres. The percent of Nestin positive cells inneurospheres had significant difference between primary and the thirdgeneration neurospheres(P＜0.05). The survival rate of NSC was up toabove 70% after cryo-preservation and revitalization. The NSC can bepreserved for a long time under profound hypothermia.Conclusions It proved that cells separated from the CNS ofneonatal rats and cultivated in suspensions were neural stem cells whichpossessed the potential of self-renewal and self-proliferation. Thecryo-preservated NSC had original characteristics after revitalization.PartⅡEffects of apoptosis by TNF-αin the neural stem cellsObjective To observe the effects of apoptosis by TNF-αin theneural stem cells of neonatal SD rats and discuss the mechanism ofapoptosis.Methods the 3rd generation NSC cultivated in vitro were dividedinto control and experimental groups with the same density. Inexperimental groups, 3 sub-groups were divided according to thedifferent concentration of TNF-αand cultivated 12h, 24h, 48h and 72hrespectively. The livability of NSC was demonstrated by MTT screeningexperiment and the apoptosis of NSC were quantitive and qualitativeinvestigated by TUNEL, FCM.Results When concentration of TNF-αwas higher than 0.5ng/ml,the livability, apoptosis amounts and percent of apoptosis of NSC had significant difference between the control and experimental groups(P＜0.05). With the same concentration of TNF-α(＞0.5ng/ml), apoptosisstem cells of the experimental groups which cultivated in longer-time(＞24h) were more than that in shorter-time (P＜0.05). In the cultivatedtime, there were no significant difference in other sub-groups.Conclusions TNF-αcan induce apoptosis on the neural stemcells. The concentration of TNF-αwas the important cause of apoptosison neural stem cells.