Long Noncoding RNA Regulating The Effect Of Sevoflurane On Proliferation Of Neural Stem Cells In Rat

An increasing number of animal studies have showed exposure to general anesthetics(GA)during pregnancy and early postnatal stage leads to neurotoxicity in developing brain.To date,there is no substitute drug more effective and safe than GA.Thus,it is an urgent issue how to repair general anesthetics-induced neurotoxicity in developing brain.When brain suffers damage,quiescent neural stem cells(NSC)are reactivated into proliferation and differentiation to generate new neurons for repairing damage region.The proliferation of NSC is the first crucial stage of endogenous repairment.Therefore,the study focusing on the effect of GA on NSC proliferation will produce a large mount of clinical implicationsA majority of long noncoding RNA(lncRNA)involved in cell type,developmental process and human disease were detected in the subventricular zone and olfactory bulb,as well as dentate gyrus.Previous study has demonstrated lncRNA served as competitive endogenous RNA to determine NSC proliferation in primates.However,it is unclear which and how IncRNA regulates the effect of sevoflurane on proliferation of NSC in rat.chapter 1:The construction of NSC model exposed to sevoflurane suitable for microarray analysisObjective:To build NSC model exposed to sevoflurane suitable for detecting IncRNA expression profile using microarray.Methods:After primary culture,NSC derived from hippocampus of neonatal rat was exposed to sevoflurane and control gas for o,3,6,12 h.The proliferative capacity was analyzed using CCK-8 kit.The survival rate of NSC was detected by flow cytometry(FCM).The quality of NSC sample was identified using agarose gel electrophoresis and spectrophotometer.Results:Compared with those of control group,the proliferation and survival of NSC exposed to sevoflurane were inhibited in concentration and time-dependent manner.The sample quality of NSC exposed to 2.4%sevoflurane for 6 h was consistent with requirment of microarray analysis.Conclusion:The cultrue NSC exposed to 2.4%sevoflurane for 6 h is suitable for microarray analysis.Chapter 2:The investigation of IncRNA expression profile in NSC exposed to sevofluraneObjective:To investigate the IncRNA expression profile in NSC exposed to sevoflurane,identifying target IncRNA associated with proliferation of culture NSC.Methods:The IncRNA expression profile in NSC exposed to sevoflurane was detected using IncRNA microarray.The 10 lncRNAs and 10 mRNAs were randomly selected to be validated using quantity PCR(qPCR).Based on bioinformatics analysis,MRAK080926 and MRAK157758 with their nearby genes were choosed to be identified using qPCR.Results:The expression data of 10 lncRNAs and 10 mRNAs by qPCR was in line with the expression profile using microarray.The nearby genes,Wnt5a and Gadd45a,were related to NSC proliferation.The positive correlation between upregulated MRAK080926 and Wnt5a was the same as upregulated MRAK157758 and Gadd45a.Conclusion:MRAK080926 and MRAK157758 are associated with the effect of sevoflurane on NSC proliferation.Chapter 3:The effect of sevoflurane on NSC proliferation was determined by RAK080926Objective:To investigate MRAK080926 regulating the effect of sevoflurane on NSC proliferation,preliminarily illustrating approach underlying MRAK080926.Methods:After knockdown of MRAK080926 with lentivirus vector,the proliferation of NSC exposed to sevoflurane was detected using cell clone test,CCK-8 kit as well as EDU assay.The survival of NSC was also analyzed by FCM and Tunel assay.The FCM was used to detect cell cycle and ROS level.Meanwhile,MRAK080926 and Wnt5a mRNA were analyzed by qPCR,Western blot was employed to detect the protein level of Wnt5a,RYK and BAX.Results:Knockdown of MRAK080926 promoted proliferation of NSC exposed to sevoflurane,downregulating the MRAK080926 level and the viability of Wnt5a/RYK-ROS.NSC in the G0 stage re-entered into cell cycle,lenghtening S phase.Conclusion:Knockdown of MRAK080926 rescues proliferation of NSC inhibited by sevoflurane,which is invloved in downregulating Wnt5a/RYK-ROS and compelling quiescent NSC to re-enter into cell cycle.Summary:Taken together,the proliferation of NSC is restrained by sevoflurane.Compared with that of control group,the expression profile of IncRNA differentiates in NSC exposed to sevoflurane,wheras,MRAK080926 was significantly upregulated.Knockdown of MRAK080926 promotes the proliferation of NSC,which is involved in downregulating Wnt5a/RYK-ROS signaling pathway.