Study Of Acupuncture Ren Meridian Effect On Differentiation And Proliferation Of Neural Stem Cells By Retrovirus Plasmid PLXSN-EGFP Tracer Technique In Brain Ischemia Rats

Objective:1.Succeed to clone plasmid pLXSN-EGFP and inject it into cerebral ventricle,to do this,we try different dosage and time,to get appreciate program in the tracing technique in active body with the plasmid pLXSN-EGFP.2.To study proliferation and differentiation of neural stem cells in brain ischemia rats treated with acupuncture on Ren meridian.Methods:1.Clone the plasmid pLXSN-EGFP with the method of CaCl₂,and transfect it with the method of Lipo2000,then observe the number of the EGFP cells and the death rate to choose the best dosage and time to inject the plasmid into the cerebral ventricle,2.Wistar rats,Male,were divided into 4 groups randomly:Normal group,Sham operation group(Sham),MCAO group,Electroacupuncture on Ren vessel group (Acu),Moxibustion on Ren vessel group(Mox).6 rats each group and each group was averagely divided into 7 days group,14 days group and 28 days group at random. EGFP positive cell and EGFP/Nestin double labeled cell expression on ischemic lateral cerebral ventricle,EGFP positive cell and EGFP/NSE double labeled cell expression on ischemic hippocampus dentate gyrus,were detected by immunocyleochemistry method,laser confocal microscopy.Results:1.The best dosage and time to inject the plasmid into the cerebral ventricle is 6ul and 7days.2.The NSCs were traced by retrovirus recombinant plasmid pLXSN-EGFP labeling.The light spectrum feature laser confocal microscopy was consistent with the EGFP labeling protein.Our experiment shows that the green fluorescent labeling was detected in the cellular membrane and cytoplasm.It indicated the transfect activity of EGFP carried by retrovirus vector was stabile.The EGFP expression was not seen in Norm and Sham group,but enhanced in ventricle of Acu and Mox group,also in DG,and less with the time longer,and compared with model group,increased more obviously.1)The number of EGFP/Nestin labeled cells accrescenced in ischemic lateral cerebral ventricle of Acu and Mox group.Compared with Acu group,the EGFP/Nestin double labeled cells in ischemic lateral cerebral ventricle increased in Mox group in the 28^thday.It implies that acupuncture and moxibustion Ren Meridian could enhance the NSCs proliferation in the ischemic lateral cerebral ventricle.2)The number of EGFP/NSE labeled cells accrescenced in DG of Acu and Mox group.Compared with Acu group,the EGFP/NSE double labeled cells in ischemic DG increased in Mox group in the 28th day.It implies that acupuncture and moxibustion Ren Meridians could enhance the NSCs proliferation and differentiation in the ischemic DG.Conclusions:1.NSCs could be marked in active body by retrovirus recombinant plasmid pLXSN-EGFP.2.Acupuncture and Moxibustion on Ren Meridian could promote the proliferation and differentiation of neural stem cells in MCAO rats.