The Study Of The Neural Stem Cells Transplantation For The Treatment Of Vascular Dementia Rats

Part1The experimental study of rat neural stem cells culture andthe transfection of GFP mediated by lentivirusObjectiveThe neural stem cells were primary proliferated and passaged stably in vitro, andwere transfected by GFP mediated by lentivirus. Then the NSCs can be used as seedcells for the transplantation.MethodsThe primary neural stem cells were isolated from the healthy pregnant Wistarrat(P15),cultured and passaged in vitro,and monoclonal cells.The immunofluo-rescence and immunohistochemistry SP method were used to identify the neural stemcells. Lentivirus mediated transfection The NSCs were transfected by GFP mediatedby lentivirus.Result1．The primary cultured cells formed the small neurosphere made of dozens ofcells. After7days, the neurospheres had grown hundreds of large clones of cells canbe passaged. After another7days NSCs had become hundreds of large clones, whichwere passaged again as the same pattern.2．The primary cultured neurosphere were dispersed and re-amplified a largenumber of new neurospheres, and the nestin staining of the passaged neurosphere waspositive; the passaged neurosphere differentiated into NSE、CNP、GFAP positive cellsdetected by immunofluorescence and immunohistochemical SP methods. 3．After NSCs were transfected by GFP mediated by lentivirus, NSCs carryinggreen fluorescence were visible observed by the fluorescence microscope.Conclusion1．Neural stem cells isolated from rat embryonic brain tissue(E15) continue toproliferated and passaged stably in vitro, which can be used in transplanting.2．NSCs were transfected by GFP mediated by lentivirus. The labeled NSCswere facilitated follow-up study as the transplanted seed cells in animals. Part2The experimental study of neural stem cells transplantationfor treating the vascular dementia ratsObjectiveThe NSCs labled with GFP mediated by lentivirus were transplantedstereotactically into the hippocampus of vascular dementia rats, so that the number ofNSCs around the hippocampus increased. The learning and memory ability of ratswere detected by navigation test and space exploration test in Morris water maze afterNSCs transplantation.Materials and MethodsThe healthy adult Wistar rats at the age of12to15months and the weightbetween300~400g were made the vascular dementia model(2VO). The NSCs ofWistar rats marked by GFP when passaged to the sixth generation were transplantedstereotactically into the hippocampus as the seed cells after the2VO model weremade for35days. The hippocampal tissue slices of the three groups rats were Nisslstaining and observed the pathological change. Before and after transplanted4w、8w、12w, the rats in each group were test by Morris water maze, and the results ofnavigation test and space exploration were analyzed. Result1．After one week of NSCs transplantation, there was the green fluorescence inthe hippocampal frozen slice of which proved NSCs could survive and proliferate inthe hippocampus.2．After the transplantation4w、8w、12w, the escape latency of NSCs group wassignificantly shorter than in the control group(P <0.05), but it was still significantlyprolonged compared with the sham group(P <0.05). The number of platform crossingincreased significantly than the control group(P <0.05), but still significantlydecreased compared with the sham group(P <0.05). The platform quadrant residencetime ratio increased significantly than the control group(except12w, P <0.05), anddecreased compared with the sham group(P <0.05).3． The NSCs group Nissl staining result showed the neuron loss anddegenerative conditions had improved significantly compared with the control group,there was more complete morphology neuron and abundant Nissl bodies in thecytoplasm relatively.ConclusionThe neural stem cells transplanted into the VaD rats model could improve thelearning and memory ability of rats to some extent. Part3The study of Notch pathway in neural stem cellstransplantation for treating the vascular dementia ratsObjectiveTo detect the dynamical chang expression of Notch1and Hes1on VaD ratshippocampus after NSCs were transplanted. To research if the learning and memoryof rats improved were related to the change of Notch1and Hes1expression inhippocampal. To investigate whether the Notch signaling pathway involved in theNSCs transplantation for treating the VaD rats. MethodsThe healthy adult Wistar rats at the age of12to15months and the weightbetween300~400g were made the vascular dementia model(2VO). The NSCs ofWistar rats marked by GFP when passaged to the sixth generation were transplantedstereotactically into the hippocampus as the seed cells after the2VO model weremade for35days. Hippocampus brain tissue protein samples of the three groups weredetected by western blot.ResultAfter the transplantation4w、8w、12w, the western blot was used to detectNotch1and Hes1expression. It suggest that the NSCs group expressed higher levelof Notch1and Hes1than the control group, but lower than the sham group. Theexpression of Notch1and Hes1of NSCs group decreased over time.ConclusionThe transplanted NSCs activated Notch1-Hes1pathway, which might take partin the NSCs transplantation for repairing the learning and memory abilities of VaDrats. Part4The synaptic plasticity changes of neural stem cellstransplantation for treating the vascular dementia ratsObjectiveTo study whether there are the morphology and structure of dendritic anddendritic spines changes at the process of the NSCs transplantation for treating theVaD rats and whether the improvement of learning and memory ability in VaD rats isassociated with the morphology and structure of dendritic and dendritic spineschanges. MethodsThe healthy adult Wistar rats at the age of12to15months and the weightbetween300~400g were made the vascular dementia model(2VO). The NSCs ofWistar rats marked by GFP when passaged to the sixth generation were transplantedstereotactically into the hippocampus as the seed cells after the2VO model weremade for35days. We analyzed the dendritic branches and the dendritic spine densityin hippocampal CA1area of the rats in the three groups.ResultThe number of dendritic branch of the control group and NSCs transplantationgroups rats were significantly decreased compared with the sham group,(P <0.05),but the decline is not obvious with time. The number of dendritic branch of NSCsgroup increased in comparison with the control group. The difference havestatistically significant (P<0.05). The dendritic spine density of hippocampal CA1pyramidal neurons of the control group and NSCs group decreased significantly,(P<0.05), the density of dendritic spines of the NSCs group increased compared withthe control group,(P <0.05).ConclusionNSCs transplantation increased the dendritic complexity and dendritic spinesdensity of hippocampal CA1neuron of VaD rats. which might be associated with theimprovement of vascular dementia rats learning and memory.