| Background and Objective:Ischemic stroke is a leading cause of long-term neurological disability due to extensive loss of neurons and their connections in demaged regions of the brain.Current therapy for ischemic stroke is limited to acute measures designed to restore perfusion and,at least experimentally,to protect ischemic cells from death in acute phase.Yet,the rapid course of neuronal death and the brain's limited regenerative capacity make treatment of stroke and related disorders difficult.One approach to this problem involves the administration of agents which target down stream mediators of pathways involved in delayed neuronal death.Another approach focuses on neural stem cells,which are found in the adult central nervous system through the process of neurogenesis and might be capable of replacing dying cells in the post-ischemic brain.The efficacy of endogenous NSCs in promoting brain repair is limited,about 80%of migrating newborn neurons would die within 6 weeks,and only abpout 0.2%of demaged cells could be replaced via neurogenesis.Although transplantation of stem cells(such as NSCs and BMSCs) can bring neurological function improvement to animals with cerebral ischemia,more and more researcher think that transplantation of neural stem cells may protect the central nervous system from inflammatory damage through a 'bystander' mechanism that is alternative to cell replacement.Recently,efforts have been extended to stimulating the formation and preventing the death of neurons and glial cells produced by endogenous stem cells within the adult CNS.And NSCs have already been used as gene delivery tools for carrying therapeutic genes to disease tissues. Transplantation of modified NSCs may have promise in cell replacement therapy for stroke.The study tested the hypothesis that transplantation of human neurotrophin-3 (hNT-3) over-expressing neural stem cells(NSCs) into rat striatum after a severe focal ischemia would have better functional recovery compared with controls,as well as promote proliferation and neuronal differentiation of the endogenous progenitor cells in the dentate gyrus(DG) of hippocampus and striatal penumbra of rats.We investigated the putative mechamism of modified NSCs transplantation to cerebral ischemia in order to find a new way for cell-based therapy.Methods The study consisted of three parts.PartⅠNeural stem cells were extracted from rat embryonic brain(E14),then grown,expended and propagated in serum-free medium.Passage 5～6 neural stem cells were seeded at a density of 1×10~5/500μl/well in 24-well plates and exposed to lentiviral vector(LV) coding for GFP reporter gene(LV/GFP) at multiplicities of infection(MOIs) of 0,1,5,10,15,20 respectively.There were six groups totally,and each group had six wells seeded.The cells were incubated in a humidified atmosphere with 5%CO2 at 37℃.2～3 days later,the cells were observed under inverted fluorescence microscope.And 3 days later,neurospheres were counted followed by flow cytometer examination to get the percentage of transgene positive NSCs. PartⅡThe lentiviral vector(LV) coding for Flag-tagged hNT-3(LV/hNT3) gene was constructed by the co-transfection of 293T cells with transfer plasmid coding for hNT-3(pGC-E1/hNT-3) and two help plasmids(pHelper 1.0 and Helper 2.0) using Lipofectamine 2000.The hNT-3 mRNA code sequences within the transduced cells was subsequently tested by RT-PCR and sequencing,the biological activity of LV/hNT-3 was demonstrated by western-blot analysis of total proteins from hNT-3 producer cells.PartⅢNSCs,transduced by hNT-3 gene mediated by LV,were transplanted into the post-infarct brain striatum of adult rats 7 days after 2-h occlusion of the middle cerebral artery(MCAO).From 1 day before transplantation to 2 weeks after transplantation,Neurological Severity Scores(NSS) were calculated and the levels of neurotrophin-3 as well as TRKC in the graft sites were studied.Immunofluorencence staining was used to label NSCs-hNT3(Flag-positive) and newly proliferated endogenous cells (BrdU-positive and DCX-positive). Results:PartⅠ7～8 days after isolated from rat embryonic brain,NSCs grew as free-floating nestin-positive neurospheres.2～3 days after GFP gene transduction,GFP-expressing cells cound be seen except in the control group, Over 90%cells were GFP-positive at 3 days after transduction by LV at MOI of 10.A significant dose-response was observed with increasing virus titer for MOI 0 to 10(P＜0.05),whereas the amount of neurospheres decrease with increased virus load for MOI 10 to 20(P＜0.05).PartⅡIn the predent study,correct hNT-3 mRNA expression was demonstrated by RT-PCR analysis of total RNAs from hNT-3 producer cells,and RT-PCR products were also sequenced and verified by NCBI blast analysis to be correct.Furthermora,the biological activity of LV/hNT-3 was demonstrated by western-blot analysis of total proteins from hNT-3 producer cells.PartⅢAfter transplantation,the NSCs-hNT3 survived the transplantation procedures could migrate in the ischemic area and express significantly higher levels of hNT-3 protein in the graft sites compared with controls(P＜0.001).And ischemic rats received modified cells had a significantly higher number of newly proliferated cells(BrdU positive) in both the hippocampal dentate gyrus(DG) ipsilateral to the lesion(P＜0.05) and striatal penumbra(P＜0.05).Of the Brdu-positive cells,over 90%in the DG and one third in the penumbra were immature neurons(DCX positive).Furthermore, transplantation of NSCs-hNT3 could bring better functional recovery compared with controls.Conclusion(1) LV is an ideal vector for gene transduction and it can transduce a foreign gene into NSCs with high efficency of over 90%at the MOI of 10.(2) Therapeutic genes could be carried to disease tissues through transplantation of modified NSCs and express effectively.(3) Over-expression of hNT-3 in the ischemic area might promote the survival of newly proliferated cells and have values for enhancing functional recovery after stroke.
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