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Background and Objectives: neural stem cells (Neural Stem Cells, NSCs) transplantation and gene therapy is the treatment of ischemic cerebrovascular disease are two important avenue of research. In recent years, neural stem cells have been used as gene therapy vectors can carry therapeutic genes into the lesion, play a role in the local expression of the therapeutic gene in ischemic injury, is expected to bring new hope for the treatment of stroke. The purpose of this study: to slow virus (lentivirus LV), mediated hNT-3 gene transferred NSCs cultured in vitro to explore the characteristics of the the NSCs-hNT3 on the in vitro differentiation and expression of NT-3 time, for in vivo transplantation the NSCs-hNT3 research and clinical applications provide the necessary in vitro experimental basis. METHODS: The experiment is divided into two parts. Part I: from 14 days pregnant Sprague-Dawley (SD) rat embryonic brain tissue isolated neural stem cells, serum-free culture method, were cultured in vitro amplification and identification by immunofluorescence staining (nestin). Cap 5-6 substituting transmitted from neural stem cells, 1 × 10 ~ 5 cells/500μl / well were seeded in 24-well plates, respectively, are multiplexed infection index (Multiplicities of infection, MOIs value) 0,1,5,10, 15,20 lentivirus carrying GFP reporter gene vector (lentiviralvector-GFP LV / GFP) dilutions, each titer add tin, a total of six groups. 2 to 3 days in the inverted fluorescence microscope observation groups GFP expression efficiency neural stem cells ball count, and three days later, observe the proliferation of LV / GFP NSCs. Parallel flow cytometry, GFP-positive transfection efficiency derived NSCs. Part II: Building expression HNT-3 gene lentiviral vectors (lentiviral vector-hNT-3, LV/hNT-3), the experimental group based on the best MOI use LV/hNT-3 transfer dye NSCs, the control group does not turn NSCs stained virus. Two groups of cells to serum-free culture by ELISA and immunofluorescence staining methods to detect two NSCs in vitro differentiation and NT-3 expression after 48h to 96h. Results: The first part: NSCs cell suspension ball growth, the nestin staining positive. After transfection with GFP gene, in addition to the control group of the MOI value of 0, 2 to 3 days of each well were GFP expression. The MOI value from 0 to 10, the positive expression rate of the cells gradually increased (P <0.05), MOI of 10 groups can be obtained> 85% transfection efficiency. The MOI value increased from 10 to 20, the formation of neural stem cells ball number was gradually reduced. The second part: NT-3 modified neural stem cells early parental morphological difference between 3-4 days after the experimental group, the proportion of neural stem cells than the control group was significantly higher, differentiated cells compared with control group in the form of: cell processes and the number of synaptic connections between cells. Turn NT-3 group of neural stem cells differentiation into neurons was significantly higher than that in control group. Conclusion: 1) LV is exogenous gene transferred to an ideal vehicle for the neural stem cells. Titer of MOI 10, LV exogenous gene can be efficiently transferred to the neural stem cells. 2) Lentiviral-mediated transfection neurotrophin -3 genes in neural stem cells cultured in vitro, efficient expression of NT-3, increase the proportion of differentiation into neurons, can form protrusions longer number.
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