Experiment Study Of HRE Regulating VEGF-expression Transfected Neural Stem Cells

Objective:Acquire high VEGF-expression gene engineering NSCs with hypoxia induced, Explore if hypoxia-response element (hypoxia-reponsive element, HRE) can improve the expression of VEGF gene in hypoxia condition, in order to seek a safe and efficient gene therapy method for hypoxic ischemic brain damage.Method:Firstly, we isolated NSCs from the brain of SD fetal rats, then NSCs were cultured and amplified by non-blood serum culture technique. NSCs and differentiated cells were identified using immunofluorescent histochemical method。Secondly, transfected VEGF165and HRE-VEGF165into NSCs respectively mediated by Lentivirus. Cells cultivate under the different oxygen concentration by grouping. Through a fluorescence microscope and flow cytometry instrument detecte the infection efficiency, RT-PCR method to detect transgenic VEGF gene expression, Western blot and Elisa to detect expression of vascular endothelial growth factor, MTT method detect NSCs proliferation activity genetic engineering NSCs.Results:The cells isolated from brain tissue of fetal rats can proliferate for a long time, both primary and passage culture of NSCs can express specific antigen of NSCs-Nestin, and after induced differentiation of cells can express specific antigen of astrocytes and Glial fibrillary acidic protein (GFAP) and specific antigen of neurons-Neuron specific enolase (NSE). The tranfection rate of VEGF-NSCs is50%,while of HRE-VEGF-NSCs in hypoxia is80%; Through Western-blot、Elisa、 RT-PCR detection, it shows that VEGF-NSCs can express VEGF, HRE-VEGF-NSCs also can express VEGF in hypoxia condition, HRE-VEGF-NSCs group express VEGF significantly higher in hypoxia condition than VEGF-NSCs group(P<0.05), and in the HRE-VEGF-NSCs group with hypoxia time prolonged, the increased amount of VEGF protein expression(P<0.05); MTT test shows that VEGF gene transfection can promote the proliferation of neural stem cells, VEGF gene with HRE promoter upstream transfection under anoxic conditions also can promote neural stem cell proliferation, and VEGF gene with HRE promoter can significantly promote the proliferation of neural stem cells in hypoxia conditions(P<0.05).Conclusion:successfully build high VEGF-expression gene engineering NSCs with hypoxia induced,prove that NSCs-VEGF gene engineering stem cells can secrete activited VEGF protein, promote neural stem cell proliferation, HRE can enhance the expression of VEGF gene in hypoxia condition, and HRE can regulation the expression of VEGF with controling time of hypoxia, finding a safe and efficient gene therapy method for hypoxic ischemic brain damage.