Nf-¦Êb In The Bridging Role Of Hif-1¦Á Genetically Modified Nscs Increases The Expression Of Vegf

Objective: To study the NF-κB in HIF-1αincreases the expression of VEGF pathway role.Methods: Extraction the tissue of 24-hour neonatal rat hippocampus,after digestion by the machinery method,in the absence of serum, including growth factor and cell auxiliaries, as well as the growth of low cell density environment and cultivate passaged cells purified single-cell cloning and identification of NSCs. Cultured HEK293 cells, amplified AdHIF-1α- GFP and empty vector Ad-GFP, and fluorescence identification, and infectious virus titer determination plural. Through adenovirus transfection method HIF-1αgene transfer into neural stem cells, and sub-group for the NSCs, NSCs after gene transfection group, empty vector transfected NSCs group. Immunofluorescence observation and munohistochemical staining and Western blotting assay transfected gene expression in NSCs. Immunohistochemical staining and Western blotting method to detect gene transfected NSCs in VEGF and NF-κB expression and detection given the concentration of NF-κB Ossetia specific inhibitor PDTC after AdHIF-1α- GFP modified NSCs in VEGF expression, and the results were statistically analyzed.Main Results:1, from the son of rat hippocampal cells were isolated from primary and cultured to obtain high purity of neural stem cells can form cell clones, nestin-positive staining confirmed with in vitro proliferation and differentiation of many potential tropism of neural stem cells, and when passaged NSCs improved digestion method.2, AdHIF-1α- GFP and Ad-GFP in HEK293 cells as the carrier amplification, the fluorescence microscope to observe the majority of cells have bright green fluorescence, fluorescence-positive cell count and MTT titer determination of recombinant adenovirus for 1×108pfu / ml, to determine the plural of infection (MOI) value of 50:1, when the MOI value of more than 50:1, the obvious increase in transfection efficiency, compared with 75:1 and 100:1 basic peak (P <0.01).3, AdHIF-1α- GFP and Ad-GFP fluorescence after transfection NSCs can be directly observed under the microscope to the issue of green fluorescent cells; fluorescence expression continued for more than 30 days, indicating that AdHIF-1α- GFP and Ad-GFP transfected neurons effectively stem cells, and can continue to express fluorescent, NSCs are good exogenous gene vector, and the strength of gene expression obtained with the MOI and transfection time. Immunohistochemistry, Western blotting showed that transfection AdHIF-1α- GFP in NSCs after significant HIF-1αexpression, normal NSCs group and empty vector expression group, indicating successful transfection. And non-transfection group compared to transfection AdHIF-1α- GFP gene in neural stem cell activity increased, and easy to divide.4, by immunohistochemistry, Western blotting showed that transfection AdHIF-1α- GFP after neural stem cells in the VEGF and NF-κB expression was significantly higher than normal NSCs group and empty vector group (P <0.05), and both the expression of There was a positive correlation; given concentration Ossetia specific NF-κB inhibitors PDTC after AdHIF-1α- GFP-modified neural stem cells in the VEGF expression was concentration-dependent reduction, the concentration of VEGF expression between the groups were significantly different (P <0.05) .Conclusion:1, Passage of NSCs when improved methods of cell digestion, and enhance cell viability, is conducive to cell proliferation.2,The one that HIF-1αgene transfected NSCs in the NF-κB and VEGF expression are up-regulated the three was a positive correlation.3, for HIF-1αmodified NSCs different concentrations of NF-κB inhibitor PDTC after, VEGF expression was concentration-dependent reduction.4,NF-κB in HIF-1αmodified NSCs increases the expression of VEGF plays a bridging role.