Experimental Study On Construction Of Recombinant Plasmid PAcGFP1-IRES2-Bcl2 And Its Expression In Neural Stem Cells

Numerous studies now confirm that apoptosis involved in variety of nervous system diseases (eg.stroke, traumatic brain injury) and related to secondary brain dysfunction chosely. B-cell lymphoma/leukemia-2(Bcl-2) as the major inhibitor protein of apoptosis, has a significantly protective effect on brain cells. To make exogenous Bcl-2 over-expressed in neural stem cells by using genetic engineering technology can enhance ability of neural stem cells to tolerate the adverse microenvironment under the position of transplantation,and it can inhibit the rate of apoptosis in order to improve the survival rate of neural stem cell after transplanting, also,to make a good foundation for the integration between neutral stem cells and peripheral nerve tissue and the recovery of nerve function, all of these are based on the maturity in transplantation of neural stem cells.The experiment was designed to build a Recombinant Plasmid pAcGFP1-IRES2-Bcl2 which contains Bcl-2 gene and green fluorescent protein (Aequorea coerulescens green fluorescent protein, AcGFPl), then transfect the plasmid into rat embryonic cortial neural stem cells (Cortex-Derived Neural Stem Cells, cNSCs),finally,to establish Bcl-2 gene and AcGFP1 gene modified cNSCs and to provide a foundation for the further research about neural stem cell transplantation therapy for stroke, traumatic brain injury and other central nervous system diseases.Objective:To construct recombinant plasmid pAcGFP1-IRES2-Bcl2 and to establish Bcl-2 gene and AcGFPl gene modified cNSCs.Methods:To extract total RNA from rat fetal brain,the coding sequence of Bcl-2 was amplified by RT-PCR from the total RNA.The Bcl-2 was choned into the pMD19-T Simple vector after purification.The recombinant clone plasmid pMD 19-Bcl2 was identified by gel electrophoresis and DNA sequencing,then the target genes were subcloned into the eukaryotic expression vector pIRES2-AcGFP 1 and identified with restriction enzyme digestion and DNA sequencing.To select pregnant SD rats within 13 days,neural stem cells were isolated from the cerebral cortex of fetal rat and incubated in DEMED/F12 culture medium,which contains FGF-2, EGF, N2 and B27.To identify the neural stem cells with immunofluorescence when they were transmitted to the third generation.To transfect recombinant expression plasmid pAcGFP1-IRES2-Bcl2 into NSCs by Lipofectamine 2000,then the cells were cultured in the medium which contains G418 for selecting. The expression of AcGFP1 was observed with fluorescence microscope, meanwhile the expression of Bcl-2 was analyzed by western blot.Results:Recombinant plasmid pAcGFP1-IRES2-Bcl2 was constructed successfully.The cells isolated from cerebral cortex of fetal rat grown steadily and kept the continuous passage. Immunofluorescence showed that the identification of Nestin tested positive. NSE, GFAP and Ga1C also tested positive after cytodifferentiation, indicating that cells had differentiated into neurons, glial stromal cells and oligodendrocyte cells.It demonstrated that cultured cells were neural stem cells. Green fluorescent protein expressed after 24 hours, as gene was transfected into neural cells.The results of Western-Blot showed that the Bcl-2 can be correctly expressed in the neural stem cells.Conclusion:It is successful to establish Bcl-2 gene and AcGFP1 gene modified cNSCs, providing a foundation for the further research about neural stem cell transplantation therapy for stroke, traumatic brain injury and other central nervous system diseases.