Construction Of Adenovirus-mediated Ad-BDNF-EGF Vector And Its Expressing In NSCs

With the development of traffic and construction in our modem society, spinalcord injury (SCI) is commonly encountered in clinical. The number of patients withSCI is significantly increasing every day Traumatic spinal cord injury can result insevere damage, leading to paraplegia, or worse. Many strategies, including surgical,pharmacological, have been used in attempts to develop new therapies that will allowpatients to regain the use of their paralyzed limbs. One new strategy is neural stemcell transplantation into the damaged spinal cord. The cell transplanted canreplacement of damaged intemeur0ns within a structure, promote the functionalreconstruction of neuronal circuits, and recovery the function of spinal cord. Thepresent research is, through biomolecpiar technique, to constitute adenoviralvector integrated BDNF,then to infect the neural stem cells in vitro, and induce itsdifferentiation, Which provide new evidence to the clinical application of the spinalNSCs.Part One: Culture of Neural Stem Cell from spinal cord of SD Fetal ratand its differentiationObjective To study isolation and culture of Neural Stem Cells from spinal cordof SD Fetal rat and investigate their characters of self-renewing and multipotency.Methods NSCs were induced with bFGF from spinal cord 12-14d SD Fetal ratSurvival and growth of these cells were observed after plating.The proliferation ofthese cells was investigated by Brdu. At the same time the undifferentiated state anddifferentiating fates of these cells were evaluated by inmnunocytochemistry.Results Primary spheres were nestin positive. We got secondary spheres fromsingle cell colonel culture. And parts of these cells had entered cell cycle stagesindicated by Bradu staining when differention was induced by removal of the mitoticfactors, the stem cells differentiated into neurons (MAP-2immunpostive),astrocytes(GFAP immunorective).Conclusion Multipotent neural stem cells can be isolated from spinal cord of SD Fetal rat and cultured and these cells are capable of proliferation and differentiationinto neurons, astrocytes and oligodendrocytes, so the cells we cultured have thecharacter of NSCs and lay a stable foundation for our following experiment.Key Words: neural stem cells, bFGF, Fetal rat, Immunocytochemistry.Part two: The transfection of gene BDNF and EGFP into Neural Stem Cellsand its expressingObjective TO transfection NeuralStem Cells with adenovirus vector of BDNFand EGFP, and also investigate the expression of BDNF and EGFP in transfectedneural stem cells.Methods The BDNF cDNA was gained by RT-PCR from rat hippocampus Afterpackaged by HEK293.BDNF and EGFP gene was transfected into cultured neuralstem cells through pAd-EGFP-BDNE The neural stem cells with BDNF cDNAgene was examined by RT-PCR and immunoreactivity methods The result of RT-PCRwas examined. NSCs clones with BDNF gene were successfully selected by G418.Results BDNF and EGFP could be expressed in transfected neural stem cells for along time, The neural stem cells infected by adenovirus were incubated.G418resistant clones of NSCs gave off strikingly bright green fluorescence, and showedstable EGFP expression detected by immunohistochemistry staining. The quality ofBDNF was analyzed by RT-PCR and the quantity of it was detected by ELISA Theresult of RT-PCR was the same as Genbank. regenerate. It was confirmed that infectedNSC could express BDNF. The BDNF cumulant could arrive to 12.68ng/ml at 72h.Conclusion The EGFP and BDNF gene can be transfected into neural stem cells,and also can be expressed for a long time. So, EGFP is one of favorable tracers in thestudy of NSCS transplantation. Adenovirus mediated EGFP gene and BDNF genetransferring the NSCs and expressing of EGFP and BDNF gene successfully indicatethat with the development of gene technology and molecular biology, NSCs can beused as target cells of gene transfer and therapy the CNS disease by transplantation.