Experimental Study On Expression, Induced Differentiation And Neuronal Protective Effects To Glial Cells Of Rat Bone Marrow Stromal Stem Cells After NGF,BDNF Gene Transfection

Objectives: To research the different effects of the rat bone marrow stromal cells (MSCs) differentiation after NGF and BDNF gene transfection, and the protection of MSCs as seeding cells to the apoptotic astrocyte cells induced by hypoxic cultivation. We also studied the optimal condition of the MSCs cultivation and the mechanism of neurotrophic factors inducing its differention and the protective effects after those genes transfection. To provide an alternative road to the treatment and functional reconvertion of the secondary nervous system injury.Methods: MSCs was isolated from bone marrow of SD rat by density gradient centrifugation and primary cultured in DMEM. They were passaged two times by being detached and were characterized in SH3(+). On the base of the previous construction of minigenes in DNA Recombination Lab., utilizing pSVCEP-NGF-CAT or/and pSVCEP-BDNF-CAT minigenes and containing the regulatory elements of human α 1 (1) collagen(COLIA1) and NGF/BDNF cDNA, were separately extracted with 'Lysis by Alkali' methods and digested with restriction enzymes EcoRI/HindIII, Sal I/ HindIIIand HindIII /BamHI. They transfect into the 1st passage MSCs with lipofectAMINE?, meanwhile empty-carrier pSVCEP-CAT and pEGFP-C, transfected MSCs as negative control and marker of detecting transfection efficiency respectively. Then to select the positive cells clone with G418, detect NGF and BDNF expression in culture medium at 2 days, 2 weeks and 4 weeks after gene transfection with directly ELISA analysis and in MSCs with in situ hybridization method. The MSCs can express Nestin, NSE, NF and GFAP, which are the markers of the neural precursor cells, were obtained with a laser scanning confocal microscope. The astrocytes were isolated, purified and primary cultured from neonatal SD rat. They were stained immunocytochemically positively with GFAP antiboby. The MSCs after NGF and BDNF genes transfection were co-cultured with the apoptic astrocytes induced by hypoxic cultivation for 1 and 2 weeks. All of the cells were detected the Caspase-3 and Fas antigens with immunocytochemistry technique, meanwhile detected the apoptotic and necrotic cells with flow cytometry.Results: The purity of MSCs can be over 85% after isolation. The MSCs can express SH3. The plasmids were identified to contain the 390bp β -NGF and 760bp BDNF cDNA inserts.It was proved that these foreign genes were expressed in the transformed bacterial cell JM109 induced with IPTG respectively. The ratio of gene transfection is 33.3%. The cell-toxic concentration of G418 is 200μg/ml. All of the culture mediums were detected NGF and BDNF. The MSCs were positive for the neuron-specific markers Nestin, NF and NSE , also for the astroglial cells-specific GFAP. The result of co-transfection group is the most remarkable. The purity of astrocyte canbe over 95% after isolation. The astrocyte can express GFAP and be induced apoptosis after hypoxic culture for 24 hours. Treatment of the co-culture with apoptotic astrocytes and MSCs resulted in decreased numbers of apoptotic and necrotic cells and reduced expression of Caspase-3 and Fas proteins.Conclusions: (1) Analysis proved that NGF/BDNF gene driven by the promoter(-332~+98) and enhancer(+544~+855) elements of human α 1 (I) collagen (COCIA1) is ectopic-expressed effectively in MSCs mediated by lipofectin. (2) MSCs are capable of differentiating into neuronal precursor cells that express several neural proteins and resemble immature neurons or glial cells by the induction of neurotrophic factor. (3) The MSCs after lipofectin-mediated neurotrophic factor genes transfection have protective effect to the apoptotic astrocyte. (4) NGF and BDNF genes have a combinable and cooperative effect of inducing differentiation to the MSCs.