Nurr1 Gene On The Differentiation Of Neural Stem Cells Into Dopaminergic Neurons

ObjectiveTo observe the effects of Nurrl gene in vitro on the dopaminergic neurons differentiation of neural stem cells derived from SD rat midbrain.Methods1. Cloning of rat Nurrl gene by gene synthesis method, and digested with the restriction enzyme, the cDNA were connected into pCDH-MCS-T2A-copGFP-MSCV plasmid vector and the recombinant pCDH-Nurrl were Successful constructed on the process of transformation、screening and identification.2. Neural stem cells were isolated and cultured from E14 SD rat midbrain. Immunofluorescence detection the neural stem cells of the embryonic origin and differentiation potential take P2 generation.3. The neural stem cells were divided into the natural differentiation group, transfected with empty plasmid vector pCDH group, recombinant plasmid vector pCDH-Nurrl group. And the expression of dopaminergic neurons specific marker TH、DAT and was determined by Western blot、Real-time PCR、 immunofluorescence.Results1. The successful construction of recombinant plasmid vector pCDH-Nurrl in SD rats.2. Neural stem cells can proliferate stably and continuously passage Isolated from E14 days SD rats brain, identified by immunofluorescence of Nestin are positive cell. After the identification, dentification of GFAP are all positive, which indicated the cultured cells were neural stem cells and has a ability of differentiation.3. The transfection conditions was observed under fluorescence microscope, ransfection gene 24h, most of the cells transfected with pCDH and recombinant plasmid pCDH-Nurrl transfection group were positive for copGFP expression, while the natural differentiation group cells showed noexpression of fluorescent protein Transfection of 48h, fluorescent protein expression peaked, plasmid pCDH transfection group and pCDH-Nurr1 transfection group and the transfection efficiency were 34.3% and 36.5%, there was no significant difference between the two groups.4. Western blot、real-time PCR、immunofluorescence showed that: the expression of TH、DAT,natural differentiation group, pCDH group, the difference was not significant, pCDH-Nurrl transfection group than the natural differentiation group, expression of pCDH group increased significantly.ConclusionOverexpression of Nurrl gene can promote neural stem cells to differentiate into dopaminergic neurons, provide a theoretical basis for further cell transplantation in the treatment of PD in vivo.