Effect Of MMS2 In The Process Of Angiotensin Ⅱ Induced Neural Stem Cells Differentiated To Dopaminergic Neurons

Objective:To transfect Methyl Methanesulfonate Sensitive 2(MMS2) siRNA into rat neural stem cells (NSCs) by liposome transfection and to investigate its inhibitory effect, and then to explore the function of MMS2 in the possible effects of angiotensin II induced the differentiation of dopaminegic phenotype neurons from NSCs.Methods :(1) NSCs isolated from the brain of newborn rats were cultured in the serum-free medium. Immunocytochemical technique was applied to detect the expression of neuroepithelial stem cell protein(Nestin), glial fibrillary acidic protein(GFAP), neuron special enolase(NSE) and cyclic nucleotide phosphohydrolase(CNP); (2) To divide the second generation of NSCs into the following six groups: A,control; B,AII; C,AT1 antagonist ( ZD7155 ) ; D,ZD7155+AII; E,AT2 antagonist (PD123319); F,PD123319+AII. To detect the expression of MMS2 mRNA level by Real-Time PCR; (3) Three siRNAs were designed and synthesized according to the mRNA sequence of rat MMS2 gene, and transfected into NSCs in culture by Cationic liposomes. To assess the efficiency of siRNA knockdown, the gene expression of MMS2 was detected by real-time PCR; (4) According to the above experiment groups, the NSCs including MMS2-undeleted and MMS2-deleted were cultured in the medium with serum to differentiate into dopaminegic neurons, then the real-time PCR was applied to detect the expression of TH mRNA level in these differentiated cells.Result:(1) We could find Nestin-positive cells and the differentiated cells were detected GFAP,NSE,CNP positive,demonstrated the multi-direction differentiation potency of NSCs;(2) Real-Time PCR revealed that the MMS2 mRNA expression of group B and D were significantly higher than the control group(P < 0.05);(3) The maximum inhibitory effects of MMS2-siRNA transfection on MMS2 expression was at 36h.The MMS2 gene was knocked down by the three siRNAs in different degrees. siRNA₃46 was the most effective siRNA,and could be used for the subsequent experiment;(4) Real-Time PCR revealed that the TH mRNA expression of group B and D differentiated from MMS2-undeleted NSCs were significantly higher than the control group(P<0.05),there was no significant difference in groups of TH mRNA expression in the cells differentiated from MMS2-deleted NSCs.Conclusion:(1)The cells isolated from the brain of newborn rats have biological characteristics of NSCs;(2) The MMS2 gene in rat NSCs can be knocked down effectively by RNAi;(3) AII increased the expression of MMS2 mRNA of NSCs and induced the differentiation of NSCs into DA neurons via AT2 recepter, MMS2 may play an important role in the process of angiotensin II induced NSCs differentiating into dopaminegic neurons.