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Objective: To establish effective liposome transient transfection method, transfected with small interfering RNA (small interfering RNA, siRNA), silent rat neural stem cells (neural stem cells, NSCs) methyl sulfonic acid-sensitive protein 2 (Methyl Methanesulfonate Sensitive 2, MMS2) expression, and then explore the directed differentiation process MMS2 in angiotensin II (Angiotensin II, AII) induced NSCs to dopamine (Dopamine, DA) neurons. Methods: (1) in vitro isolated and cultured neonatal rat brain sources NSCs detected by immunocytochemistry specific marker of neuroepithelial stem cell protein ((neuroepithelial stem cell protein, Nestin) expression and identification of its multi-differentiation potential detection NSCs differentiated cells of glial fibrillary acidic protein (glial fibrillary a diet which protein, GFAP), neuron-specific enolase (neuron special enolase, NSE) and 2,3 - Central nucleotide di-lipase (cyclic nucleotide phosphohydrolase, CNP ) expression; (2) the second generation of NSCs experimental design divided into six groups: A: control group, B: AII group, C: AT1 receptor antagonist ZD7155 group D: AT1 receptor antagonist ZD7155 AII group E: AT2 receptor antagonist PD123319 group, F: AT2 receptor antagonist PD123319 AII group, by real-time quantitative PCR assay groups NSCs MMS2 mRNA expression; (3) design and chemical synthesis MMS2 gene sequence of siRNA molecules, cationic liposomes were transiently transfected NSCs, using real-time quantitative PCR detection NSCs MMS2 gene mRNA expression; (4) above experimental groups before and after transfection of NSCs induced differentiation as DA neurons, transfected cells were induced to differentiate cells in each group of tyrosine hydroxylase (tyrosine hydroxylase, TH) mRNA expression were detected by real-time quantitative PCR method. Results: (1) cells isolated and cultured in vitro, in the the NSCs dedicated medium was growth of suspended gathered formula, the cell mass expression of Nestin and differentiation of cells expressing GEAP, NSE and CNP cells isolated and cultured NSCs with multiple differentiation potential; (2) real-time PCR method to detect group B, D group cell the MMS2 gene expression level was significantly higher, respectively, 4.96 times and 3.58 times that of the control group, the difference was statistically significant (P lt; 0.05), while the C, E and F group MMS2 gene expression levels and the control group, the difference no three pairs of specific significant difference (P gt; 0.05); 3) MMS2-siRNA the MMS2 gene of rat NSCs silent 36h after transfection maximum inhibition; MMS2-siRNA sequences were effectively suppressed MMS2 gene expression of silent efficiency were the siRNA 2 34 (64 ± 6)% the siRNA 3 46 (84 ± 3)% the siRNA 4 16 (58 ± 5)%, compared with the blank control group, there were significant differences (P lt; 0.05); siRNA 3 46 the silence most efficient, can be used for subsequent experiments; (4) real-time PCR assay untransfected NSCs induced to differentiate for 10 days significantly increased in group B and group D TH gene expression levels, were 4.56 times and 3.41 times that of the control group, the difference was statistically significance (P lt; 0.05) , C, E, and F TH gene expression levels with the control group showed no significant difference (P gt; 0.05); MMS2-siRNA transfected NSCs 10 days after the induction of differentiation, TH gene expression levels of each experimental group compared to the control group, there was no significant difference in the sex difference (P gt; 0.05). Conclusion: (1) in vitro isolation and culture of neonatal rat brain cells NSCs biological characteristics; (2) siRNA molecules targeting MMS2 gene fragments can effectively suppress NSCs MMS2 gene expression; (3) AII AT2 receptor MMS2 elevated in NSCs expression and induce NSCs to DA neurons differentiation, MMS2 in AII induced NSCs to DA neurons directed differentiation process may play an important role.
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