MMS2在血管紧张素II诱导神经干细胞向多巴胺能神经元分化过程中的作用

Effect of MMS2 in the process of angiotensin II induced neural stem cells differentiated to dopaminergic neurons

目的探讨甲基磺酸敏感蛋白2（MMS2）在血管紧张素II（AII）诱导神经干细胞（NSCs）向多巴胺（DA）能神经元定向分化过程中的作用。方法体外分离培养新生鼠大脑来源的NSCs，通过巢蛋白免疫细胞化学方法对神经前体细胞进行鉴定；在此基础上，将第2代的NSCs按实验设计分成6组：A：对照组；B：All组；C：ATl受体拮抗剂ZD7155组；D：ATI受体拮抗剂ZD7155＋AII组；E：AT2受体拮抗剂PD123319组；F：AT2受体拮抗剂PD123319＋AII组。通过实时荧光定量PCR检测各组细胞MMS2及THmRNA的表达；转染MMS2基因的小干扰RNA（siRNA）片段到NSCs上沉默MMS2在NSCs的表达，再按以上分组将其诱导分化为DA能神经元，通过实时荧光定量PCR方法检测转染后诱导分化的各组细胞THmRNA的表达。结果体外分离培养的NSCs，在培养基中呈悬浮生长，神经球表达Nestin。实时荧光定量PCR法检测B组、D组细胞的MMS2及THmRNA的表达高于对照组，其差异有统计学意义（P〈0．05）；C、E、F组细胞的MMS2及THmRNA的表达与对照组比较无统计学意义（P〉0．05），转染MMS2-siRNA后诱导分化的各组细胞间THmRNA的表达无统计学意义（P〉0．05）。结论AII通过AT2受体使MMS2在NSCs的表达升高并诱导NSCs向DA能神经元分化，MMS2在AII诱导NSCs向DA能神经元定向分化的过程中可能发挥重要作用。

Objective To explore the possible effects of methyl methanesulfonate sensitive 2 （MMS2） in the process of angiotensin II inducing differentiation of neural stem cells （NSCs） into dopaminegic phenotype neurons. Methods NSCs were isolated from the brain of newborn rats and were cultured in the serum-free medium. Identification of neural precursor cells was done by Nestin immunocyt ochemical staining. Then the second generation of NSCs was divided into the following six groups： A, control; B, AII; C, AT1 antagonist ZD7155; D, ZD7155＋AII; E, AT2 antagonist PD123319; F, PD123319＋AII. The detection of expression of MMS2 and TH mRNA level was done by real-time PCR. The silence of the expression of MMS2 in NSCs was brought about via the transfection of MMS2-siRNA, and then the NSCs were induced to differentiate into dopaminegic neurons. The expression of TH mRNA level in the cells of the groups after transfeetion was detected by real-time PCR. Results Nestin-positive cells were observed in suspended growth in the medium. Real-Time PCR revealed that the MMS2 and TH mRNA expression of group B and D were significantly higher than that of the control group（P〈0.05 ）, There was no significant difference in MMS2 and TH mRNA expression between group C, E, F and the control, respectively. Conclusion AII increased the expression of MMS2 mRNA in NSCs and induced the differentiation of NSCs into DA neurons via AT2 recepter. MMS2 may play important roles in the process of angiotensin II inducing NSCs to differentiate into dopaminergic neurons.