血管紧张素II体外诱导神经干细胞分化为多巴胺能神经元的机制研究

The mechanism study of angiotensin II induces Neural stem cells differentiated into dopaminergic neurons

目的探讨血管紧张素II(AngiotensinII,AII)诱导神经干细胞(Neural stem cells,NSCs)定向分化为多巴胺(Dopamine,DA)能神经元的过程中血管紧张素1型(AT1)受体和血管紧张素2型(AT2)受体所起的作用。方法在无血清培养基中分离培养NSCs,通过巢蛋白(nestin)免疫细胞化学对神经前体细胞进行鉴定;在此基础上,将第二代的NSCs在含10%胎牛血清的培养基中按照实验设计分为6组,分别是,A:对照组,B:AII组,C:AT1受体拮抗剂ZD7155组,D:AT1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,观察NSCs向DA能神经元定向诱导分化情况。通过酪氨酸羟化酶(TH)免疫细胞化学进行DA能神经元鉴定,并观察各组DA能神经元突触的长度变化,通过实时荧光定量RT-PCR检测各实验组中TH基因表达水平。结果细胞团中可以看到nestin免疫阳性着色,实时荧光定量RT-PCR法检测B组、D组的TH基因表达水平高于对照组,差异有统计学意义(P<0.05),C、E、F组的TH基因表达水平与对照组比较无差异(P>0.05),各组DA能神经元的突触长度没有明显差别(P>0.05)。结论AII诱导产生DA能神经元的作用是由AT2受体介导,激活AT1、AT2受体均不能诱导DA能神经元突触延长或抑制突触延长。

Objective To explore the function of angiotensin type 1 and type 2（AT1 and AT2）recepters in the possible effects of angiotensin II induced the differentiation of dopaminegic phenotype neurons from neural stem cells（NSCs）. Methods NSCs were cultured in the serum-free medium. NSCs were determined by Nestin immunocytochemical staining. On this basis,we difined 6 groups,A,control;B,AII;C,AT1 antagonist ZD7155;D,ZD7155＋AII;E,AT2 antagonist PD123319;F,PD123319＋AII. Differentiated cells were detemined by TH immunocytochemical staining to investigate the process length and real-Time PCR to detect the expression of TH mRNA level. Results We could find Nestin-positive cells. Real-Time PCR revealed that the TH mRNA expression of group B and D were significantly higher than the control group（P0.05）. There was no significant difference in groups of process length of TH＋ positive cells. Conclusion AII induced the differentiation of NSCs into DA neurons via AT2 recepter. AT1/AT2 recepter did not involve in the increasing of the length process of TH＋ positive cells.