胶质细胞系源性神经营养因子促进大鼠中脑多巴胺能神经元存活与分化的机制

THE INTRACELLULAR MECHANISMS FOR GDNF TO PROMOTE THE SURVIVAL AND DIFFERENTIATION OF MIDBRAIN DOPAMINERGIC NEURONS

目的探讨胶质细胞系源性神经营养因子(GDNF)促进中脑多巴胺(DA)能神经元存活和分化过程中,磷脂酰肌醇3激酶(P13K)信号通路和丝裂原活化蛋白激酶(MAPK)信号通路的可能作用。方法生后大鼠中脑脑片培养,并依培养基内加入的不同物质分为空白对照组、GDNF组、PI3K通路阻断组、MAPK通路阻断组。培养6 d后,进行酪氨酸羟化酶(TH)免疫组织化学染色,光镜观察,计算机辅助图像分析,统计学处理;同时用Westernblotting方法检测脑片中TH的表达。结果GDNF组TH表达阳性神经元的形状更趋向成熟,其TH表达阳性神经元的密度、胞体大小和TH的表达水平均显著高于空白对照组;P13K通路阻断组TH表达阳性神经元几乎消失,其TH表达水平显著低于GDNF组而与空白对照组无显著差别;MAPK通路阻断组TH表达阳性神经元的密度及TH表达水平与GDNF组无显著区别,但TH表达阳性神经元胞体显著小于GDNF组。结论P13K通路参与介导GDNF对DA能神经元的促存活作用,而MAPK通路参与介导其促形态学分化作用。

Objective To explore the possible roles of PI3K pathway and MAPK pathway in mediating the survival and differentiation promoting effect of glial cell line-derived neurotrophic factor （GDNF） on midbrain dopaminergic neurons. Methods Midbrain slices of early postnatal rats were cultured, the slices were divided into four groups according to the different substances added into the culture medium： i.e. blank control group, GDNF group, Wortmanin plus GDNF group, and PD98059 plus GDNF group. On the 6th day, some slices were fixed, embedded and sectioned. The sections were processed for tyrosine hydroxylase（TH） immunohistochemistry, and then examined under microscope to determine the morphologic index for statistic analysis （n = 6）. Meanwhile, some slices were taken for Western blot to examine the expression of TH protein in midbrain slices （n = 4）. Results GDNF group slices showed the morphologic maturity, the density and the diameter of TH positive neurons, as well as the level of TH expression, were all significantly higher than that of the control groups. The survival effect of GDNF on DA neurons was almost abolished when the PI3K pathway was blocked with Wortmannin; while the MAP kinase and ERK kinase （MEK） inhibitor PD 98059 was added to block the MAPK pathway, the diameter of TH positive neurons was decreased significantly. Conclusion The PI3K pathway might mediate the survival effect of GDNF, while the MAPK pathway seems to be involved in the differentiation process.