生长相关蛋白-43影响神经细胞分裂方向作用的研究

Growth Associated Protein-43 Influences the Orientation of Cell Division in the Brain

目的研究生长相关蛋白-43(growth associated protein-43,GAP-43)与G蛋白对神经细胞分裂方向的调控,探讨GAP-43参与神经发生的机制。方法 6只C57BL/6J GAP-43高表达的转基因孕鼠(E13.5 d和E17.5 d的胎鼠均为24只)为实验组,6只野生型孕鼠(E13.5 d和E17.5 d的胎鼠均为24只)为对照组。分别取E13.5 d的3只实验组及对照组胎鼠,脑室区进行免疫荧光染色测定GAP-43的表达位置;再分别取E13.5 d的12只实验组及对照组胎鼠,向脑中加入裂解液分别提取膜蛋白和细胞质蛋白,通过Western blot测定GAP-43的表达位置及表达量;取E17.5 d的3只GAP-43高表达的转基因胎鼠的鼠脑进行GAP-43和G蛋白的免疫荧光共染色,观察二者的共定位情况;取E17.5 d的9只GAP-43高表达的转基因胎鼠的鼠脑进行免疫共沉淀(coimmunoprecipitation,co-IP)检测GAP-43与Gαi间的相互作用。取E13.5 d和E17.5 d的9只实验组胎鼠计数转基因胚胎鼠脑中神经前体细胞(neuro progenitor cell,NPC)中沿水平和垂直不同方向分裂的细胞数量,并与对照组小鼠进行对照。结果 E13.5 d实验组胎鼠的GAP-43蛋白的表达量高于对照组(P<0.05);GAP-43特异性表达于细胞膜上;免疫荧光共染色结果表明GAP-43与Gαi共定位于细胞膜上,进一步通过co-IP证明GAP-43和Gαi相互结合;E13.5 d时,转基因组与对照组相比,细胞沿水平、中间和垂直角度分裂的细胞数量差异有显著性(P<0.05)。结论 GAP-43可调控神经细胞分裂方向,这可能是GAP-43参与神经发生的机制。本研究为进一步研究GAP-43对卒中后神经损伤修复的作用机制提供基础。

Objective To study the mechanism of growth associated protein-43（GAP-43） involved in neurogenesis,in order to verify the hypothesis that GAP-43 may influence the orientation of cell division through interacting with G proteins.Methods The experiment was divided into two groups,the experimental and control groups.Six C57BL/6J GAP-43 transgenic pregnant mice（24 fetuses of E13.5 and E17.5 days） were as the experimental group,and six wild-type pregnant mice（24 fetuses of E13.5 and E17.5 days） were as the control group.The localization of GAP-43 was determined by immunofluorescence and Western blot,while the interaction of GAP-43 and Gαi were detected by co-immunoprecipitation（co-IP）.Furthermore,in order to compare the orientation of cell division in fetal brains of the transgenic mice with that in wild type mous,we measured the exact angle of the dividing chromatics in regard to the ventricular zone during neurogenesis.Results Respectively selected 24 embryonic mice of embryonic period of 13.5 and 17.5 in each group（transgenic and wild type groups）.The Western blot and immunofluorescence results showed that the expression of GAP-43 was higher in transgenic groups than those in wild type groups and was on the membrane in both two groups（P〈0.05）.The co-IP result showed that GAP-43 interacted with Gai.The analysis of the exact angle of the dividing chromatics revealed a significant difference in dividing cells in the transgenic group compared with the wild type group of E13.5（P〈0.05）.Conclusion The GAP-43 is involved in the orientation of cell division through Gai and this may be an important mechanism for neurogenesis in the mammalian brain.This study provides the foundation of the mechanism of GAP-43 in neurogenesis and stroke for further study.