中国卒中杂志
中國卒中雜誌
중국졸중잡지
CHINESE JOURNAL OF STROKE
2015年
2期
128-134
,共7页
生长相关蛋白%转基因小鼠%Gαi%细胞分裂平面%神经发育
生長相關蛋白%轉基因小鼠%Gαi%細胞分裂平麵%神經髮育
생장상관단백%전기인소서%Gαi%세포분렬평면%신경발육
Growth associated protein-43%Transgenic mice%Gαi%Orientation of cell division%Neurogenesis
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。
<br> 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（co-immunoprecipitation，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 cel，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。
<br> 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）。
<br> 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。
目的研究生長相關蛋白-43（growth associated protein-43，GAP-43）與G蛋白對神經細胞分裂方嚮的調控，探討GAP-43參與神經髮生的機製。
<br> 方法6隻C57BL／6J GAP-43高錶達的轉基因孕鼠（E13.5 d和E17.5 d的胎鼠均為24隻）為實驗組，6隻野生型孕鼠（E13.5 d和E17.5 d的胎鼠均為24隻）為對照組。分彆取E13.5 d的3隻實驗組及對照組胎鼠，腦室區進行免疫熒光染色測定GAP-43的錶達位置；再分彆取E13.5 d的12隻實驗組及對照組胎鼠，嚮腦中加入裂解液分彆提取膜蛋白和細胞質蛋白，通過Western blot測定GAP-43的錶達位置及錶達量；取E17.5 d的3隻GAP-43高錶達的轉基因胎鼠的鼠腦進行GAP-43和G蛋白的免疫熒光共染色，觀察二者的共定位情況；取E17.5 d的9隻GAP-43高錶達的轉基因胎鼠的鼠腦進行免疫共沉澱（co-immunoprecipitation，co-IP）檢測GAP-43與Gαi間的相互作用。取E13.5 d和E17.5 d的9隻實驗組胎鼠計數轉基因胚胎鼠腦中神經前體細胞（neuro progenitor cel，NPC）中沿水平和垂直不同方嚮分裂的細胞數量，併與對照組小鼠進行對照。
<br> 結果 E13.5 d實驗組胎鼠的GAP-43蛋白的錶達量高于對照組（P<0.05）；GAP-43特異性錶達于細胞膜上；免疫熒光共染色結果錶明GAP-43與Gαi共定位于細胞膜上，進一步通過co-IP證明GAP-43和Gαi相互結閤；E13.5 d時，轉基因組與對照組相比，細胞沿水平、中間和垂直角度分裂的細胞數量差異有顯著性（P<0.05）。
<br> 結論 GAP-43可調控神經細胞分裂方嚮，這可能是GAP-43參與神經髮生的機製。本研究為進一步研究GAP-43對卒中後神經損傷脩複的作用機製提供基礎。
목적연구생장상관단백-43（growth associated protein-43，GAP-43）여G단백대신경세포분렬방향적조공，탐토GAP-43삼여신경발생적궤제。
<br> 방법6지C57BL／6J GAP-43고표체적전기인잉서（E13.5 d화E17.5 d적태서균위24지）위실험조，6지야생형잉서（E13.5 d화E17.5 d적태서균위24지）위대조조。분별취E13.5 d적3지실험조급대조조태서，뇌실구진행면역형광염색측정GAP-43적표체위치；재분별취E13.5 d적12지실험조급대조조태서，향뇌중가입렬해액분별제취막단백화세포질단백，통과Western blot측정GAP-43적표체위치급표체량；취E17.5 d적3지GAP-43고표체적전기인태서적서뇌진행GAP-43화G단백적면역형광공염색，관찰이자적공정위정황；취E17.5 d적9지GAP-43고표체적전기인태서적서뇌진행면역공침정（co-immunoprecipitation，co-IP）검측GAP-43여Gαi간적상호작용。취E13.5 d화E17.5 d적9지실험조태서계수전기인배태서뇌중신경전체세포（neuro progenitor cel，NPC）중연수평화수직불동방향분렬적세포수량，병여대조조소서진행대조。
<br> 결과 E13.5 d실험조태서적GAP-43단백적표체량고우대조조（P<0.05）；GAP-43특이성표체우세포막상；면역형광공염색결과표명GAP-43여Gαi공정위우세포막상，진일보통과co-IP증명GAP-43화Gαi상호결합；E13.5 d시，전기인조여대조조상비，세포연수평、중간화수직각도분렬적세포수량차이유현저성（P<0.05）。
<br> 결론 GAP-43가조공신경세포분렬방향，저가능시GAP-43삼여신경발생적궤제。본연구위진일보연구GAP-43대졸중후신경손상수복적작용궤제제공기출。
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 inlfuence the orientation of cell division through interacting with G proteins.
<br> 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 immunolfuorescence 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.
<br> 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 immunolfuorescence 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 Gαi. The analysis of the exact angle of the dividing chromatics revealed a signiifcant difference in dividing cells in the transgenic group compared with the wild type group of E13.5 (P<0.05).
<br> Conclusion The GAP-43 is involved in the orientation of cell division through Gαi 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.
