SHH-N基因腺相关病毒表达载体的构建及其对神经干细胞增殖相关基因的影响

基础医学与临床 ›› 2011, Vol. 31 ›› Issue (3): 291-296.

SHH-N基因腺相关病毒表达载体的构建及其对神经干细胞增殖相关基因的影响

刘东升¹,崔岩¹,申仑¹,杜延平¹,李桂林²,王任直²,王岩³,张波¹

1. 大连医科大学附属第一医院
2. 中国医学科学院北京协和医院神经外科
3. 清华大学玉泉医院

收稿日期:2010-08-23 修回日期:2010-12-21 出版日期:2011-03-05 发布日期:2011-03-14
通讯作者: 张波 E-mail:zhangbodl@126.com
基金资助:
大连市科技计划项目（2008J99JH268）;市自然科学基金

Construction and Identification of SHH-N Gene Adeno-associated Virus Vector and Its Effection on Genes Related to Proliferation In Neural Stem Cells

LIU Dong-sheng ¹,CUI_Yan ²,SHEN Lun ²,DU Yan-ping ²,Gui-lin LI³,Ren-zhi WANG³,WANG Yan ²,ZHANG Bo ¹

1. Dalian Medical University
2.
3. Dept. of Neurosurgery, PUMC Hospital, CAMS & PUMC

Received:2010-08-23 Revised:2010-12-21 Online:2011-03-05 Published:2011-03-14
Contact: ZHANG Bo E-mail:zhangbodl@126.com

摘要/Abstract

摘要： 目的构建rAAV-SHH-N–EGFP载体并检测其对神经干细胞增殖相关基因影响。方法分离并培养大鼠脑室下区神经干细胞，提取RNA、反转录、PCR得到SHH-N的cDNA，克隆入pSNAV2.0-CMV-IRES-EGFP，包装得到腺相关病毒rAAV-SHH-N – E GFP，感染神经干细胞48h后采用实时定量PCR检测SHH信号通路下游相关基因的mRNA水平变化，并观察rAAV-SHH-N-EGFP载体在神经干细胞内的表达情况。结果 (1)克隆得到SHH-N基因，测序结果与NCBI报道序列一致。(2)成功构建pSNAV2.0-CMV -SHH-N-IRES-EGFP表达载体并鉴定，包装得到rAAV-SHH-N-EGFP。(3) 实时定量PCR分析rAAV-SHH-N-EGFP感染神经干细胞48h后, rAAV-SHH-N-EGFP处理组较感染rAAV-EGFP的对照组，Gli1和Nmyc的mRNA水平上调。(4) rAAV -SHH-N-EGFP可有效感染神经干细胞，在感染14d后稳定表达SHH-N。结论成功构建rAAV-SHH-N-EGFP过表达载体并使其在神经干细胞内稳定表达。过表达SHH-N可使神经干细胞内SHH信号通路相关基因Gli1转录水平上调，并使细胞增殖相关基因Nmyc转录水平上调。

Abstract: Objective Construct and identify SHH-N gene adeno-associated virus vector and detect its effections on genes related to proliferation in neural stem cells(NSCs).Methods Isolated and cultured the neural stem cells in the subventricular zone of postnatal rat brain, SHH-N was gained by clone.pSNAV2.0-CMV-SHH-N-IRES-EGFP was established by using enzyme cutting and ligation , and then transfecting to packaging cell line 293T to acquire virus. Real time PCR was performed after SHH-N was infected neural stem cells by rAAV-SHH-N-EGFP vector for 48 hours, SHH-N gene expression of infected cells after 2 week was obversed by fluorescence microscope. Result (1)SHH-N gene was coincident with NCBI report.(2) pSNAV2.0-CMV-SHH-N-IRES-EGFP expression vector and rAAV-SHH-N-EGFP vector was successful established and packaged.(3) Real time PCR was performed after SHH-N infection for 48 hour, induction of Nmyc and Gli1 in rAAV-SHH-N-EGFP -treated group was enhanced compared to control group.Conclusion rAAV-SHH -N-EGFP vector had been successfully established and it can stably express in neural stem cells. Forced expression of SHH-N in NSCs resulted in enhanced induction of Nmyc and Gli1.

中图分类号:

刘东升崔岩申仑杜延平李桂林王任直王岩张波. SHH-N基因腺相关病毒表达载体的构建及其对神经干细胞增殖相关基因的影响 [J]. 基础医学与临床, 2011, 31(3): 291-296.

LIU Dong-sheng CUI_Yan SHEN Lun DU Yan-ping Gui-lin LI Ren-zhi WANG WANG Yan ZHANG Bo. Construction and Identification of SHH-N Gene Adeno-associated Virus Vector and Its Effection on Genes Related to Proliferation In Neural Stem Cells [J]. Basic & Clinical Medicine, 2011, 31(3): 291-296.