大鼠胶质细胞源性营养因子及内皮素B受体基因共重组腺病毒的构建及表达  

Construction of recombinatant adenovirus carrying GDNF and EDNRB gene and its co-expression in neural stem cells

作　　者：孙念峰[1] 舒晓刚[1] 张景辉[1] 陈景波[1] 王国斌[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院普外科,武汉430022

出　　处：《中华小儿外科杂志》2006年第12期654-658,共5页Chinese Journal of Pediatric Surgery

基　　金：国家自然基金(编号:30371397)

摘　　要：目的构建胶质细胞源性营养因子(GDNF)及内皮素B受体基因(EDNRB)共表达腺病毒并观察其在神经干细胞(NSC)中的表达。方法利用AdEasy腺病毒表达系统构建GDNF及EDNRB基因共表达腺病毒Ad-GE,体外感染NSC后通过观察绿色荧光蛋白表达及RT-PCR、流式细胞仪法鉴定外源基因的表达。结果NSC经Ad-GE感染后24h可观察到绿色荧光蛋白(GFP)表达,RT-PCR、流式细胞仪法证实外源性GDNF及EDNRB基因均可在NSC中表达。结论成功构建GDNF及EDNRB基因共表达重组腺病毒,证实了其在NSC的表达,为先天性巨结肠的联合基因治疗打下基础。Objective To construct the recombinant adenovirus expressing the glial cell line derived neurotrophic factor （GDNF） and endothelin receptor B （EDNRB） gene and investigate its coexpression in neural stem cells. Methods The AdEasy system was used to generate the recombinant Adenovirus Ad-GE coexpressing GDNF and EDNRB gene. The expression of exogenous genes in neural stem cells after infection was determined with the green fluorescence protein （GFP） expression and confirmed by reverse transcription polymerrase chain reaction （RT-PCR） method and real-time PCR. Results GFP expression could be observed under fluorescent microscope 24h later after infection and coexpression of GDNF and EDNRB gene was confirmed by RT-PCR method and real-time PCR. Conclusions Exogenous GDNF and EDNRB gene can be over-expressed in neural stem cells. This offers the possibility of a novel approach to local combination gene therapy of Hirschsprung disease.

关 键 词：干细胞 DNA 重组 腺病毒 

分 类 号：R346[医药卫生—基础医学] R726.5