【作者】 孙念峰；

【导师】 王国斌；

【作者基本信息】 华中科技大学 ， 外科学， 2007， 博士

【摘要】 目的:构建大鼠胶质细胞源性营养因子(GDNF)及内皮素B受体(EDNRB)基因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Ad-Easy腺病毒表达系统构建GDNF及EDNRB基因共表达腺病毒PAd-GE,体外感染NSC后通过观察绿色荧光蛋白表达及RT-PCR、实时荧光定量PCR.流式细胞仪法鉴定外源基因的表达。结果:NSC经重组腺病毒PAd-GE感染后24h可观察到绿色荧光蛋白(GFP)表达,RT-PCR、实时荧光定量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。更多还原

【Abstract】 Objective To construct the recombinant adenovirus bearing glialcellline derived neurotrophic factor (GDNF) and endothelin receptor B (EDNRB) gene and investigate its coexpression in neural stem cells. Method The AdEasy system was used to gene vate the recombinant Adenovirus Ad-GE coexpressing GDNF and EDNRB gene . The expression of exogenous genes in neural stem cells after infection was determine serving the expression of the green fluorescence protein(GFP) and detected by reverse transcription polymerrase chain reaction (RT-PCR) method and real-time fluorogenic quantitative PCR. Result 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 fluorogenic quantitative PCR. Conclusion Exogenous GDNF and EDNRB gene can be coexpressed in neural stem cells , which may offer the possibility of a novel approach to local combination gene therapy of Hirschsprung disease .更多还原