重组腺相关病毒转染神经干细胞球的实验研究  

THE EXPERIMENTAL STUDY OF rAAV2 TRANSFECTING NEURAL STEM CELLS-DRIVED NEUROSPHERES

作　　者：窦万臣[1] 王任直[1] 李桂林[1] 王欣[2] 李学坤[2] 张波[1] 田士强[1] 姚勇[1] 

机构地区：[1]中国医学科学院中国协和医科大学北京协和医院,北京100730 [2]中国医学科学院中国协和医科大学基础医学研究所,北京100730

出　　处：《中国应用生理学杂志》2004年第2期166-170,共5页Chinese Journal of Applied Physiology

摘　　要：目的 :探讨重组腺相关病毒 2型 (rAAV2 )对神经干细胞球的转染能力。方法 :①将FITC标记的rAAV2(FITC rAAV2 )分成两组 ,A组直接与神经干细胞球混合 ,B组与肝素混匀后再与神经干细胞球混合 ,孵育 30min后在荧光显微镜下观察 ;②含有GFP报告基因的rAAV2 (rAAV2 GFP)与神经干细胞球孵育 30min后 ,分成两组 :A组继续在培养箱内培养 ,B组分散成单细胞后移植到大鼠脑内 ,一个月后分别在荧光显微镜下观察神经干细胞球和大鼠脑组织切片中报告基因的表达情况 ;③将含有低氧启动子 (低氧应答元件 ,HRE)、VEGF和GFP的rAAV2(rAAV2 HRE VEGF GFP)转染神经干细胞球后分为两组 :A组在低氧条件下培养 ,B组在常规条件下培养 ,72h后观察报告基因的表达情况。结果 :①FITC rAAV2转染神经干细胞球的结果 :A组有明亮的绿色荧光 ,B组基本无绿色荧光 ;②rAAV2 GFP转染神经干细胞球后一个月 ,A、B两组均可以看到绿色荧光 ;③rAAV2 HRE VEGF GFP转染神经干细胞球后 72h ,A组可见绿色荧光 ,B组无绿色荧光。结论 :rAAV2可以与神经干细胞球特异性结合 ,rAAV2携带的外源基因在体内和体外均可以有效表达 ,rAAV2携带外源基因的表达可以人为调控。Aim: To investigate the abilities of recombinant adeno-associated virus type 2 (rAAV2) transfecting neurospheres. Methods: The rAAV2 conjugated with FITC (rAAV2-FITC) was added into the culture medium of neurospheres and 30 minutes later the neurospheres were detected with a fluorescence microscopy to determine if the AAV can combine with neurospheres. The rAAV2 containing GFP reporter gene (rAAV2-GFP) was incubated with the neurospheres for a month and then detected the ability of transfe- cting neurosphers. The neurospheres transfected with rAAV2-containing GFP were transplanted to the brain of rats. A month later the rats were sacrificed and the brains were removed to detect if there are expressions of the reporter gene. The neurospheres were transfected with rAAV2 containing hypoxia responds elements (HRE) and vascular endothelium growth factor(VEGF) gene and reporter gene GFP (rAAV2-HRE-VEGF-GFP) and then cultured in low oxygen density environments. Seventy-two hours later the neurospheres were detected through a fluorescence microscopy. Results: The neurospheres incubated with rAAV2-FITC present bright green fluorescence. GFP, the reporter gene, can be seen clearly 1 month after being transfected with rAAV2-GFP. The same green fluorescence protein can be observed ex vivo as well. The fluorescence can be seen in neurospheres transfected by rAAV2-HRE-VEGF-GFP only in low oxygen density. Conclusion: The rAAV2 can transfect neurospheres specifically and efficiently. Reporter gene can be expressed in the neurospheres in vivo and ex vivo. Expression of reporter gene can be adjusted by HRE.

关 键 词：RAAV2 转染 神经干细胞球 外源基因 

分 类 号：Q782[生物学—分子生物学]