携带酪氨酸羟化酶基因的质粒和逆转录病毒在大鼠成纤维细胞和脑胶质细胞中表达效率的比较  

THE EXPRESSION EFFICIENCY OF THE TRANSGENE, TYROSINE HYDROXYLASE, MEDIATED BY PLASMID AND RETROVIRUS IN PRIMARY FIBROBLIASTS AND GLIAL CELLS OF THE RAT

作　　者：陈晴[1] 段德义[1] 杨慧[1] 孙成三[1] 陈立南[1] 徐群渊[1] 

机构地区：[1]首都医科大学北京神经科学研究所北京市神经再生修复研究重点研究室,北京100054

出　　处：《解剖学报》2005年第1期76-80,共5页Acta Anatomica Sinica

摘　　要：目的　为提高体外转基因 (exvivo)治疗帕金森病大鼠模型的疗效 ,本实验拟比较两种基因表达载体介导的外源基因在两种不同运载细胞的瞬时表达效率。　方法　分别将酪氨酸羟化酶 (TH)基因的重组表达质粒和重组逆转录病毒导入原代培养的大鼠成纤维细胞和脑胶质细胞 ,用免疫组织化学检测TH的表达效率 ;Westernblot法检测样品中TH特异性条带并转换成A值 ,以比较其在不同细胞中表达的相对含量。　结果　免疫组织化学检测表明 ,表达质粒的转染率为 5 %～ 7% ,逆转率病毒的感染率为 18%～ 2 0 % ;根据GDNF标准品蛋白条带含量与Westernblot结果的相应A值的标准曲线 ,推断出质粒转染和逆转率病毒感染的成纤维细胞中单个阳性细胞表达的TH相对平均含量分别为 4 76× 10 - 2 A和 4 35× 10 - 2 A ,质粒转染和逆转率病毒感染的脑胶质细胞分别是 5 4 5×10 - 2 A和 5 0 5× 10 - 2 A。　结论　不论是质粒载体还是逆转录病毒载体所携带的外源基因在两种细胞的单个阳性细胞中的表达无显著性差异。但由于逆转录病毒载体的感染率高于质粒载体 ,对于原代培养的成纤维细胞和脑胶质细胞 ,逆转率病毒载体是一种更好的表达载体。Objective Two types of cells transfered by two different types of vectors containing gene of interest were used to compare their expression efficiency in vitro, in order to provide an effective protocol for ex vivo gene therapy in a rat model of Parkinson disease. Methods The recombinant plasmid and retrovirus containing tyrosine hydroxylase (TH) gene were transferred into primary rat fibroblasts and glial cells respectively. The expression efficiency of TH in vitro was detected by immunocytochemistry and Western bloting. Results The transfection efficiency of the plasmid vector is 5%-7% while the transduction efficiency of retrovirus is 18%-20%. The relative quantities of TH protein in every single positive cell are: 4.76×10 -2 A (plasmid, fibroblast), 4.35×10 -2 A (retrovirus, fibroblast), 5.45×10 -2 A (plasmid, glial cell) and 5.05×10 -2 A (retrovirus, glial cell).Conclusion There were no significant differences in the levels of transient expression (48 hours) of TH protein in different cells transferred with different vectors. The retrovirus, however, might be a better expression system for the primary fibroblasts and glial cells compared with plasmid, since it showed to have a much higher transduction efficiency.

关 键 词：帕金森病 酪氨酸羟化酶 质粒载体 逆转录病毒载体 成纤维细胞 脑胶质细胞 

分 类 号：Q786[生物学—分子生物学]