腺病毒载体介导的GFP在鸡胚成纤维细胞中的表达及RNA干涉  

The expression and RNAi of GFP gene adenovirus vector mediated in chick embryo fibroblast cells

作　　者：傅安静[1] 汤承[1] 李定霏[2] 谢青[3] 岳华[1] 

机构地区：[1]西南民族大学生命科学与技术学院 [2]成都市农林科学院水产研究所,四川成都610072 [3]四川省兽药监察所,四川成都610041

出　　处：《西南民族大学学报（自然科学版）》2008年第2期276-280,共5页Journal of Southwest Minzu University(Natural Science Edition)

基　　金："十一五"国家科技支撑计划重大项目资助(2006BAD06A08);四川省应用基础资助项目(No.05JY029-007-5)

摘　　要：鸡胚成纤维细胞(CEF)是多种动物及人类病毒的易感细胞;也是研究鸡的基因组功能的理想材料.本试验的目的是研究非复制型腺病毒载体对CEF的转染效率及安全性;并建立在CEF细胞中进行RNAi的技术平台.采用GFP重组非复制型腺病毒载体(Ade-GFP)转染CEF细胞;结果证明0.1-2000 MOI Adv-GFP均可转染CEF细胞并表达GFP;转染后16h开始出现GFP阳性细胞;胞核与胞浆可见明亮的荧光;荧光细胞数量及荧光强度在24～36h达到高峰;以后逐渐衰减;约180h全部消失;转染效率最高为22.3%;形态学观察及流式细胞仪测定结果显示;Adv-GFP转染的CEF细胞未见细胞病变;多数转染组细胞活力不受影响;说明用Adv-GFP携带外源转染CEF细胞是安全可行的;用脂质体转染试剂转染化学合成的GFP-siRNA;成功地干涉了腺病毒载体介导的GFP基因在鸡胚成纤维细胞的表达;干涉效率为85%;证明了鸡胚成纤维细胞中存在RNAi机制;为进一步利用非复制型腺病毒载体递送siRNA在CEF细胞内进行RNAi的研究奠定了基础.Adenovirus vectors, expressing siRNAs in cell are the alternative delivery methods, which could block the target gene expression for longer time than that siRNAs transfected directly. To evaluate the transfect efficiency of replication deficiency adenovirus vectors and establish the RNA interference （RNAi） technical platform in chick embryo fibroblast （CEF）, the transfeet efficiency of GFP recombined adenovirus vector （adv-GFP） mediated in CEF cells is evaluated in this study. The results show that GFP gene expression is observed in CEF cells, with the delivery of adv-GFP from 0.1 - 1000 MOI. GFP positive cells are seen 16h post infection. GFP presents both in caryon and cytoplasm. The numbers of GFP positive cells and brightness of fluorescence reacted peak value during 24 - 36h ,decayed subsequently and disappeared in about 180h. The highest transfect efficiency, 22.3%, is achieved with 1 MOI adv-GFP delivery. The results in this experiment show that adv-GFP is safe and feasible to transfect CEF cells with no cytopathy and cell activity lost. GFP gene expression mediated by adv-GFP in CEF is repressed observably, with 85% interference efficiency, by synthesized anti-GFP siRNA transfected with cationic liposome reagent. We find that the RNAi mechanism is present in CEF cells like in other type cell. RNAi technique platform in CEF cells is established successfully in this study.

关 键 词：RNA干涉 腺病毒载体 鸡胚成纤维细胞 绿荧光蛋白 

分 类 号：Q81[生物学—生物工程]