重组hTRAIL腺病毒载体的构建及其体外抗肿瘤效应的初步观察  

Construction of Ad.hTRAIL and preliminary study on its in vitro anti-tumor effect

作　　者：张瑞萍[1] 云琳[2] 金增强[3] 倪海东[1] 樊克兴[1] 周倩[1] 郭亚军[1] 

机构地区：[1]第二军医大学基础医学部国际合作肿瘤研究所,上海200433 [2]郑州铁路职业技术学院,郑州450052 [3]第二军医大学长征医院口腔科,上海200003

出　　处：《第二军医大学学报》2005年第10期1144-1147,共4页Academic Journal of Second Military Medical University

基　　金：国家杰出青年科学基金(39925021)

摘　　要：目的:利用AdEasy载体系统构建含人肿瘤坏死因子相关凋亡诱导配体(TNF-related apoptosis inducing ligand,TRAIL)全长基因的重组腺病毒载体,并初步观察其体外对肿瘤细胞的杀伤作用。方法:利用RTPCR法从人外周血单个核细胞(PBMCs)克隆人TRAIL全长基因。将hTRAIL cDNA克隆入穿梭质粒pAdTrack CMV中,与腺病毒骨架结构pAdEasy1共同转化入大肠杆菌BJ5183中进行同源重组,重组子经酶切鉴定正确后,用脂质体转染入人胚肾293细胞中,包装获得重组腺病毒颗粒,Westernblot检测TRAIL蛋白的表达。将重组腺病毒颗粒体外感染对TRAIL敏感的3种人肿瘤细胞(淋巴瘤细胞Jurkat、前列腺癌细胞PC3、宫颈癌细胞HeLa)后,进行AnnexinⅤFITC/PI染色流式细胞术检测、细胞基因组的DNA电泳和锥虫蓝染色,以观察TRAIL对肿瘤细胞的杀伤作用。结果:利用RTPCR法从人PBMCs中扩增出843bp的cDNA片段,测序证实为人TRAIL基因。重组子经酶切鉴定正确,Western印迹证实感染TRAIL腺病毒的293细胞中有TRAIL蛋白的正确表达。将重组腺病毒颗粒体外感染HeLa、PC3和Jurkat细胞,4h后用AnnexinⅤFITC/PI染色,流式细胞术检测AnnexinⅤ+PI-细胞群分别占(41.15±3.12)%、(46.20±4.07)%、(38.56±3.45)%;24h后,抽提Jurkat细胞的基因组进行DNA电泳,出现细胞凋亡时特有的DNA条带;36h后用锥虫蓝对HeLa、PC3、Jurkat细胞染色,细胞死亡率分别为(75±5)%、(82±7)%、(70±5)%。结论:成功构建了hTRAIL腺病毒载体,体外感染HeLa、PC3、Jurkat肿瘤细胞后,能通过诱导细胞的凋亡而杀伤肿瘤细胞;为下一步的基因治疗和基础研究打下了基础。Objective：To clone hTRAIL full-length cDNA and construct its recombinant adenovirus by AdEasy vector system, and to explore the anti-tumor effect of Ad. hTRAII, in vitro. Methods, hTRAIL cDNA was amplified by RT-PCR from peripheral blood mononuclear cells （PBMCs） and cloned into shuttle vector pAdTrack-CMV; the product was subsequently cotransformed into E. coli BJ5183 with an adenovirus backbone plasmid pAdEasy-1; then the recombinant adenoviral vector was generated by homologous recombination. After confirmed by restriction endonuclease analysis, the linearized recombinant plas mid was transfected into 293 cells by lipofectamine. The hTRAIL recombinant adenovirus was verified by Western blot. The anti-tumor effects of Ad. hTRAII, on 3 sensitive tumor cell lines HeLa, PC-3 and Jurkat were observed by Annexin V-FITC/PI staining, DNA electrophoresis and trypan blue exclusion. Results： An 843 bp cDNA was amplified by RT-PCR from PBMCs, and its sequence was confirmed as expected. Western blotting indicated that hTRAIL recombinant adenovirus vector was successfully constructed. Flow cytometry showed that the percentages of AnnexinV- PI- cells on HeLa, PC-3 and Jurkat were （41. 15±3.12）%,（46.20±4.07）% and （38. 56±3.45）%, respectively. Genome DNA electrophoresis of Jurkat cells showed the characteristic DNA ladder of apoptosis. Trypan blue exclusion displayed that cell death rate of HeLa,PC-3 and Jurkat were respectively（75±5）%, （82± 7）% and （70±5）% 36 h after infected with Ad. hTRAIL. Conclusion： Human hTRAIL gene has been cloned and its recombinant adenovirus（ Ad. hTRAIL） has been constructed. Ad. hTRAIL can kill tumor cells in vitro through induction of apoptosis, which provides a basis for gene therapy and basic study of tumors.

关 键 词：人肿瘤坏死因子相关凋亡诱导配体基因 腺病毒载体 凋亡 肿瘤 

分 类 号：R73-362[医药卫生—肿瘤]