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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 construct a recombinant adenovirus vector containing the full length of TNF-related apoptosis inducing ligand (TRAIL) by using AdEasy vector system and to observe its killing effect on tumor cells in vitro. Methods: Human TRAIL full-length gene was cloned from human peripheral blood mononuclear cells (PBMCs) by RTPCR. The hTRAIL cDNA was cloned into the shuttle plasmid pAdTrack CMV and transformed into adenovirus backbone structure pAdEasy1 into E. coli BJ5183 for homologous recombination. The recombinants were identified by restriction enzyme digestion and transfected into human embryonic kidney 293 cells The recombinant adenovirus particles were packaged and the expression of TRAIL protein was detected by Western blot. The recombinant adenoviruses were infected in vitro by three kinds of human TRAIL-sensitive human tumor cells (Jurkat, PC3 and HeLa), then analyzed by flow cytometry with AnnexinⅤFITC / PI, DNA electrophoresis And trypan blue staining in order to observe the killing effect of TRAIL on tumor cells. Results: The 843bp cDNA fragment was amplified from human PBMCs by RTPCR and sequenced to be human TRAIL gene. The recombinants were identified by restriction enzyme digestion and Western blotting confirmed the correct expression of TRAIL protein in 293 cells infected with TRAIL adenovirus. The recombinant adenovirus particles were infected in HeLa, PC3 and Jurkat cells in vitro and were stained with Annexin V FITC / PI after 4 h. The percentage of Annexin V + PI-cell population detected by flow cytometry was (41.15 ± 3.12)%, (46.20 ± 4.07)%, 38.56 ± 3.45)%. After 24h, the genomic DNA of Jurkat cells was extracted and subjected to DNA electrophoresis. DNA bands specific to apoptosis were observed. After 36 hours, the cells were stained with trypan blue for HeLa, PC3 and Jurkat cells. The cell death rates were (75 ± 5)%, (82 ± 7)%, (70 ± 5)%. CONCLUSION: The hTRAIL adenovirus vector was successfully constructed. After in vitro infection of HeLa, PC3 and Jurkat tumor cells, it can kill tumor cells by inducing apoptosis of cells. It laid the foundation for further gene therapy and basic research.