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目的 :构建携带人肿瘤坏死因子相关凋亡诱导配体 (h TRAIL )基因的辐射诱导表达载体p Egr- h TRAIL ,并研究其在人肺腺癌细胞株 A 5 4 9中的表达及促凋亡作用。方法 :采用常规分子生物学方法构建表达质粒 p Egr- h TRAIL ,体外通过脂质体转染 A5 4 9细胞 ,经 G4 18筛选稳定转染细胞 A 5 4 9- sh TRAIL ,利用逆转录聚合酶链反应 (RT- PCR)法检测目的基因的表达 ,采用 Annexin- V- FITC凋亡检测试剂盒检测细胞早期凋亡。结果 :经限制性内切酶酶切鉴定 p Egr- h TRAIL表达质粒构建正确 ;稳定转染细胞 A 5 4 9- sh TRAIL的 h TRAILm RNA表达量明显增加 ;稳定转染细胞 A 5 4 9- sh TRAIL的凋亡细胞百分数明显增加 ,是 A5 4 9细胞的 1.8倍(P<0 .0 5 )。结论 :成功构建 p Egr- h TRAIL表达质粒 ,体外稳定转染细胞 A5 4 9- sh TRAIL具有明显诱导凋亡作用。
OBJECTIVE: To construct the radiation-induced expression vector p Egr-h TRAIL carrying human TRAIL gene and study its expression in human lung adenocarcinoma cell line A 5 49 Dead effect. METHODS: The expression plasmid p Egr-h TRAIL was constructed by conventional molecular biology method. A549 cells were transfected by liposomes in vitro, and then A549 cells were transfected with A549 cells by G418 selection. The expression of TRAIL was detected by reverse transcriptase polymerase The expression of the target gene was detected by reverse transcription-polymerase chain reaction (RT-PCR). The apoptosis of the cells was detected by Annexin-V-FITC apoptosis detection kit. Results: The recombinant plasmid pEGFP-TRAIL was identified by restriction endonuclease digestion. The expression of TRAILm RNA in stable transfected A549-9sh TRAIL cells was significantly increased. The stable transfected cells A549-9- sh TRAIL significantly increased the percentage of apoptotic cells, 1.8 times that of A549 cells (P <0.05). CONCLUSION: The pEGF-TRAIL expression plasmid was successfully constructed and the apoptosis-inducing effect of A549 9-sh TRAIL in vitro was significantly inhibited.