辐射诱导表达载体pEgr-hTRAIL的构建及其对肿瘤细胞的体外诱导凋亡作用

目的：构建携带人肿瘤坏死因子相关凋亡诱导配体（hTRAIL）基因的辐射诱导表达载体pEgr-hTRAIL，并研究其在人肺腺癌细胞株A549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pEgr-hTRAIL，体外通过脂质体转染A549细胞,经G418筛选稳定转染细胞A549-shTRAIL，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pEgr-hTRAIL表达质粒构建正确；稳定转染细胞A549-shTRAIL的hTRAIL mRNA表达量明显增加；稳定转染细胞A549-shTRAIL的凋亡细胞百分数明显增加，是A549细胞的1.8倍（P<0.05）。 结论：成功构建pEgr-hTRAIL表达质粒，体外稳定转染细胞A549-shTRAIL具有明显诱导凋亡作用。

Construction of pEgr-hTRAIL expression vector induced by irradiation and its apoptosis in tumor cells in vitro

Objective To construct the radiation-inducible expression vector pEgr-hTRAIL containing human TNF related apoptosis inducing ligand (hTRAIL) gene and study its expression and function of inducing apoptosis in A549 human lung adenocarcinoma cells. Methods Expression vector pEgr-hTRAIL was constructed with DNA recombinant technique. pEgr-hTRAIL plasmids were packaged with lipofectin to transfect into A549 cells in vitro. Stably transfected cell line A549-shTRAIL was selected through G418. The expression of hTRAIL gene was detected by RT-PCR. The early stage apoptosis of A549 cells was detected by Annexin-V-FITC apoptosis detecting kit. Results Expression vector pEgr- hTRAIL was constructed correctly by identification with restriction enzyme digestion. The expression of hTRAIL mRNA in stably transfected cell line A549-shTRAIL was increased significantly. The percentage of apoptotic A549-shTRAIL was increased significantly; it was 1.8 times as much as A549 cells (P<0.05). Conclusion Expression vector pEgr-hTRAIL is constructed successfully,which can increase the apoptosis of the stably transfected cell line A549-shTRAIL significantly.