人端粒酶催化亚单位启动子调控自杀基因HSV-TK肿瘤细胞特异性表达载体的构建

Construction of Tumor Cells Specific Expression Vector of Suicide Gene of HSV-TK Driven by hTERT Promotor

将自杀基因TK插入质粒pGL3--hTp和pGL3-Control中,取代其上的荧光素酶基因,分别构建hTERT启动子和SV40启动子调控的TK基因表达质粒pGL3-hTp-TK和pGL3-SV40-TK,酶切和PCR鉴定结果显示重组质粒pGL3-hTp-TK和pGL3-SV40-TK构建成功;用脂质体法将pGL3-hTp-TK和pGL3-SV40-TK瞬时转染端粒酶阳性的人肺腺癌细胞株A549及端粒酶阴性的人胚肺成纤维细胞株MRC-5,RT-PCR显示转染pGL3-SV40-TK的细胞A549和MRC-5均有TK mRNA表达,转染pGL3-hTp-TK的A549细胞中也有TK mRNA表达,但转染pGL3-hTp-TK的MRC-5细胞无TK mRNA表达.提示hTERT启动子可以调控自杀基因HSV-TK在肺癌细胞中靶向表达,可能是肿瘤靶向性基因治疗中比较理想的转录调控元件.

Suicide gene was inserted into plasmids of pGL3-hTp and pGL3-control respectively to replace luciferase gene, so that expression vectors of TK gene driven by hTERT promoter （pGL3-hTp-TK） and SV40 promoter （pGL3-SV40-TK） were constructed. Enzyme digestion and PCR suggested that recombinant plasmids of pGL3-hTp-TK and pGL3-SV40-TK were successfully constructed. Then, recombinants of pGL3-SV40-TK and pGL3-hTp-TK were transiently transfected into telomerase-positive human lung adenocarcinoma cell line A549 and telomerase-negative human fetal lung fibroblast cell line MRC-5. RT- PCR suggested that pGL3-SV40-TK transfected A549 and MRC-5 cells expressed TK mRNA , so did pGL3-hTp-TK-transfected A549 cells. However, pGL3-hTp-TK-transfected MRC-5 cells did not express TK mRNA. The results suggested that hTERT promoter could drive TK gene express selectively in lung cancer cell lines and it might be an ideal transcriptional control element for tumor targeting gene therapy.