端粒酶RNA亚基模板突变抑制HeLa肿瘤细胞的生长

在端粒酶阳性的肿瘤细胞中引入带突变模板的人端粒酶RNA亚基（MT-hTR）是一种新颖的抑 制肿瘤细胞生长的方法.本研究通过构建MT-hTR真核表达重组体，初步观察其对人宫颈癌He La细胞生长的影响.通过分子克隆技术及PCR介导的定点突变技术构建野生型及突变型hTR表 达重组体，将空载体及重组体分别与质粒pEGFP-C1通过脂质体共转染HeLa细胞，经药物G418的筛选得到稳定表达外源基因的细胞.RT-PCR检测目的基因的表达，TRAP分析细胞的端粒酶活性，并通过MTT法绘制各组细胞的生长曲线.结果发现，重组体在细胞内能成功表达目的基因，并且在Mutant1转染细胞中检测到相应的突变型端粒酶活性.构建的4个MT-hTR重组体中，有3个能使HeLa细胞生长速度减缓.以上结果提示，在HeLa细胞内表达MT-hTR能抑制细胞的生长速度，并且突变模板的设计可能会影响到抑制效应的发挥.

Inhibition of Mutant Template Human Telomerase RNA on HeLa Cell Growth

The induction of mutant template human telomerase RNA (MT-hTR) leads to rapid growth arrest in telomerase positive cancer cells. This study aimed to build recombinants expressing MT-hTR，then observed the influence on the growth of HeLa cells by MT-hTR. The recombinants expressing wild_type hTR(WT-hTR) or MT-hTR were constructed by molecular cloning and PCR based mutagenesis technique. HeLa cells were cotransfected with pEGFP-C1 vector and recombinants expressing WT-hTR or MT-hTR. Stable cell clones expressing extrinsic genes were obtained by G418 screening. hTR expression level was detected by RT-PCR and telomerase activity was analyzed by TRAP. The cell growth velocity was measured by MTT. The results revealed that the recombinants could successfully express WT-hTR or MT-hTR in HeLa cells，and corresponding mutant telomerase activity was detected in mutant1 transfection group. Three of four mutant transfection groups grew slower th an control group. These results demonstrated that MT-hTR could inhibit the growth of HeLa cells，and the design of mutant template might influence the inhibition effectiveness.