【摘要】 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HeLa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.更多还原

【Abstract】 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 than 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.更多还原