hTERTp/TK/pGL3靶向抑制端粒酶活性及其对鼻咽癌干细胞的杀伤作用

［摘 要］ 目的:探讨hTERTp/TK/pGL3载体靶向抑制端粒酶活性及其杀灭鼻咽癌CD133+干细胞的作用机制。方法:将已构建的hTERTp/TK/pGL3载体及其对照处理因素（CMV/TK/pGL3和TK/pGL3载体）转染至鼻咽癌CD133+干细胞、CD133－肿瘤细胞、人脐静脉内皮细胞(ECV)(对照组)和鼻咽癌SUNE未分选细胞中，采用Stretch PCR 法检测4种细胞端粒酶活性改变。MTT法测定CD133+干细胞和ECV细胞存活率。结果:CD133+鼻咽癌干细胞体外培养7 d后细胞逐渐增多，CD133+鼻咽癌干细胞体内成瘤实验阳性。CD133+鼻咽癌干细胞转染hTERTp/TK/pGL3或CMV/TK/pGL3后端粒酶活性降低；而ECV细胞端粒酶为阴性表达；CD133+鼻咽癌干细胞分别转染TK/pGL3、CMV/TK/pGL3和hTERTp/TK/pGL3后细胞存活率平均为（87.4±0.4）％、（20.5±0.4）％和（27.9±0.2）％，ECV对照细胞组转染TK/pGL3、CMV/TK/pGL3和hTERTp/TK/pGL3后细胞存活率平均为（90.7±0.1）％、（18.1±0.2）％和（86.2±0.1）％，hTERTp/TK/pGL3杀灭鼻咽癌CD133+干细胞效率明显高于ECV细胞组（P<0.01）。结论:hTERTp/TK/pGL3载体可以通过下调端粒酶活性靶向抑制端粒酶阳性的鼻咽癌CD133+干细胞。

Targeted inhibition of hTERTp/TK/pGL3 on telomerase activity and its killing effect on nasopharyngeal cancer stem cells

Objective To explore the mechanism of targeted inhibition of hTERTp/TK/pGL3 vector on the telomerase activity and its killing effect on nasopharyngeal cancer stem cells.Methods The nasopharyngeal carcinaoma CD133+ stem cells,CD133-tumor cells,human umbilical vein endothelial cells(ECV cells)(control group) and nasopharyngeal carcinaoma SUNE unsorted cells were transfected by hTERTp/TK/pGL3 vector and its control vectors(CMV/TK/pGL3 and TK/pGL3).The telomerase avtivities of nasopharyngeal carcinaoma CD133+ stem cells and ECV cells were detected by Stretch PCR.The survival rates of CD133+stem cells and ECV cells were detected by MTT method.Results After culture for 7 d in vitro,the number of nasopharyngeal carcinaoma CD133+ stem cells was increased,the tumorigenicity test of CD133+ stem cells in vivo was positive.The telomerase activity of nasopharyngeal carcinaoma CD133+ stem cells was decreased after transfected by hTERTp/TK/pGL3 or CMV/TK/pGL3;the telomerase activity of EVC cells was negative.After transfected by TK/pGL3,CMV/TK/pGL3,and hTERTp/TK/pGL3,the survival rates of CD133+ stem cells were 87.4%±0.4%,20.5%±0.4%,and 27.9%±0.2%,respectively.After transfected by TK/pGL3,CMV/TK/pGL3,and hTERTp/TK/pGL3,the survival rates of ECV cells were 90.7%±0.1%,18.1%±0.2%,and 86.2%±0.1%,respectively.The efficiency of hTERTp/TK/pGL3 vector in killing CD133+ stem cells was higher than that of ECV cells(P<0.01).Conclusion hTERTp/TK/pGL3 vector can targetedly inhibit the telomerase-positive nasopharyngeal carcinaoma CD133+ stem cells by down-regulation of telomerase activity.