端粒酶反义hTR和反义hTERT合用对宫颈癌HeLa细胞凋亡的影响  

Study on the apoptosis of cervical cancer HeLa cells transfectead by comol.L-1 bination of telomol.L-1erase anti-hTR and anti-hTERT

作　　者：陈忠东[1] 雷厉秀[1] 

机构地区：[1]南华大学第一附属医院妇产科,湖南衡阳421001

出　　处：《中国药理学通报》2005年第12期1490-1494,共5页Chinese Pharmacological Bulletin

摘　　要：目的探讨端粒酶反义hTR和反义hTERT联合作用于宫颈癌HeLa细胞,对HeLa细胞凋亡的影响。方法实验分空白对照组、脂质体对照组、正义hTR组、正义hTERT组、反义hTR组、反义hTERT组及反义hTR+反义hTERT组。分别采用四甲基偶氮唑蓝法(MTT)、逆转录聚合酶链反应技术-端粒重复序列扩增(TRAP)酶联免疫法(ELISA)、吖啶橙染色法、流式细胞术检测宫颈癌HeLa细胞转染反义hTR和反义hTERT后细胞的端粒酶活性、形态学、凋亡和周期的改变。结果宫颈癌HeLa细胞转染端粒酶反义核酸后,出现明显的细胞凋亡形态学改变,端粒酶活性抑制率、凋亡率增高及细胞周期阻滞,与空白对照组、脂质体对照组及正义核酸组比较,统计学上差异有显著性(P<0.01)。HeLa细胞转染0.2μmol·L-1反义hTR+反义hTERT72h后,端粒酶活性抑制率、细胞凋亡率分别为80.5%、28.6%,与反义hTR组和反义hTERT组比较(端粒酶活性抑制率、细胞凋亡率分别为hTR:62.7%、13.2%;hTERT:66.5%、13.7%),差异有显著性(P<0.01,q值分别为0.919、1.075)。反义hTR+反义hTERT组比反义hTR组和反义hTERT组细胞凋亡的形态学改变更明显,反义hTR+反义hTERT组G0/G1期细胞比例增加(G1期细胞比例为57.8%,空白对照组G1期细胞比例为50.7%)。结论端粒酶反义核酸能通过抑制端粒酶活性诱导细胞凋亡,端粒酶反义hTERT和反义hTR有协同效应。Aim To investigate the apoptosis of cervi- cal cancer HeLa cells cultured in vitro transfectead by combining telomerase anti-hTR and anti-hTERT. Methods The activity of telomerase was tested by polymerase chain reaction-telomeric repeat amplification protocolenzyme-linked immunosorbent assay （PCR- TRAP- ELISA ）. Cell morphologies were observed by fluorescence microscope stained with acridine orange. Apoptosis and cell cycle were examined by Flow Cytometer method （FCM）. Results The telomerase activity of HeLa cells transfected by antisense oligonucleotides were significantly decreased compared with those of nontreated control, sense oligonucleotides and oligofectamineTM （ P 〈 0.01 ）. Meanwhile, the telomerase activity of HeLa cells transfected by anti-hTR ＋ anti-hTERT was ignificantly decreased compared with those of anti-hTR and anti-hTERT alone （P 〈 0.01 ） ; likewise, apoptotic rates was higher significantly （P 〈0. 01 ）. Transfected by 0.2 μmol · L^-1 antisense oligonucleotides after 72 h, telomerase activity inhibitory rates, apoptotic rates of HeLa cells were antihTR： 62.7%, 13.2% ; anti-hTERT： 66. 5%, 13.7% ; anti-hTR ＋ anti-hTERT：80. 5%, 28. 6% respectively （q = 0. 919,1. 075 ）. Apoptotic morphological characteristic of HeLa cells transfected by antisense oligonucleotides was observed. Moreover, HeLa cells ransfected by anti-hTR ＋ anti-hTER were more significant; percentage of G0/G1 phase of HeLa cells transfected by anti-hTR ＋ anti-hTERT was increased （ percentage of cells in G0/G1 phase was 57.8% , that of control was 50. 7% ）. Conclusions Apoptosis of cervical cancer HeLa cells probably douwn-regulate telomase activity by telomerase anti-hTR and anti-hTERT , Combination of telomerase anti-hTR and anti-hTERT have significantly synergistic effects on induction of ap optosis in HeLa cells.

关 键 词：宫颈癌 端粒酶 反义核酸 凋亡 

分 类 号：R273[医药卫生—中西医结合]