端粒酶RNA反义核酸联合其催化亚单位反义核酸对子宫颈癌Hela细胞生长的抑制作用

Combination of antisense human telomerase RNA and antisense human telomerase catalytic subunit inhibits cervical cancer Hela cell growth

目的 探讨端粒酶RNA反义核酸 (反义hTR)联合端粒酶RNA催化亚单位反义核酸(反义hTERT) ,对宫颈癌Hela细胞生长的抑制作用及作用机理。方法 实验分为空白对照、脂质体对照、正义hTR、正义hTERT、反义hTR、反义hTERT及反义hTR +反义hTERT组。分别采用四甲基偶氮唑蓝 (MTT)法、PCR 端粒重复序列扩增 (TRAP) 酶联免疫吸附实验 (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后细胞的增殖、端粒酶活性及细胞形态学、细胞凋亡和细胞周期阻滞的变化。结果 宫颈癌Hela细胞转染端粒酶反义核酸后 ,出现明显的细胞增殖抑制、端粒酶活性下降、细胞凋亡形态学改变、细胞凋亡率升高及细胞周期阻滞。反义hTR、反义hTERT组与空白对照、脂质体对照及正义hTR、正义hTERT组比较 ,差异有统计学意义 (P <0 0 1)。Hela细胞转染反义核酸 (0 2 μmol/L) 3d后 ,反义hTR +反义hTERT组 ,细胞增殖抑制率、相对端粒酶活性 (相对于空白对照组 )、细胞凋亡率分别为 6 9 3%、19 6 %、2 8 6 % ,与反义hTR组和反义hTERT组比较 ,差异均有统计学意义 (P <0 0 1) ,Q值分别为 0 86 7、0 919、1 0 75。反义hTR +反义hTERT组G0 /G1期细胞比例增加。结论 端粒酶反义核酸能通过特异性抑制端粒酶活性。

Objective To investigate the inhibitory effect of combination of antisense human telomerase RNA (anti hTR) and antisense human telomerase catalytic subunit (anti hTERT) on proliferation of cervical cancer Hela cell line cultured in vitro Methods Cervical cancer Hela cells were transfected by anti hTR, anti hTERT, anti hTR + anti hTERT, sense hTR, sense hTERT with Oligofectamin TM reagent The proliferation activity of cervical cancer Hela cells was determined using methyl thiazolyl tetrazolium (MTT) The activity of telomerase was tested by polymerase chain reaction telomeric repeat amplification protocol enzyme linked immunosorbent assay (PCR TRAP ELISA) Cell morphologies were observed under fluorescence microscope with acridine orange staining Apoptosis and cell cycle were examined by flow cytometer method (FCM) Results The inhibitory rates on proliferation and apoptotic rates of Hela cells transfected by antisense oligonucleotides were significantly higher compared with those of control, sense oligonucleotides and Oligofectamin TM (all P < 0 01) Meanwhile, the inhibitory rates on proliferation and apoptotic rates of Hela cells transfected by anti hTR + anti hTERT were significantly higher compared with those transfected by anti hTR or anti hTERT alone( P < 0 01); telomerase activity was significantly decreased( P < 0 01) Transfected by antisense oligonucleotides at 0 2 μmol/L for 3 d,inhibitory rates on proliferation , telomerase activity(relative to control), apoptotic rate of Hela cells was 69 3%, 19 6%, 28 6% respectively(Q = 0 867,0 919,1 075) Percentages of G 0/G 1 phase cells were increased Conclusions Telomerase anti hTR and anti hTERT inhibit Hela cell growth probably through suppressing telomerase activity, inducing apoptosis and blocking cell cycle Combination of anti hTR and anti hTERT has synergistic action on cervical cancer Hela cells