全硫代反义寡核苷酸对卵巢癌细胞生长抑制作用  

Effects of telomerase antisense oligodeoxy nucle-otides on HO-8910 ovary carcinoma cell

作　　者：刘学[1] 单伟[1] 曾瑞霞[1] 房艳[1] 李德华[1] 秦书俭[1] 

机构地区：[1]辽宁医学院解剖学教研室,锦州121001

出　　处：《中国实用医药》2009年第8期12-16,共5页China Practical Medicine

摘　　要：目的采用针对人端粒酶hTR基因的反义寡聚脱氧核苷酸,探讨端粒酶反义寡聚脱氧核苷酸(antisense oligodeoxy nucleotides,ASODN)对卵巢癌HO-8910细胞端粒酶活性及细胞增殖的影响。方法将实验分为空白对照组、脂质体对照组、端粒酶全硫代正义寡聚脱氧核苷酸(Phosphorathioatesense oligodeoxy nucleotides,PS-SODN)组和不同剂量的全硫代反义寡聚脱氧核苷酸(Phosphorat-hioate an-tisense oligodeoxy nucleotides,PS-ASODN)组;②脂质体介导的细胞转染后,PS-ASODN和PS-SODN分别作用于卵巢癌HO-8910细胞后并培养24、48、72h,分别采用酶联免疫吸附法(ELISA)、吖啶橙染色法、四甲基偶氮唑蓝比色法(MTT)、流式细胞术检测HO-8910细胞的端粒酶活性、细胞形态、体外增殖、细胞凋亡和细胞周期的改变。结果ELISA法检测结果显示端粒酶PS-ASODN作用卵巢癌HO-8910细胞72h后端粒酶活性表达为阴性,说明端粒酶PS-ASODN能够抑制端粒酶活性;②吖啶橙染色观察细胞形态:PS-ASODN作用的卵巢癌HO-8910细胞有明显凋亡现象,凋亡细胞体积缩小,染色质浓缩,说明PS-ASODN能够促进卵巢癌HO-8910细胞凋亡;③MTT实验:PS-ASODN明显抑制卵巢癌HO-8910细胞的增殖(P<0.0l),并呈一定剂量和时间依赖关系;④流式细胞仪检测细胞凋亡和周期:与空白对照组比较,PS-ASODN组G0/G1期细胞明显增多,差异显著(P<0.01);在G0/G1期前出现亚二倍体凋亡峰,表明细胞被阻止在G1/S期。结论PS-ASODN作用于卵巢癌HO-8910细胞后,卵巢癌HO-8910细胞增殖受到明显抑制,并出现凋亡;②PS-ASODN对端粒酶活性的抑制率与PS-ASODN的浓度和作用时间呈依赖关系,即抑制率随着反义寡核苷酸的浓度和作用时间的增加而增大;③以端粒酶RNA模板区为靶点的PS-ASODN明显抑制卵巢癌HO-8910细胞的增殖,其机制可能是通过降低细胞的端粒酶活性而诱发细胞的凋亡,PS-ASODN对卵巢癌的治疗具有重要价值。Objective In this study, we apply Oligonucleotide aimed directly at human telomerase RNA （hTR）, To study the effects of telomerase Phosphorathioate antisense oligodeoxy nueleotides （PS-ASODN） on telomerase activity and proliferation of H0-8910 ovary carcinoma cell. Methods （1）HO-8910 ovary carcinoma Cell was transfected by PS-ASODN, Phosphorathioate sense oligodeoxy nucleotides （PS-SODN）mediated by Lipotap Liposomal Transfection Reagen. The experiments were classified into normal group,PS-ASODN groups at varied concentrations ,PS-SODN group and oligofectamineTM alone. （2）The proliferation activity of HO-8910 o- vary carcinoma cell line was determined by using methyl thiazolyl tetrazolium assay. The telomerase activity was determined by using enzyme-linked immunosorbent assay. The cell morphology was observed by fluorescence mi- croscope stained with acridine orange. Flow cytometry was adopted to examine apoptotic rate and cell cycle. Restilts （1）With ELISA,Telomerase of H0-8910 ovary carcinoma cell was repressed by telomerase PS-ASODN af- ter 72 h later, it showed that telomerase PS-ASODN could lead inhibition of telomerase activity. （2）The cell morphology was observed by fluorescence microscope stained with acridine orange：Apoptotic morphological charac- teristic of HO-8910 ovary carcinoma cell transfected by antisense oligonucleotides was observed. Moreover, the apoptotic cell physical volume contracts. The HO-8910 ovary carcinoma cell growth was inhibited by Phosph-orathioate antisense Oligonucleotide and appeared the apoptosis. （3）PS-ASODN caused significant （P 〈 0. 01 ） inhibition of cell growth, and the rate of inhibition has the discrepancy between the different dose and different time （P 〈0. 01 and P 〈0. 01 ）. （4）The apoptotie peak was detected with FCM and cells were stayed in G1/S stage and a sub-G1 stage cell apoptotic peak appear in front of GO/G1 stage （P 〈 0. 01 ）. Typical apoptotic morpholog- ic feature was discovered under light microscope.

关 键 词：端粒酶全硫代反义寡聚脱氧核苷酸 卵巢癌HO-8910细胞 端粒酶 细胞凋亡 

分 类 号：R737.31[医药卫生—肿瘤]