全硫代反义寡核苷酸对卵巢癌细胞生长抑制作用

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。

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

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. Conclusion （1）The HO-8910 ovary carcinoma cell growth was inhibited by Phosphorathioate antisense Oligonucleotide and appeared the apoptosis. （2）It showed that the rate of inhibition has the discrepancy between the different dose and different time. In other words, the rate of inhibition enhance along with the augment of the dose and time. （3）PS-ASODN inhibits strongly the proliferation of HO- 8910 ovary carcinoma cell by inhibiting telomerase activity which induce cell apoptosis. PS-ASODN might be a new target to ovary carcinoma therapy.