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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 To investigate the effect of antisense oligodeoxy nucleotides (ASODN) on the telomerase activity of human ovarian cancer cell line HO-8910 and the antisense oligodeoxynucleotides of human telomerase hTR gene Effect of cell proliferation. Methods The experiment was divided into blank control group, liposome control group, telomerase total thio-sense oligodeoxy nucleotides (PS-SODN) group and different doses of total thio antisense oligodeoxydeoxy PS-ASODN and PS-SODN were treated with liposome-mediated transfection of cells and cultured The expression of telomerase in HO-8910 cells was detected by enzyme-linked immunosorbent assay (ELISA), acridine orange staining, MTT assay and flow cytometry Activity, cell morphology, proliferation in vitro, apoptosis and cell cycle changes. Results The results of ELISA showed that telomerase activity was down-regulated by PS-ASODN in telomerase HO-8910 cells after 72 hours, indicating that telomerase PS-ASODN could inhibit telomerase activity; ② acridine orange staining Morphology PS-ASODN induced apoptosis in ovarian cancer HO-8910 cells, the apoptotic cells were reduced in size and chromatin condensed, indicating that PS-ASODN could promote the apoptosis of HO-8910 ovarian cancer cells; ③TT-test: PS-ASODN (P <0.01), and in a dose-and time-dependent manner; ④The apoptosis and the cycle of the cells were detected by flow cytometry: Compared with the blank control group, the G0 / G1 phase cells significantly increased (P <0.01); sub-diploid apoptotic peak appeared before G0 / G1 phase, indicating that the cells were blocked in the G1 / S phase. Conclusions PS-ASODN inhibits the proliferation of HO-8910 ovarian cancer cells and induces apoptosis after it is treated with PS-ASODN. ②The inhibitory rate of telomerase activity and the concentration of PS-ASODN and the action time The inhibition rate increased with the increase of antisense oligonucleotide concentration and time; ③ PS-ASODN targeting telomerase RNA template region significantly inhibited the expression of HO-8910 Proliferation, its mechanism may be by reducing the telomerase activity of cells and induce apoptosis, PS-ASODN is of great value for the treatment of ovarian cancer.