端粒酶反义寡核苷酸对膀胱癌治疗作用的体外实验

Experimental study on therapeutic effects of antisense oligonucleotides of telomerase on BIU87 cell of bladder cancer in vitro

目的 :探讨端粒酶反义核苷酸对膀胱癌的治疗作用。方法 :用 TRAP-银染方法检测 2 5例膀胱癌组织和 4例癌旁组织中端粒酶活性的表达。以端粒酶 RNA模板区为靶点 ,不同剂量的序列为 TAGGGTTAGACAA的硫代磷酸修饰的反义寡核苷酸 (PS- ASON )作为实验组 (A、 B、 C) ,以硫代磷酸修饰 1 3个核苷酸的随机引物作为对照组 (D) ,仅加入培养液作为空白对照组 (E) ,与人膀胱癌细胞株 BIU87细胞共同孵育 1～ 30 d,计细胞数 ,光镜及电镜观察 ,DNA凝胶电泳检测、流式细胞仪测定和分析细胞的凋亡和坏死。结果 :2 5例膀胱癌组织中 ,2 4例(96 % )表达端粒酶活性 ,4例癌旁组织无端粒酶活性表达。与随机引物及空白对照组比较 ,实验组的 ASON能明显减少细胞增殖数目 ,实验组细胞在第 2、 8天及 8天后出现较明显的凋亡峰和 DNA梯状电泳带 ,并出现退化、老化、凋亡和坏死的形态学变化 ,凋亡率明显增高 (P<0 .0 0 1 ) ,并显示剂量的依赖性 (P<0 .0 1 )。结论 :以端粒酶 RNA模板区为靶点的反义寡核苷酸引起膀胱癌 BIU87细胞增殖抑制 ,退化、老化、凋亡和坏死 。

Objective To investigate the therapy effects of the antisense oligonucleotides of telomerase on the BIU87 cell derived from bladder cancer. Methods Telomerase activities in 25 tissue specimens of bladder cancer and 4 normal ones were detected by TRAP AgNO 3 stain.The phosphorothioate antisense oligonucleotides (PS ASON) of differential doses with sequence TTAGGGTTAGACAA targeted to telomerase RNA template (as experiment group A,B, and C), PS random primer with 13 nucleotides (as control group,D),and culture medium only (as empty control group,E) were incubated with the BIU87 cell lines. The number of cells were counted from 1 to 30 days, The apoptotic rate was assayed by flow cytometry. Apoptosis was detected by DNA ladder electrophoresis. The cell form was observed with light and electron microscope. Results Of 25 cases of bladder cancer, the activities of telomerases were expressed in 24 cases (96%), there was no expression in 4 normal tissues. ASON in experiment group reduced the number of cells compared with group D and E. The transparent apoptotic cusp at flow cytometry and revealed ladder electrophsis zone were presented in experiment groups at 2nd, 8th day and 8 days later. Degeneration, aging, necrosis, and apoptosis occurred under the microscope. The efficacy depended on the doses of ASON ( P <0 01) and it was of sequence selective specifity. Conclusion Inhibition of BIU87 cell proliferation, aging,necrosis,and apoptosis induced by ASON targeted hTR may have potential significance in tumor therapy.