端粒酶hTERT基因反义核酸对肿瘤坏死因子-α诱导膀胱癌T24细胞凋亡的影响

Inhibition of telomerase with hTERT antisense enhances TNF-α-induced apoptosis in bladder cancer cells T24

目的探讨人类端粒酶反转录酶催化亚单位(hTERT)基因的全硫代修饰反义寡核苷酸抑制膀胱癌T24细胞端粒酶活性后,肿瘤坏死因子-α(TNF-α)对膀胱癌T24细胞凋亡的增敏作用。方法采用端粒重复序列扩增-多聚酶链反应-酶联免疫吸附测定法检测膀胱癌细胞的端粒酶活性;采用噻唑蓝比色试验观察hTERT基因的全硫代修饰反义寡核苷酸与TNF-α共同作用对膀胱癌T24细胞生长活力的影响;通过流式细胞仪测定凋亡细胞的百分率。结果hTERT基因的全硫代修饰反义寡核苷酸作用于膀胱癌T24细胞48 h,其端粒酶活性下降;作用72 h,其端粒酶活性受到抑制,与反义核酸组、空白对照组相比差异有统计学意义(P<0.05)。hTERT基因的全硫代修饰反义寡核苷酸作用于膀胱癌T24细胞后,加入TNF-α作用48 h,T24细胞的抑制率为39%,与对照组、正义核酸组、全硫代修饰反义寡核苷酸组、TNF-α组及正义核酸+TNF-α组相比,差异有统计学意义(P<0.05)。hTERT基因的全硫代修饰反义寡核苷酸作用于膀胱癌T24细胞后加入4μg/mL TNF-α作用48 h,凋亡细胞的百分率为35.18%;与对照组、正义核酸组、全硫代修饰反义寡核苷酸组、TNF-α组及正义核酸+TNF-α组相比,差异有统计学意义(P<0.05)。结论通过hTER工基因的全硫代修饰反义寡核苷酸能降低膀胱癌T24细胞端粒酶活性;对TNF-α诱导膀胱癌T24细胞凋亡有增敏作用。

Objective To investigate the effect of inhibition of telomerase with human telomerase reverse transcriptase（hTERT） antisense on bladder cancer cells to tumor necrosis factor-α（TNF-α）-induced apoptosis.Methods Antisense phosphorothioate oligodeoxynucleotide was synthesized and purified.Telomerase activity was measured by telomeric repeat amplification protocol telomerase polymerase chain reaction-enzyme linked immunosorbent assay kit;cell viability was detected by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl tetrazohum bromide assay;cell apoptosis was determined by flowcytometry.Results hTERT antisense phosphorothioate oligodeoxynucleotide could significantly inhibit telomerase activity.The cell viability was 39%after hTERT antisense phosphorothioate oligodeoxynucleotide combined with TNF-α treatment for 48 h;the cell viability decreased with time after hTERT antisense phosphorothioate oligodeoxynucleotide combined with TNF-α treatment.The percentage of apoptosis was 35.18%after hTERT antisense phosphorothioate oligodeoxynucleotide combined with TNF-α treatment for 48 h;the percentage of apoptosis increased with time after hTERT antisense phosphorothioate oligodeoxynucleotide combined with TNF-α treatment.There was no difference in cell viability and the percentage of apoptosis between sense phosphorothioate oligodeoxynucleotide and the control.Conclusion Inhibition of telomerase with hTERT antisense can enhances TNF-α-induced apoptosis in T24 cells.