端粒酶逆转录酶在脐静脉内皮细胞耐受曲古抑菌素A诱导凋亡中的作用  

Role of human telomerase reverse transcriptase in apoptosis of human umbilical vein endothelial cells induced by trichostatin A

作　　者：吴鹏[1] 奚玲[1] 陈刚[1] 王蓓蓓[1] 罗丹枫[1] 卢运萍[1] 周剑锋[2] 马丁[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院妇产科,武汉430030 [2]华中科技大学同济医学院附属同济医院血液科,武汉430030

出　　处：《中华肿瘤杂志》2007年第5期334-337,共4页Chinese Journal of Oncology

基　　金：国家自然科学基金资助项目（30371657、30500596）;国家973重大基础研究项目（2002CB513107）

摘　　要：目的观察曲古抑菌素A（TSA）对脐静脉内皮细胞及官颈癌细胞凋亡、端粒酶逆转录酶（hTERT）表达的影响,并探讨hTERT在脐静脉内皮细胞耐受TSA中的作用。方法磺酰罗丹明B （RSB）法检测药物动力学特征;流式细胞仪检测周期改变和凋亡;RT-PCR检测hTERT和p21^Waf1基因表达变化;免疫荧光结合流式细胞术检测hTERT蛋白表达变化;转染hTERT质粒后,PCR-TRAP- ELISA法检测转染细胞端粒酶活性;AnnexinV／PI检测转染细胞在TSA作用下的早期凋亡。结果在大剂量TSA作用脐静脉内皮细胞后,增殖抑制、周期阻滞,但凋亡并不显著;HeLa细胞在相同剂量的TSA作用下凋亡明显。脐静脉内皮细胞经TSA诱导后,hTERT表达上调,p21^Waf1则无明显变化;而HeLa细胞p21^Waf1表达上升,hTERT表达下降。转染显性负突变hTERT的脐静脉内皮细胞,其端粒酶活性显著低于对照组。TSA作用转染不同质粒的脐静脉内皮细胞的凋亡率与对照组差异有统计学意义。结论脐静脉内皮细胞可以耐受大剂量TSA诱导的凋亡,hTERT表达上调可能是脐静脉内皮细胞耐受TSA诱导凋亡的重要机制之一。Objective The aim of this study was designed to investigate the effect of TSA on human umbilical vein endothelial cells and to reveal its possible mechanisms and relationship between apoptosis and activity of telomerase reverse transcriptase. Methods sulforbodamine B method was employed to determine the growth rate of umbilical vein endothelial cells. The cell apoptotic rate was measured by flow cytometry （FCM）. The hTERT and p21^Waf1 mRNA expression before and after TSA treatment were detected by semiquantitative reverse transcription polymerase chain reaction （RT-PCR）. The quantitative of bTERT protein expression in cells were detected by flow cytometry. After transfection, the cell telomerase activity was detected by PCR and telomeric repeat amplification protocol assay （PCR-TRAP-ELISA）and early apoptosis was measured by Annexin V/PI stain and flow cytometry. Results After being treated with TSA, the proliferation of umbilical vein endothelial cells was inhibited. Slight apoptosis and cell cycle arrest were detected. However, the same concentration of TSA induced serious apoptosis in HeLa cells. Up-regulation of hTERT mRNA expression was observed within 48 h after TSA treatment, but the change of p21^Waf1 expression was not significant. The umbilical vein endothelial cells bTERT protein expression level was increased within 24 b. After transfection of the dominant negative, wild type and control bTERT plasmid, a significant difference of telomerase activity in these cells was observed by PCR-TRAP-ELISA assay. WT-hTERT- transfected cells were more resistant to apoptosis induced by tricbostatin A. Conclusion Human umbilical vein endothelial cells could be resistant to apoptosis induced by high concentrate TSA, and hTERT might play an important role in this process.

关 键 词：曲古抑菌素A 凋亡 端粒酶逆转录酶 P21^WAF1 

分 类 号：R686[医药卫生—骨科学]