人端粒酶逆转录酶下调p53促进胃癌细胞抵抗氧化应激下的死亡

Human telomerase reverse transcriptase downregulates p53 and inhibits the death of gastric carcinoma cells under oxidative stress

目的:利用慢病毒包装技术构建人端粒酶逆转录酶(h TERT)过表达载体lenti-h TERT,在胃癌细胞MKN45细胞中探讨过表达h TERT对氧化应激下细胞死亡的影响及其相关机制。方法:在低表达h TERT的MKN45胃癌细胞中转染lentih TERT和空载体lenti-NC,嘌呤霉素筛选建立稳定的细胞株,采用Western blot进行鉴定;采用叔丁基过氧化氢(TBH)氧化刺激,倒置显微镜下观察记录细胞死亡情况,台盼蓝染色检测细胞死亡比例,2’7’-二乙酰二氯荧光素检测细胞ROS含量;采用western blot和RT-PCR技术检测细胞在氧化应激条件下p53的表达。结果 :h TERT慢病毒过表达载体系统lenti-h TERT成功转染胃癌细胞株MKN45并建立稳定转染细胞株,过表达h TERT的MKN45胃癌细胞在氧化应激下可抑制细胞死亡,降低细胞内ROS水平;降低了p53蛋白表达水平,而不影响p53 m RNA表达水平。结论:胃癌细胞MKN45中h TERT过表达可通过下调p53蛋白水平抵抗氧化应激下的细胞死亡。

Objective： To establish human telomerase reverse transcriptase （hTERT） overexpression vector lenti-hTERT using the lentiviral packaging technique, and to investigate the effect of hTERT overexpression on cell death in gastric carcinoma MKN45 cells and related mechanisms. Methods： The gastric carcinoma MKN45 cells with low hTERT expression were transfected with lenti-hTERT or an empty vector, lenti-NC. Puromycin was used to select a stable cell line, and Western blot was used for identification. Tert-butyl hydroperoxide was used for oxidative stress, and an inverted microscope was used to observe and record cell death. Trypan blue staining was used to observe the proportion of dead cells, 2＇7＇-DHFA-DA was used to measure reactive oxygen species （ROS） level, and Western blot and RT-PCR were used to measure the expression of p53 under oxidative stress. Results： The gastric carcinoma MKN45 ceils were successfully transfected with lenti-hTERT, and a stable cell line was established. The gastric carcinoma MKN45 cells with hTERT overexpression showed inhibited cell death and a reduced ROS level; there was a reduction in the protein expression of p53, while there was no change in the mRNA expression of p53. Conclusions： In gastric carcinoma MKN45 cells, hTERT overexpression can downregulate the protein expression of p53 and thus inhibit cell death under oxidative stress.