放射控导的wt-p53蛋白正反馈基因环路对肺腺癌细胞周期、凋亡及放射敏感性的影响

Effect of irradiation-induced wt-p53 positive circuit on cell life cycle,apoptosis and irradiation sensitivity of lung adenocarcinoma cells

目的研究体外环境放射控导的wt-p53蛋白正反馈基因环路对肺腺癌细胞细胞周期、凋亡率及放射敏感性的影响。方法采用直线加速器8MV-X线,400MU/min、4Gy、源皮距100cm的照射条件照射治疗组pE6R4-p53-EGFP/H1299转染细胞和对照组H1299(p53-/-)、pE6-p53-EGFP/H1299和pR4-p53-EGFP/H1299转染细胞,12h后收集各组细胞,流式细胞仪分析该环路在放射线诱导下对肺腺癌细胞周期和细胞凋亡率的影响;绘制细胞放射剂量-存活曲线,计算平均致死剂量(Do)值及放射增敏比(sensitive enhancement ratio,SER)。结果4Gy8MV-X线照射治疗组和对照组细胞后12h,治疗组细胞在照射后(75.13±1.42)%发生明显G0/G1期阻滞;照射后各组细胞凋亡率均高于照射前,照射后治疗组细胞凋亡率为(23.73±0.21)%,分别是对照组H1299(p53-/-)细胞[(4.17±0.12)%]、pE6-p53-EGFP/H1299细胞[(15.67±0.32)%]、pR4-p53-EGFP/H1299细胞[(9.23±0.15)%]的5.69、1.51倍和2.57倍。治疗组pE6R4-p53-EGFP/H1299细胞和对照组pE6-p53-EGFP/H1299、H1299(p53-/-)细胞Do值分别0.91、1.073、1.413Gy,根据Do值计算SER分别为2.63和1.34。结论放射可诱导wt-p53蛋白正反馈基因环路上调wt-p53蛋白表达,从而调控肺腺癌细胞发生显著G/G1阻滞,使细胞凋亡比例增加,显著提高了肺腺癌细胞放射敏感性。

Objective To study the effects of irradiation-induced wt-p53 positive feedback circuit on cell life cycle, apoptosis rate and irradiation sensitivity of adenocarcinoma of lung in vitro. Methods The therapeutic group pE6R4-p53-EGFP/H1299 cells and the other 3 control groups H1299 （p53^-/- ） , pE6-p53- EGFP/H1299 and pR4-p53-EGFP/H1299 cells were irradiated with 8-Mev electron beam generated by a linear accelerator at a dose rate of 400 cGy/min and a source-skin distance （SSD） of 100 cm, 12 h exposure to irradiation. The cell life cycle and the rate of apoptosis were analyzed by FCM. Mean lethal dose （Do） and sensitive enhancement ratio （SER） index were calculated from the irradiation dose-survival curve. Results In 12 h post 4-Gy 8MV-X-ray irradiation, FCM analysis showed that most cells in therapeutic group were arrested at G0/G1 stage [ （75.13 ± 1.42）% ], compared with the 3 control groups [ （38.47±0.87）%, （62.57 ± 0. 76）% and （51.23 ± 2.41 ）%, respectively]. After irradiation, the cell apoptotic rate in each group was higher than that in the ceils without irradiation. The apoptotic rate in the therapeutic group was （23.73 ± 0. 21 ） %, which was 5.69, 1.51 and 2.57 folds respectively higher in the 3 control groups [ （4.17 ± 0.12 ） %, （15.67±0.32）% and （9.23±0. 15）%]. Do values were 0.91, 1.073 and 1.413 Gy respectively in pE6R4-p53-EGFP/H1299, pE6-p53-EGFP/H1299 and H1299 cells. The SER, derived from Do values, was 2.63 and 1.34, respectively. Conclusion Irradiation up-regulates wt-p53 gene expression, regulates cell cycle and induces cell apoptosis. Thus this positive feedback circuit increases the sensitivity of lung adenocarcinoma to irradiation.