二氢青蒿素联合放疗对肺癌GLC-82细胞凋亡的影响及机制研究

目的研究二氢青蒿素（dihydroartemisinin,DHA）联合放疗对人肺腺癌GLC-82细胞周期和凋亡的影响及其机制。方法 MTT法检测不同浓度DHA分别在24、48、72 h对GLC-82细胞的抑制作用;克隆形成实验分析,多靶单击拟合模型方程计算放射增敏比,评价增敏效果;流式细胞术检测DHA联合放疗对GLC-82细胞周期和凋亡的影响;Western blot检测P53、P21、Bcl-2和Bax蛋白的表达。结果不同浓度DHA（4、8、16、32、64、128μg/mL）作用24、48和72 h的IC50值分别为：38.25、20.58、10.36μg/mL,对GLC-82细胞的毒性有明显剂量和时间依赖性,抑制率较空白对照组明显增加（P〈0.01,P〈0.05）;DHA对GLC-82细胞具有放射增敏作用,其增敏比为1.4。DHA联合放疗可使GLC-82细胞周期的构成发生明显的变化并诱导细胞凋亡,凋亡率为21.5%,与空白对照组比较,差异有统计学意义（P〈0.05）。Western blot表明,DHA联合放疗作用GLC-82细胞后P53和P21的蛋白水平表达增加,同时下调Bcl-2蛋白表达（P〈0.01,P〈0.05）。结论 DHA对肺腺癌GLC-82细胞有较强的细胞毒性和放射增敏作用,其作用机制可能是使GLC-82细胞生长停滞在G0/G1期并诱导细胞凋亡,使S期细胞比例降低;使P53功能恢复,通过抑制Bcl-2蛋白的表达促使凋亡,从而起到杀伤肿瘤的作用。

Effect of DJhydroartemiainin Combined Irradiation on the Apoptosis of Human Lung Cancer GLC82 Cells and Its Mechanism Study

Objective To study the effect of Dihydroartemisinin （DHA） combined irradiation on the apoptosis of human lung cancer GLC-82 cells and to study its mechanism. Methods The growth inhibition rate of GLC-82 cells acted by different concentrations DHA was detected using MTT assay at 24, 48, and 72 h, respectively. Clone forming test was used. With multi-target single-hit model, the radiosensitization effect was assessed by calculating sensitizing enhancement ratio （SER）. The effect of DHA combined irradiation on the apoptosis of GLC-82 cell cycle distribution and apoptosis were measured by flow cytome- try. The protein expression of p53, p21, Bcl-2, and Bax were detected by Western blot. Results Different concentrations DHA （4, 8, 16, 32, 64, and 128 iJLg/mL） had cytotoxicity on GLC-82 cells. The IC50 for 24, 48, and 72 h was 38.25, 20.58, and 10.36 μg/mL, respectively, in obvious dose- and time-dependent manner. The growth inhibition rate was more significantly increased than that of the blank control group （P 〈 0.01, P 〈0.05）. DHA had sensitization enhancement effect on GLC-82 cells, with SER of 1.4. DHA combined irradiation could obviously change the structure of GLC-82 cells cell cycle and induce apoptosis （with the apoptosis rate of 21.5%）, which was significantly different from that of the blank control group （P 〈 0.05）. Western blot showed the expression of p53 and p21 protein could be increased by DHA combined irradiation, and the expression of Bcl-2 protein down-regulated （P 〈0.01, P 〈0.05）. Conclusions DHA had stronger cytotoxicity and radiosensitization on GLC-82 cells. Its mechanisms might lie in making the arrestof GLC-82 cells＇ growth at G0/G1 phase, decreasing the ratio of cells at S phase, restoring the function of p53, decreasing the expression of Bcl-2 protein, and inducing apoptosis in GLC-82 cells.