Rh2逆转耐药细胞对顺铂抗性作用的机制

Research on the mechanism of gensenoside Rh2 reversing the resistance of lung adenocarcinoma cells to cisplafin

目的探讨人参单体Rh2逆转肺腺癌耐药A549DDP细胞对顺铂抗性作用的机制。方法以Rh2单独作用为Rh2组，低效浓度的顺铂单独作用为DDP组，二者联合作用为Rh2＋DDP组，不加药物干预为对照组，紫外光分光光度计检测各组细胞的线粒体PTP开放情况，流式细胞仪检测细胞内钙离子浓度及线粒体跨膜电位的变化及细胞凋亡状况，Western印迹检测细胞色素C及Caspase-3表达；Hoechs33258染色荧光显微镜下观察细胞形态的变化。结果Rh2组和DDP组细胞内钙离子浓度与对照组的接近，Rh2＋DDP组细胞内钙离子浓度升高，与对照组比较，差异有统计学意义（t=22．47，P〈0．01）。前3组细胞的线粒体吸光度（A540nm）值接近，而Rh2＋DDP组A540nm值明显低于对照组（t=8．21，P〈0．01）。前3组线粒体跨膜电位接近，Rh2＋DDP组细胞内线粒体跨膜电位明显低于对照组（χ^2=46．01，P〈0．01）。前3组细胞的胞质中细胞色素C和Caspase-3表达极低，Rh2＋DDP组细胞色素C和Caspase-3高表达。荧光显微镜下，前3组细胞核荧光分布均匀；Rh2＋DDP组的细胞核数目减少，荧光成团块分布，很多细胞核缩小或变形。前3组凋亡细胞百分率分别为6．32％、7．24％、7．41％，3组间差异无统计学意义，Rh2＋DDP组凋亡细胞百分率为21．96％，与对照组差异有统计学意义（t=10．92，P〈0．05）。结论无毒浓度的Rh2逆转A549DDP细胞对顺铂的耐药性作用可通过线粒体凋亡通路实现。

Objective To explore the mechanism of gensenoside Rh2 in reversing the resistance of lung adenocarcinoma cells to cisplatin. Methods In the Rh2, DDP and DDP ＋ Rh2 group A549DDP cells were treated with Rh2, cisplatin, cisplatin ＋ Rh2 respectively for 48 hours. In the control group the A549DDP cells were not treated with any kind of drugs. The state of mitochondrial permeability transition pore （PTP） was evaluated by uhraviolate spectrofluorometer. Concentration of calcium in cells, membrane potential of mitochondrion and apoptosis cells were determined by flow cytometry. The expression of cyt-c and Caspase-3 was estimated by Western blots . Cellular shapes were observed by fluorencent microscopy. Results The concentrations of calcium were similar among the former three groups, but was obviously increased in the Rh2 ＋ DDP group （ t = 22.47, P 〈 0. 01 ）. A54o of mitoehondrion among the former three groups were not different , but decreased significantly in the Rh2 ＋ DDP group （ t = 8. 21, P 〈 0. 01 ）. The membrane potentials of mitochondrion showed no difference in the former groups, that was remarkably lower in the Rh2 ＋ DDP group than in the control group （ χ^2 = 46.01, P 〈 0. 01 ）. There was little expression of cty-c and Caspase-3 in the former groups , but high expression of those in the Rh2 ＋ DDP group. Fluorenscence was distributed equably on the cells nucleus in the former groups. In the Rh2 ＋ DDP group many cell nucleus shrank or were distorted. The apoptosis rate of the cells was 6. 32% ,7. 24% ,7.41% and 21.96% in the four groups respectively. Which in Rh2 ＋ DDP group was higher than that in the control group （ t = 10. 92, P 〈 0. 05 ）. Conclusions The effect of gensenoside Rh2 reversing resistance of lung adenocarcinoma A549DDP cells to cisplatin can be performed by apoptotic mitochondria pathway.