小白菊内酯对人肺癌A-549细胞周期变化和凋亡通路的影响

The research of parthenolide on changing the cell cycle and influencing the apoptosis pathways of human lung cancer A-549 cell line

目的:研究小白菊内酯对人肺癌A-549细胞株的抗增殖和诱导凋亡作用及其机制,为研发新型抗肿瘤中药奠定坚实的基础。方法:应用MTT法测定小白菊内酯对肺癌细胞的毒性作用和抑制增殖作用;应用倒置显微镜、Annexin V-FITC/碘化丙锭(PI)染色荧光显微镜观察药物作用前后人肺癌A-549细胞的形态学变化;应用活性氧(ROS)检测药物作用后细胞内活性氧的水平,以及活性氧含量与细胞凋亡之间的关系;应用免疫细胞化学技术检测药物作用前后蛋白阳性表达变化的情况并进一步探讨小白菊内酯对人肺癌A-549细胞株抑制增殖和诱导凋亡的作用机制。结果:(1)MTT法检测结果显示:小白菊内酯在不同浓度(5mg/L、10 mg/L、20 mg/L、40 mg/L、50 mg/L、55 mg/L、60 mg/L、65 mg/L)对人肺癌A-549细胞有不同程度的生长抑制作用,并呈现出浓度和时间梯度依赖性,当药物终浓度为55 mg/L作用时间为48h时,对A-549细胞的抑制率为51.02%。(2)Annexin V-FITC/PI染色荧光显微镜观察发现实验组细胞可见大量绿色荧光细胞和红色凋亡细胞。(3)活性氧检测发现:实验组细胞内活性氧的水平出现明显升高。(4)免疫细胞化学检测法结果显示对照组Caspase-3、Caspase-8、Caspase-9、Fas和Bax分别为10.07%、11.42%、14.09%、9.75%和11.87%,加入小白菊内酯后(实验组)细胞中Caspase-3、Caspase-8、Caspase-9、Fas和Bax蛋白的表达为89.24%、85.75%、84.46%、85.14%和84.09%,与对照组相比明显增加,有显著差异。结论:小白菊内酯作用于人肺癌A-549细胞株后,诱导其出现细胞凋亡的改变,它通过死亡受体通路和线粒体通路来完成诱导凋亡的机制,小白菊内酯作用后活性氧水平的增加可能参与诱导细胞凋亡的机制。

Objective： To investigate the effect of parthenolide on inhibiting proliferation and inducing apoptosis of human lung cancer A-549 cell line in vitro,and to provide the evidence of experiment and theory for the new anti-tumor Chinese medicine. Methods： It was applied that training human lung cancer A-549 cells in vitro. MTT assay was used to measure the toxic effect and the growth inhibition rate of human lung cancer A-549 cells which were treated with parthenolide by different concentrations（ 5 mg / L,10 mg / L,20 mg / L,40 mg / L,50 mg / L,55 mg /L,60 mg / L,65 mg / L）,when the concentration of drug eventually is 55 mg / L and the duration of 48 h,the inhibition rate achieve 51. 02%on the A-549 cells; Morphological changes of the tumor cells were observed by Annexin V-FITC / PI double staining; The method was used that testing the level of reactive oxygen species in cells and discussing the relationship between ROS with the apoptosis of cells; Immunocytochemistry was used to detect the changes of protein expression and in order to further explore the mechanism research of parthenlide on inducing the proliferation and apoptosis of human lung cancer A-549 cell line. Results：（ 1） MTT assay showed that parthenolide by different concentrations had different levels of growth inhibition on human lung cancer A-549 cell line and the effect was in a doseage and time gradient dependence.（ 2） Annexin V-FITC / PI double staining and fluorescence microscope showed the control group cells almost colored,but the experimental group cells appeared amount of green and red fluorescent apoptosic cells.（ 3） Reactive oxygen species assay detected the level of the ROS in cells increased significantly that compared with the control group.（ 4） Parthenolide up-regulated the expression of Caspase-3,Caspase-8,Caspase-9,Fas,Bax from 10. 07%,11. 42%,14. 09%,9. 75% and 11. 87% to 89. 24%,85. 75%,84. 46%,85. 14% 和 84.09%,and they were markedly different from the control groups（ P〈0. 05）. Conclusions： The A-549 cells after were roled with parthenolide show the obvious changes of apoptosis,the mechanism of apoptosis which parthenolide induce is completed by the death receptor pathways and the mitochondrial pathways,the increase of the level of the ROS that were treated with parthenolide may participate in inducing the apoptosis mechanism.