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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 study the anti-proliferation and apoptosis-inducing effect of parthenolide on human lung cancer cell line A-549 and its mechanism, and to lay a solid foundation for the development of new anti-tumor traditional Chinese medicine. Methods: MTT assay was used to determine the toxicity of parthenolide on lung cancer cells and its inhibitory effect on proliferation of human lung cancer cell line A-549 was observed with inverted microscope and Annexin V-FITC / propidium iodide (PI) staining. The changes of intracellular reactive oxygen species (ROS) and the content of reactive oxygen species (ROS) in cells were detected by reactive oxygen species (ROS) assay. The changes of protein expression before and after drug treatment were detected by immunocytochemistry And further explore the parthenolide human lung cancer cell line A-549 proliferation and apoptosis induction mechanism. Results: (1) The results of MTT assay showed that the effects of parthenolide on the proliferation of ovarian epithelial cells in different concentrations (5mg / L, 10mg / L, 20mg / L, 40mg / L, 50mg / L, 55mg / L, / L, 65 mg / L) could inhibit the growth of human lung cancer A-549 cells to a certain extent and showed a concentration-dependent and time-dependent manner. When the drug concentration was 55 mg / L for 48 h, The inhibition rate of -549 cells was 51.02%. (2) Annexin V-FITC / PI staining Fluorescence microscopy showed that a large number of green fluorescent cells and red apoptotic cells were observed in the experimental group. (3) The detection of reactive oxygen species (ROS) showed that the levels of reactive oxygen species in the experimental group were significantly increased. (4) Immunocytochemistry results showed that Caspase-3, Caspase-8, Caspase-9, Fas and Bax in control group were 10.07%, 11.42%, 14.09%, 9.75% and 11.87% The expression of Caspase-3, Caspase-8, Caspase-9, Fas and Bax in experimental group was significantly higher than that in control group (89.24%, 85.75%, 84.46%, 85.14% and 84.09% difference. Conclusion: Parthenolide can induce the apoptosis of A-549 cell line in human lung cancer cell line A-549. It induces apoptosis through the death receptor pathway and mitochondrial pathway. The activity of parthenolide Increased oxygen levels may be involved in the mechanism of apoptosis induction.