水飞蓟宾诱导肺腺癌Anip973细胞凋亡的分子机制研究

摘要

目的:探讨水飞蓟宾诱导肺腺癌Anip973细胞系细胞凋亡的分子机制.方法:采用MTT法、倒置显微镜和电子显微镜等形态学检测以及流式细胞仪(FCM)技术检测、DNA Ladder分析、凋亡分子PARP的表达检测细胞凋亡,同时进行凋亡相关蛋白Bax、Bcl-2、caspase-3和caspasc-9表达活性分析.结果:(1)水飞蓟宾对人肺腺癌Anip973细胞系细胞的增殖有显著抑制作用;(2)水飞蓟宾作用Anip973细胞48h后,随着浓度的增加,倒置显微镜下可见细胞数目减少,胞体变小、变圆,到高浓度时出现较多的死亡细胞;(3)扫描电镜观察发现,随着水飞蓟宾作用浓度的增加,Anip973细胞中出现增多的凋亡细胞,凋亡细胞表现出典型的超微结构特征;(4)流式细胞仪检测的结果发现,随着药物作用时间的延长,Anip973细胞的G1期细胞比例增多,S期细胞明显减少,G2期细胞略有减少,并出现明显的凋亡峰.(5)水飞蓟宾作用后的Anip973细胞出现明显的DNA Ladder和PARP降解增加等凋亡特征;(6)水飞蓟宾作用后,Anip973细胞中的凋亡相关蛋白Bax表达增加、caspase-3和caspase-9酶活性增加.而Bel-2表达降低.结论:水飞蓟宾在体外有抑制人肺腺癌细胞Anip973的增殖作用,并通过激活线粒体依赖的caspase凋亡通路,诱导其凋亡.%Objective: To investigate the molecular mechanism of Sliybinin-induced apoptosis of Lung Cancer Cell Anip973.Methods: The cancer cell apoptosis was detected by methyl thiazolyl tetrazolium (MTT) colorimetric assay, morphological observations with inverted microscope and electron microscope, flow cytometry, DNA ladder analysis, expression analysis of PARP as well as the expression and activity analysis of some apoptosis-related proteins including Bax, Bcl-2, caspase-3 and caspase-9.Results: (1) Sliybinin had significant inhibiting effect on the cellular proliferation of Anip973 cells; (2) When treated by Sliybinin for 48 hours, with the increasing of the concentrations, the number of Anip973 cells decreased, the cell body became smaller and more circular under light microscope,and the number of dead cells escalated with an increased concentration of silymarin treatment (3) More Anip973 cells presented apoptosis and these cells appeared typical ultrastructural features with the increasing of Sliybinin concentration; (4) The proportion of Anip973 cells in Gl phase increased, and the number of cells in S phase decreased significantly, which reduced slightly in G2 phase and also presented significant apoptotic peak with the prolongation of drug action; (5) The Anip973 cells treated with Sliybinin presented distinct apoptotic features including DNA ladder, PARP degradation, etc; (6) In Anip973 cells treated with Sliybinin, the expression of Bax and the enzyme activities of caspase-3 and caspase-9 increased, while the expression of Bcl-2 decreased.Conclusions: Sliybinin can inhibit the proliferation of human lung adenocarcinoma cell line Anip973 and induce its apoptosis by activating the mitochondria-dependent caspase cascade pathway.