【作者】 安巍巍；

【导师】 王敏伟； 池岛乔；

【作者基本信息】 沈阳药科大学 ， 药理学， 2004， 博士

【摘要】 本文对去甲斑蝥素体外抗肿瘤活性进行了较为系统的研究，主要阐述了去甲斑蝥素在三种肿瘤细胞：人黑色素瘤A375-S2细胞，人宫颈癌HeLa细胞，小鼠纤维瘤L929细胞中诱导细胞死亡的机制。 实验结果表明去甲斑蝥素对A375-S2、L929和HeLa细胞均具有明显的细胞毒活性，抑制细胞生长呈明显的时间和剂量依赖性。形态学方法、核荧光染色、DNA片段化分析、及LDH活力分析实验证实了去甲斑蝥素诱导A375-S2、L929和HeLa细胞死亡通过诱导细胞凋亡的机制。 去甲斑蝥素诱导A375-S2细胞凋亡的信号转导途径中多种基因、蛋白酶以及各种激酶参与了凋亡的调控。Bcl-2/Bax或Bcl-x_L/Bax的蛋白表达比率在给药后明显降低，促进了细胞色素c的释放，进而激活了caLspase-9，caspase-9的激活引发了caspase的级联反应，导致下游的caspase-3的激活，caspase-3切开ICAD和CAD的复合物，释放的CAD切断DNA致使核基质崩塌导致细胞凋亡。同时，去甲斑蝥素诱发了p38和JNK激酶的激活，蛋白激酶C在去甲斑蝥素激活MAPK过程中起重要的调节作用。 去甲斑蝥素作用L929细胞后，caspase家族抑制剂不抑制去甲斑蝥素诱导L929细胞的死亡，而促进L929细胞对去甲斑蝥素的敏感性，但caspase-3抑制剂可时间、剂量依赖性的抑制去甲斑蝥素对L929细胞的作用。去甲斑蝥素作用24h后PARP蛋白发生降解，即激活成有活性的片断，同时ICAD也发生了降解。60μmol/L去甲斑蝥素作用细胞12h时Bax与Bel-2的表达比率开始升高，在24，36h时非常明显，而Bcl-2家族另一抗凋亡蛋白Bcl-x_L表达在36h表达降低，同时PKC抑制剂staurosporine可抑制去甲斑蝥素诱导的Bcl-x_L表达的降低，而对Bax和Bcl-2蛋白表达影响较弱；MAPK家族中ERK和JNK蛋白的激活参与了去甲斑蝥素对L929细胞的作用，而p38蛋白的激活较弱；60μmol/L去甲斑蝥素处理L929细胞，随着作用时间的增加，p53蛋白的磷酸化增加，P13-K的抑制剂wortmannin在12h时有效抑制了p53的激活，提示P13-K参与了去甲斑蝥素对L929细胞中p53激活的调控。 在去甲斑蝥素诱导HeLa细胞凋亡的过程中，激活了caspase-8，caspase-8的激活引发了caspase的级联反应，导致下游的caspase-3的激活，caspase-3裂解ICAD和CAD的复合物，释放的CAD切断DNA致使核基质崩塌导致细胞凋亡。去甲斑蝥素更多还原

【Abstract】 This dissertation reports that norcantharidin (NCTD) induced cell death through different mechanisms in A375-S2, HeLa, and L929 cells.The studies demonstrate that NCTD inhibited the proliferation of human melanoma A375-S2, mouse fibrosarcoma L929 and human cervical HeLa cells in a dose- and time-dependent manner. MTT assay, photomicroscopical observation and DNA agarose gel electrophoresis showed that NCTD induced cell apoptosis in A375-S2, L929 and HeLa cells.In NCTD-treated A375-S2 cells, cysteine aspartic specific proteases: caspase-3 and -9 were activated, and the expression of ICAD and PARP was decreased in a time-dependent manner. NCTD increased the expression of the apoptosis inducer, Bax, decreased the expression of the anti-apoptotic protein, Bcl-xL and Bcl-2, promoted the release of cytochrome c, and activated down-stream caspase-9 in mitochondrial apoptotic pathway. The change of Bcl-2/Bax or Bcl-xL/Bax expression was reversed by caspase-3 inhibitor, and caspase-3 inhibitor had no significant effect on the release of cytochrom c. These observations indicated that appropriate dose of NCTD activated the mitochondrial apoptotic pathway that involved the Bcl-2 family including Bcl-2, Bcl-xL and Bax. At the same time, the participation of caspase-9 and caspase-3 was required in this apoptotic pathway. The activity of caspase-8 increased at 28 h and suggested that caspase-8 was involved in the later stage of apoptosis. The inhibitory effect of NCTD on A375-S2 cells was partially reversed by the inhibitors of MAPK inhibitors and PKC inhibitors. The expression of JNK phosphorylation and p38 phosphorylation was increased after treatment with NCTD and the inhibitor of JNK and p38 had significant inhibitory effects on the protein up-regulation of phosphorylated JNK and p38 expression. Simultaneously, PKC family-inhibitor, staurosporine, blocked the up-regulation of phosphorylated JNK and phosphorylated p38 and had little effect on the ERK expression. Taken together, NCTD induced A375-S2 cell apoptosis by activating JNK and p38 pathways, increasing the ratio of Bax/Bcl-xL or Bax/Bcl-2 protein expression, and promoting the release of cytochrome c更多还原