caspase-9在重离子辐射诱导的丝裂灾变致死效应中的作用研究

Mitotic catastrophe(MC) is an oncosuppressive cascade that precedes cell death and senescence. The molecular bridges that link mitotic aberrations and cell death in lethal mechanism of MC are still largely elusive. Caspase-9 is the key initiator caspase in mitochondria apoptosis pathway. The preliminary studies of our research group discovered that caspase-9 was activated after irradiation high LET carbon ion on metaphase HeLa cells, when MC was effectively induced. We proposed that activation of caspase-9 is an critical link that contacts MC with apoptosis. To confirm this assumption, the study of caspase-9 function will contribute to the understanding of cohesive mechanism between MC and apoptosis. p53 is the key molecular of apoptosis induction in the case of DNA damages. In this application project, taking tumor cells with different p53 status as research object, siRNA technique and recombinant of eukaryotic expression vector for human p53 will be used to confirm whether p53 status in tumor cells influrences the apoptotic pathway following MC induced by heavy ions. And siRNA technique, specific inhibitor of caspase-9 and site-directed mutagenesis technique will be used to exploit the function as link of caspase-9 in connection between MC and lethal apoptosis induced by heavy ions. This study will contribute to understanding the lethal effects of MC induced by heavy ions and establishing foundations for promoting cancer curative effects of heavy ions.

丝裂灾变（MC）是发生在凋亡或坏死前的肿瘤抑制机制。MC致死效应机制中联系有丝分裂异常与细胞死亡的分子纽带尚未阐明。本课题组前期研究显示，重离子诱导肿瘤细胞发生MC的同时，线粒体凋亡通路的关键起始蛋白酶caspase-9被活化。因此推测caspase-9活化是联系重离子诱导的MC与凋亡的重要环节。为证实该假说，研究caspase-9功能对理解MC与凋亡的衔接机制将有很大裨益。p53是细胞在DNA 损伤后诱导凋亡的关键因子。本项目拟以不同p53状态肿瘤细胞为研究对象，采用siRNA技术和人重组真核表达载体，明确细胞p53状态对重离子诱导MC致死效应中凋亡通路的影响；并利用siRNA技术、caspase-9抑制剂及定点突变法，明确caspase-9在重离子诱导的MC和凋亡间的纽带作用。本研究是对重离子诱导肿瘤细胞MC致死效应的有益探索，为进一步提高重离子治疗疗效奠定理论基础。

与低LET辐射相比，碳离子辐射已经被证实能诱导更高频率的复杂性DNA损伤，包含DNA双链断裂（DSBs）和非DSB簇性损伤。我们好奇于高LET碳离子辐射诱导的有丝分裂期DNA损伤能否引起二次DNA损伤并关注在此过程中caspase-9是否参与丝裂灾变（mitotic catastrophe, MC）介导的凋亡。研究发现用高LET碳离子辐照经同步化到有丝分裂前中期的HeLa细胞后，有丝分裂后G1和S检查点被削弱以及有丝分裂后G2-M检查点被废除，因此无法阻止损伤DNA的错误复制和染色体错误分离。同时，有丝分裂滑脱只与nocodazole的使用有关，胞质分裂失败和细胞融合共同促进双核细胞形成。Chk1和Cdh1活性在大量多倍体出现时受到抑制，显示出染色体核型和数量的改变。细胞在经过同步化和辐照后增殖能力完全被抑制并且细胞发生强烈的凋亡。由于增加的染色体损伤导致更多的H2AX磷酸化，因此我们认为碳离子辐照有丝分裂细胞诱导了二次H2AX磷酸化，并且该二次损伤提供了最后的机会对染色体不稳定的危险细胞进行清除。此外，活化的caspase-9仅仅在单纯同步化细胞和碳离子辐照的同步化细胞中发现。结合碳离子辐照的同步化细胞在药物释放的0h就发生了强烈的急性的MC和细胞凋亡（此后也一直维持高水平的凋亡），而单纯辐照细胞则发生了延迟的高水平MC和凋亡，我们推测caspase-9参与急性MC介导的凋亡而非延迟的凋亡。具体的机制仍有待在更多不同p53状态的细胞中进行研究。

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