SERCA分子参与二氢青蒿素诱导结肠癌细胞凋亡的机制研究

Dihydroartemisinin (DHA) may induced a complex reaction of the apoptotic signaling network in colon cancer cells by direct inhibition of the endoplasmic reticulum calcium pump SERCA, and the inhibition by DHA on SERCA may be iron dependent. . .This subject intends to select the key protein DHA induced apoptosis in colon cancer cells by screening siRNA library. To analyze the artemisinin-induced expressional-wild type and mutant type (DHA non-sensitive) of the SERCA colon cancer cell apoptosis genes differentially expression by real-time PCR microarray analysis; To identify the influence on reaction kinetics by external resolution of DHA and ferri ions on the of the wild-type and mutant type SERCA. . .Therefore, to determine the important signaling molecules on DHA induced apoptosis in colon cancer cells and establish a more comprehensive understanding on DHA-induced colon cancer cell apoptosis network, as well as to interprete the role of SERCA in the mechanism of the DHA molecular and cellular on a leveraged level. And it is also a strong expectation that new ideas and thoughts on the research of anti-tumor medicine would continously come up by in-depth and profound studying and analyzing the artemisinin mechanism of drug action research.

二氢青蒿素（DHA）可能通过直接抑制内质网钙泵SERCA诱导结肠癌细胞复杂的凋亡信号网络反应，而且DHA对SERCA的抑制可能具有铁离子依赖性。本课题拟通过siRNA文库筛选DHA诱导结肠癌细胞凋亡的关键蛋白；利用实时PCR微阵列分析青蒿素诱导的表达野生型和突变型(DHA非敏感型)SERCA结肠癌细胞的凋亡基因差异表达；体外解析DHA和铁离子对野生型和突变型SERCA反应动力学的影响。从而确定DHA诱导结肠癌细胞凋亡的重要信号分子，建立对DHA诱导结肠癌细胞凋亡网络较全面的认识，解读SERCA在DHA分子和细胞水平作用机制中所承担的角色。并期待在纵深拓展青蒿素药物作用机制研究的同时，为抗肿瘤中药的研究提供新思路和开辟新途径。

二氢青蒿素（DHA）可能通过直接抑制内质网钙泵SERCA诱导结肠癌细胞复杂的凋亡信号网络反应，而且DHA对 SERCA的抑制可能具有铁离子依赖性。目前其确切机制不清楚，有待探讨。本课题拟通过siRNA文库筛选DHA诱导结肠癌细胞凋亡的关键蛋白；利用实时PCR微阵列或Western分析青蒿素诱导的表达野生型与突变型（DHA非敏感型）SERCA结肠癌细胞的凋亡基因差异表达。期望通过本研究全面解析DHA在HCT116细胞诱导凋亡信号网络的关键蛋白、途径及期间的关系；从细胞水平及分子水平阐明SERCA在DHA促凋亡机制中承担的角色。从而确定DHA诱导结肠癌细胞凋亡的重要信号分子，建立对DHA诱导结肠癌细胞凋亡网络较全面的认识，解读SERCA在DHA分子和细胞水平作用机制中所承担的角色。并期待在纵深拓展青蒿素药物作用机制研究的同时，为抗肿瘤中药的研究提供新思路和开辟新途径。.方法：应用ELISA 检测不同浓度DHA处理HCT116细胞后SERCA活性;:利用Western分析青蒿素诱导的表达野生型与突变型（DHA非敏感型）SERCA结肠癌细胞的凋亡基因差异表达; 构建siRNA文库；siRNA文库筛选DHA诱导结肠癌细胞凋亡的关键蛋白。.结果:.①.DHA诱导HCT116细胞凋亡。.②.CHOP在DHA诱导HCT116细胞凋亡中高表达。.③.siRNA沉默CHOP表达后，细胞凋亡被抑制。.④.DHA诱导HCT116细胞凋亡时SERCA2b活性被抑制。.⑤.余见正文..结论:DHA诱导HCT116细胞凋亡涉及内质网应激、死亡受体介导和线粒体途径等复杂的分子机制. 本研究揭示内质网应激是DHA诱导HCT116细胞凋亡的始动机制!!但二氢青蒿素（DHA）与SERCA分子结合位点及SERCA分子调控的上下游关系.凋亡的信号网络等有待进一步探讨。