【作者】 杨杰；

【导师】 蒋宇扬；

【作者基本信息】 清华大学 ， 化学， 2005， 硕士

【摘要】 磷在生命科学中扮演着十分重要的角色,它不仅是生命体的结构组成部分,而且是调节生命体新陈代谢等重要生理生化功能的基本元素。本文对具有诱导肿瘤细胞凋亡活性的N-磷肽甲酯进行了构效关系的研究,并对促凋活性最好的化合物诱导K562细胞凋亡的机理进行了较深入的探讨。以（DIPP-Leu）₂-LysOMe、（DIPP-Trp）₂-LysOMe两种N-磷肽甲酯为模型化合物,研究其类似物对K562细胞增殖抑制活性的影响,构效关系表明:氨基酸构型和磷酯结构均对化合物的细胞增殖抑制活性有较大影响。含L型氨基酸的N-磷肽甲酯活性高于含D型氨基酸的,其中赖氨酸构型的改变对化合物活性的影响最大;含二异丙氧基磷酯结构的化合物活性最高,改变磷酯结构中烷氧链的长度,化合物对K562细胞增殖抑制活性急剧下降。凋亡机理研究表明:（DIPP-L-Leu）₂-L-LysOMe是通过线粒体依赖的通路诱导K562细胞凋亡。在凋亡早期,化合物的作用上调促凋亡蛋白Bax的表达,下调抗凋亡蛋白Bcl-2和Bcl-x_L的表达,由此改变了线粒体渗透转运孔（PTP）,导致线粒体膜电位（MMP）下降、膜的通透性增大,造成细胞色素c （Cyto c）从线粒体释放到细胞质中,从而激活下游的caspase-9和caspase-3,启动线粒体调控的凋亡通路,诱导K562细胞凋亡。此外,该化合物还能导致细胞内Ca²⁺浓度升高,以及活性氧（ROS）产生,这与细胞凋亡的生化指标相符。这一系列事件表明（DIPP-L-Leu）₂-L-LysOMe是通过线粒体路径启动细胞凋亡的,而且调控Bcl-2家族蛋白和诱导线粒体功能异常可能是其引起细胞凋亡发生的主要作用机理。更多还原

【Abstract】 Phosphorus plays a crucial role in life science. Many phosphoryl proteinsand phosphoryl peptides have important biological activities. In the presentdissertation, the synthesis and activities of inhibiting K562 cells’ （humanchronic myelogenous leukemia （CML） cell line） proliferation of a series ofN-phosphoryl peptide methyl esters have been investigated, and theapoptosis-inducing mechanism of （DIPP-L-Leu）₂-L-Lys-OMe on K562 cellshas been further studied.Study on the structure-activity relationship of the analogues of（DIPP-Leu）₂-LysOMe and （DIPP-Trp）₂-LysOMe showed the configurations ofamino acids and the phosphoryl groups of N-phosphoryl peptide methyl estersaffect the compounds’ activities of inhibiting K562 cells’ proliferation. TheN-phosphoryl peptide methyl esters synthesized by L-amino acides have thehigher activities than those synthesized by D-amino acids. The configurationof Lysine has the greater influence on the compounds’ activities than that ofother amino acids. The result also indicated that （N-diisopropyosyl-L-Leu）₂-L-LysOMe has the higher activity than other substituted alkyloxy（N-phosphoryl-L-Leu）₂-L-LysOMe.（DIPP-L-Leu）₂-L-LysOMe was known to induce apoptosis of K562 cells.Studies on the molecular and cellular mechanism involved in this processshowed that （DIPP-L-Leu）₂-L-LysOMe-induced apoptosis is associated withcytosolic accumulation of cytochrome c （Cyto c）, sustained loss ofmitochondrial transmembrane potential （MMP）, transient generation ofreactive oxygen species （ROS） and elevation of intracellular Ca²⁺concentration. Immunofluorescence analysis indicates that （DIPP-L-Leu）₂-L-LysOMe induced upregulation of pro-apoptotic Bax and downregulation ofanti-apoptotic Bcl-2 and Bcl-x_L. Together with previous research that（DIPP-L-Leu）₂-L-LysOMe cause the activation of caspase-9 and -3 but notcaspase-8, present findings suggest that （DIPP-L-Leu）₂-L-LysOMe induce theapoptosis through mitochondria-dependent pathway in K562 cells. Theregulation of Bcl-2 family members and mitochondrial dysfunction aredirectly responsible for the pro-apoptotic effects of （DIPP-L-Leu）2-L-Lys-OMe.更多还原