【作者】 郑燕彬；

【导师】 李祺福；

【作者基本信息】 厦门大学 ， 细胞生物学， 2007， 硕士

【摘要】 本文在鉴定姜黄素(curcumin, Cur)诱导人成骨肉瘤MG-63细胞凋亡的基础上,应用选择性抽提整装透射电镜和蛋白质组学分析技术,对MG-63细胞诱导凋亡过程中核基质-中间纤维系统的构型变化和核基质蛋白组成的变化进行系统研究。分析鉴定与肿瘤细胞凋亡相关的特异核基质蛋白,并探索它们在肿瘤细胞凋亡过程中的调控作用,以期能够在较为整体的水平上进一步认识肿瘤细胞凋亡及其机理,从而找出肿瘤细胞凋亡研究的新方向。实验结果显示,经7.5μg/mL姜黄素处理之后,人成骨肉瘤MG-63细胞的增殖受到明显抑制,发生了G0/G1期阻滞,出现明显的亚G0/G1期凋亡峰,光镜和透射电镜观察结果显示,经姜黄素诱导处理后的MG-63细胞出现了细胞形态规则、核质比例减小、细胞核固缩、核内染色质凝聚、线粒体肿胀、内质网网腔扩大、出现凋亡小体等显著的凋亡特征;而且凋亡相关基因bcl-2和突变型p53的表达下调、bax和fas基因的表达上调,并且出现细胞凋亡特征性DNA降解梯状条带;选择性抽提整装光镜和电镜观察显示,经姜黄素处理的MG-63细胞的核基质和中间纤维比对照组明显稀疏,且分布更加不均匀,并分别与核纤层连系,形成趋于断裂但相对还较为完整的网状结构;双向凝胶电泳分析显示在姜黄素诱导MG-63细胞凋亡前后存在27个差异表达的核基质蛋白,经质谱分析,鉴定了其中的21个蛋白,其中在凋亡的MG-63细胞中表达上调的蛋白鉴定为:DNA断裂诱导转录子DNA-damage-inducible transcript 4-like、盘状结构域Discoidin、DNA聚合酶zeta等7种蛋白;表达下调的蛋白质为:Prohibitin、Glutamate dehydrogenase、Vimentin、Human Nucleophosmin、Heat-shock 70等14种蛋白。其中除Vimentin、Prohibitin、Nucleophosmin和Hsp70外,其余均为首次在核基质中发现的蛋白质。研究结果表明,7.5μg/mL姜黄素对人成骨肉瘤MG-63细胞的凋亡具有显著诱导作用,在MG-63细胞凋亡过程中不仅其核基质-中间纤维系统构型产生了与其相应非肿瘤细胞相对的凋亡特征性变化,而且其核基质蛋白组成也相应发生了明显改变。由此证实了与肿瘤细胞凋亡诱导相关特异核基质蛋白的存在及其对肿瘤细胞凋亡诱导的调控作用。更多还原

【Abstract】 In this study, the curcumin (Cur) was used to induce the human osteosarcoma MG-63 cells into apoptosis, and its effects were investigated by selective extraction-whole mount optic and transmission electron microscopy and techniques of proteomic. The differentially expressed nuclear matrix proteins were analyzed in order to explore the molecular mechanisms in a system level.The results revealed that the osteosarcoma MG-63 cells were induced into apoptosis after treated with 7.5μg/mL Cur as the proliferation of MG-63 cells were inhibited, and the cell cycle were arrested in G0/G1 phase. The malignant morphological and ultrastructural characteristics were changed, while the expression level of apoptosis promote gene was upregulated, and the expression level of apoptosis suppressor gene was downregulated. And at the same time,the configuration of nuclear matrix-intermediate filament(NM-IF)was altered. Nuclear matrix proteins(NMPs), selectively extracted from MG-63 cells treated with or without Cur, were subjected to proteomic analysis.There were 21 of 27 differentially expressed spots were identified, including the DNA damage inducible transcript, Discoidin, DNA ploymase zeta, prohibitin, glutamate dehydrogenase, Vimentin, Human Nucleophosmin, Heat-shock 70 etc. associated with remodeling of cellular configuration. Except vimentin, prohibitin, HSP70, nucleophosmin, all other proteins were found as NMPs for the first time.In conclusion, the configuration and composition of nuclear matrix were changed following the MG-63 cell apoptosis, and it is possible to get a system understanding of the molecular mechanisms from the studies of nuclear matrix.更多还原