【作者】 唐剑；

【导师】 李祺福；

【作者基本信息】 厦门大学 ， 细胞生物学， 2008， 博士

【摘要】 本研究以环六亚甲基双乙酰胺（HMBA）处理人肝癌SMMC-7721细胞,鉴定HMBA对人肝癌SMMC-7721细胞的诱导分化效果。在此基础上,综合应用蛋白质组学、免疫细胞化学、细胞分子生物学等技术,对SMMC-7721细胞诱导分化前后核基质蛋白表达变化进行系统研究,分析鉴定和确证与肿瘤细胞增殖分化相关的特异核基质蛋白,并研究特异核基质蛋白与人肝癌细胞相关癌基因、抑癌基因产物在细胞内的共定位关系与变化,探索它们在肿瘤细胞诱导分化过程中的调控作用。以期能够在较为整体的水平上阐明肝癌细胞诱导分化的调控机制,并进一步认识细胞癌变与逆转机理和细胞增殖与分化的调控原理问题。实验结果显示,5 mmol/L HMBA对人肝癌SMMC-7721细胞的增殖活动具有抑制作用,诱导处理7天后,细胞生长抑制率达50%,细胞倍增时间延长1.8倍,G₀/G₁期细胞比例由40.6%上升为70.6%,S期细胞比例由27.4%下降为6.9%,细胞周期被阻滞于G₀/G₁期;光镜和电镜观察结果显示,经5 mmol/LHMBA处理后的SMMC-7721细胞出现核质比减小,细胞体积增大,细胞表面微绒毛减少,细胞核形态趋于规则,核仁数目减少,核内异染色质减少,常染色质增多,线粒体嵴数目增多,内质网和高尔基体更为典型、发达等变化;免疫细胞化学检测结果显示,经诱导处理后,SMMC-7721细胞内癌基因c-fos、c-myc、c-erbB-2、bcl-2以及mtp53表达产物减少,抑癌基因Rb、p21、p27表达产物增多;选择性抽提整装光镜和电镜观察显示,经HMBA处理的SMMC-7721细胞核基质纤维和中间纤维数量和层次更加丰富,分布更为均匀,单丝成份增多,与核纤层连系更为紧密,形成贯穿整个核质区域的较为规则的纤维网络;双向凝胶电泳与MALDI-TOF质谱分析共鉴定出9个在HMBA诱导分化前后差异表达的核基质蛋白,其中表达上调的有4个,分别是p27^BBP、BTF2-p44 subunit、tubulin beta 2C、sin3b;表达下调的有4个,分别是突变型pystl、DKFZp434K1815 variant、laminin-binding protein、nucleophosmin:新出现的一个蛋白是SFRS1;蛋白印迹杂交、免疫荧光显微镜和免疫胶体金标记电镜观察确证了nucleophosmin和prohibitin在SMMC-7721细胞诱导分化前后细胞核基质中的表达变化及其在核基质上的定位;激光共聚焦显微镜观察显示nucleophosmin和prohibitin与癌基因c-myc、c-fos和抑癌基因p53、Rb表达产物在细胞内均存在一定的共定位关系,并且它们的表达水平和细胞定位在诱导分化前后发生了变化。研究结果表明,5 mmol/L HMBA能有效抑制人肝癌SMMC-7721细胞的增殖活动,改变SMMC-7721细胞形态与超微结构特征,下调癌基因c-los、c-myc、c-erbB-2、bcl-2、mtp53和上调抑癌基因Rb、p21、p27等的表达,从而对人肝癌细胞的分化具有显著诱导效果。在SMMC-7721细胞分化过程中,其核基质.中间纤维系统构型产生了与正常细胞相似的恢复性变化,并且出现了多种差异表达的核基质蛋白,其中nucleophosmin和prohibitin为不同诱导分化物诱导多种肿瘤细胞后出现的共同差异核基质蛋白。而nucleophosmin和prohibitin与c-Myc、c-Fos、pRb、mtp53等均存在共定位关系并在SMMC-7721细胞分化过程中出现分布和位移变化的现象。由此证实了HMBA能够干预SMMC-7721细胞相关癌基因、抑癌基因的活性,改变一些与基因表达调控和细胞信号转导相关的重要核基质蛋白的表达,进而阻滞细胞周期,促使人肝癌细胞分化。这为我们在较为整体的水平上深入研究肿瘤细胞诱导分化的调控机制以及阐明细胞癌变与逆转机理提供了科学依据和探索的新方向,并为肿瘤的诊断和抗癌药物的开发提供潜在的靶向性蛋白。更多还原

【Abstract】 In this study,5mmol/L hexamethylene biacetamide（HMBA ） was used to induce the differentiation of human hepatocarcinoma SMMC-7721 cells,and its effects were examined.On this base,by use of the methods of subcellular proteomics, immunocytochemistry and molecular biology,we studied systematically the changes of nuclear matrix proteins during differentiation of SMMC-7721 cells induced by HMBA,analysed their co-localizational relationship with products of related oncogenes and tumor suppressor genes,and speculated their functions to the induced differentiation of SMMC-7721 cells.It’s useful for us to find out the role of differential expressed nuclear matrix proteins on induced differentiation of tumor cell and explore the molecular mechanisms of carcinogenesis and malignant phenotypic reversion in a relatively systematical level.The results revealed that the proliferation of SMMC-7721 cells were inhibited after 7 days of treatment by HMBA.The reduplication time ratio of treated cells to untreated cells was 1.8.The percentage of cells in G₀/G₁ phase increased from 40.6% to 70.6%while decreased from 27.4%to 6.9%in S phase.The cell cycle of SMMC-7721 cells were arrested in G₀/G₁ obviously.Light and electric microscope observation had showed that after treated with 5 mmol/L HMBA,the ratio of nuclear to cytoplasm lessened,the volume of cell increased,the number of nucleoli decreased. The number of microvilli and the heterochromatin decreased while euchromatin and free ribosomes increased.The endoplasmic reticulum,mitochondfion and golgi body all developed.It was revealed by immunocytochemistry assay that the expression level of oncogenes including c-myc,c-fos,c-erbB-2,bcl-2,mtp53 were downregnlated,while the expression level of tumor suppressor genes such as Rb,p21 and p27 were upregulated.By cellular selective extaction and whole-mount SEM/TEM observation,we found that after HMBA inducement,the NM-IF filaments of SMMC-7721 cells were concentrated and distributed uniformly.The heterogeneous population of filaments,including highly branched utrathin filaments could also be seen in the regular meshwork.The connection between the two kinds of filaments and the relatively thin,condensed and sharply demarcated lamina composed of intermediate-sized filaments was relatively fastened.Meanwhile,9 NM proteins changed remarkably during SMMC-7721 cell differentiation.Four proteins,i.e. mutant pystl,hypothetical protein,nucleophosmin,and LBP were downregulated, whereas four other proteins,eIF6,p44 subunit,β-tubulin 2C,and sin3b were upregulated with the last one,SFRS1 only found in the differentiated SMMC-7721 cells.Two proteins-nucleophosmin and prohibitin had been found existed and localized in the nuclear matrix,as well as its altered expression,by use of western blot, immunofluorescence staining and immune electron microscopy analysis.It also showed that nucleophosmin and prohibitin were colocalized with oncogene c-fos and c-myc products,as well as tumor suppressor gene mtp53 and Rb products.Both of their expression level and localization changed during the differentiation of SMMC-7721 cells.It is concluded that the human hepatocarcinoma SMMC-7721 cells could be induced into differentiation by treatment with HMBA,the malignant morphological and ultrastructural characteristics were reversed,the configuration and composition of nuclear matrix was altered.Some nuclear matrix proteins which were importantant to gene expression and regulation or cell signal transduction have changed in their expression level and subcellular localization during the differentiation.The colocalization of nucleophosmin and prohibitin with products of related oncogenes such as c-myc,c-los and tumor suppressor genes such as Rb,p53 suggested the pathway and mechanisms in which specific nuclear matrix proteins regulated the proliferation and differentiation of the human hepatocarcinoma SMMC-7721 cells. These results indicated that HMBA could interfere the activities of some oncogenes and tumor suppressor genes,regulate the expression of important nuclear matrix proteins,then cause cell cycle arrest,and induce human hepatocarcinoma cells into differentiation.Our study provides proofs and a new way to explore the mechanisms of induced differentiation of tumor cells and cell carcinogenesis and its reversion. Besides,this study provides several potential target proteins for cancer diagnosis and cancer therapy.更多还原