【作者】 梁盈；

【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】 本论文以维甲酸（retinoic acid,RA）处理人神经母细胞瘤SK-N-SH细胞,鉴定RA对人神经母细胞瘤细胞的终末分化诱导效果。在此基础上,综合应用亚细胞蛋白质组学分析、免疫细胞化学、细胞分子生物学等技术,对在SK-N-SH细胞诱导分化过程中核基质蛋白的变化进行了较为系统的研究。分析鉴定和确证了与神经母细胞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神经母细胞瘤细胞相关癌基因、抑癌基因在细胞内的共定位关系与变化,探索它们在神经母细胞瘤细胞诱导分化过程中的调控作用,以期能够在较为整体水平上进一步认识神经母细胞瘤细胞癌变与逆转机理问题,从而找出神经细胞增殖与分化相关的靶向性蛋白。实验结果显示,经1μmol/L RA处理之后,人神经母细胞瘤SK-N-SH细胞的增殖受到抑制,细胞生长抑制率达57.93%,细胞发生了G₀/G₁期阻滞。光镜和电镜观察结果显示,经1μmol/L RA处理后的SK-N-SH细胞形态和超微结构发生恢复性变化,细胞呈极性状、伸出多个轴突树突状突起、细胞逐渐变小变圆并融合在一起形成类似神经节样结构,并出现核质比例减小,细胞体积减小,细胞表面微绒毛减少,细胞核形态趋于规则、核仁数目减少,细胞核内异染色质减少、常染色质增多,细胞内线粒体和粗面内质网增多、高尔基体结构更为典型等变化;免疫细胞化学检测结果显示,经RA诱导处理后,SK-N-SH细胞内癌基因c-myc、c-fos表达产物减少,抑癌基因p53、p27表达产物增多;同时,RA能够促进SK-N-SH细胞的神经元特异性标志物NSE、Synaptophysin、MAP2的表达。选择性抽提整装光镜和电镜观察显示,经RA处理的SK-N-SH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,与核纤层联系更为紧密,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41个在RA诱导分化前后表达差异的核基质蛋白,其中在分化的SK-N-SH细胞中表达上调的蛋白点为:Vimentin、MTMR2、SUMO-3等;表达下调的蛋白为TRPV2、Nucleophosmin、Prohibitin等;处理后消失的蛋白为ATP synthase beta chain、PSB3;处理后新出现的蛋白为LGI1、SYCE1。蛋白质印迹杂交与免疫荧光显微镜观察确证了Nucleophosmin（NPM）、Prohibitin（PHB）、Vimentin和hnRNP A2/B1在SK-N-SH细胞分化过程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NPM、PHB和hnRNP A2/B1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均发生了改变。激光共聚焦显微镜观察结果还显示NSE、SYP和MAP2与癌基因c-myc、c-fos和抑癌基因p53、Rb表达产物在细胞内也存在一定的共定位关系,它们的表达水平和细胞定位在诱导分化前后也发生了变化。研究结果表明,1μmol/L RA能有效抑制人神经母细胞瘤SK-N-SH细胞的增殖活动,改变SK-N-SH细胞形态与超微结构特征,下调癌基因c-myc、c-fos和上调抑癌基因p53、p27等的表达,从而对人神经母细胞瘤细胞的分化具有显著诱导效果。在SK-N-SH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NPM、PHB、Vimentin和hnRNP A2/B1为不同诱导分化物诱导多种肿瘤细胞分化后出现的共同差异核基质蛋白。而功能性共同差异核基质蛋白NPM、PHB、hnRNP A2/B1和神经元特异表达标志物NSE、SYP、MAP2与c-Myc、c-Fos、P53、Rb等均存在共定位关系并在SK-N-SH细胞分化过程中出现分布和位移变化的现象。由此证实了RA能够干预SK-N-SH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,促使人神经母细胞瘤细胞分化。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研究的新方向,并为神经母细胞瘤的诊断和抗癌药物的研究提供了新的靶向性蛋白。更多还原

【Abstract】 In this study,1μmol/L Retinoic Acid（RA） was used to induce the terminal differentiation of human neuroblastoma SK-N-SH cells,and its effects were investigated.On this base,by use of the methods of subcellular proteomics, immunocytochemistry and cellular molecular biology,we studied systematically the changes of nuclear matrix proteins during differentiation of SK-N-SH cells induced by RA,analysed their co-localizational relationship with products of related oncogenes and tumor suppressor genes,and speculated their functions to the induced differentiation of SK-N-SH 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 experiment results showed that after treaed with 1μmol/L RA,the proliferation rate of SK-N-SH cells was inhibited,the inhibition rate amounts to 57.93%,and the cell cycle was arrested in G₀/G₁ phase;the morphology and ultrastructure of SK-N-SH cells underwent a significant change and appeared as normal differentiated neuronal cells:cells became multipolar with several neurites,cell bodies were smaller, with long axonal processes extending from one cell to another to form ganglia;The number of microvilli and the heterochromatin decreased while euchromatin and free ribosomes increased.The endoplasmic reticulum,mitochondrion and Golgi body all developed.The immunocytochemistry assey also revealed that the expression level of oncogenes including c-myc,c-los were downregulated,and the expression level of tumor suppressor genes including p53,p27 were upregulated,and at the same time, the expression of the neuronal markers of terminal differentiation,including NSE, SYP and MAP2 increased greatly after treatment.By cellular selective extaction and whole-mount SEM/TEM observation,we found that after treatment with RA,the NM-IF filaments of SK-N-SH 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,52 nuclear matrix proteins（NMPs）,41 of which were identified changed significantly during SK-N-SH differentiation;The changes of Nucleophosmin,Prohibitin,Vimentin and hnRNP A2/B 1 differentially expressed nuclear matrix protein during differentiation induced by RA,was further confirmed by western-blot and had been found existed and localized in the nuclear matrix by use of immunofluorescence staining and immune electron microscopy analysis.The 4 specific NMPs were co-localized with oncogenes c-myc and c-fos products,as well as tumor suppressor genes p53 and Rb products. The expression level and location of them were changed following the proliferation and differentiation of SK-N-SH.The co-localization of neuronal markers with oncogenes and tumor suppressor genes also changed obviously during the differentiation.Our study showed that the human neuroblastoma SK-N-SH cells were induced into terminal differentiation after treatment with RA,as the proliferation of SK-N-SH cells were inhibited,the cell cycle were arrested in G₀/G-1,the malignant morphological and ultrastructural characteristics were reversed,the configuration of nuclear matrix-intermediate filament was altered,the expression of the neuronal phenotype markers were highly increased.Some nuclear matrix proteins which play an important role in DNA replication and repair,cell cycle regulation,gene expression and regulation,have changed in expression level and location during the differentiation. The co-localization of nucleophosmin,prohibitin vimentin and hnRNP A2/B1 with products of neuroblastoma associated oncogenes c-myc,c-fos and tumor suppressor genes p53,Rb suggested the pathway and mechanisms in which specific nuclear matrix proteins regulated the proliferation and differentiation of the human neuroblastoma SK-N-SH cells.In conclusion,1μmol/L RA can induce SK-N-SH cells into terminal differentiation by regulating the activities of some oncogenes and antioncongenes,altering the expression of some key nuclear matrix proteins,arresting cell cycle,and facilitating the expression of neuronal phenotype markers.Our findings will help to elucidate the signaling pathways and mechanisms of neuroblastoma cell growth and differentiation as well as carcinogenesis,and provides a new way to explore the mechanisms of induced differentiation of tumor cells and cell carcinogenesis and its reversion.Further study of the proteins identified here will be useful for the development of clinical therapies targeting neuroblastoma.更多还原