【作者】 赵雯；

【导师】 史景泉； 卞修武；

【作者基本信息】 第三军医大学 ， 病理学与病理生理学， 2002， 博士

【摘要】 恶性胶质瘤具有生长迅速和高度侵袭等生物学特性，其机制尚未阐明，也给治疗带来很大难度。近些年发现，细胞骨架的构成和不同成分之间的相互作用与肿瘤的生物学特性可能有密切联系。我们既往研究也发现，NDGA对恶性胶质瘤具有诱导分化治疗作用，而在这一过程中，作为细胞骨架重要成分之一的中间丝增多，其结构蛋白GFAP表达明显上调，GFAP基因甲基化状态也发生改变。然而，NDGA对胶质瘤细胞整个骨架系统的影响和GFAP基因在诱导分化中的作用及其意义仍然不清。 本研究分四个部分，分别从恶性胶质瘤细胞骨架成分和NDGA的影响、正义GFAP cDNA真核表达载体和反义GFAP逆转录病毒表达载体的构建与鉴定及其对胶质瘤细胞生物学特性的影响，以及NDGA对反义GFAP cDNA封闭后的胶质瘤细胞GFAP基因表达的诱导作用等方面，探讨了细胞骨架蛋白和GFAP基因功能状态在恶性胶质瘤增殖、分化和NDGA诱导分化中的可能作用及其意义。主要研究内容和结果如下： 1．应用免疫荧光细胞化学、激光共聚焦显微镜、原位包埋、选择性抽提和高压透射电镜技术，首次对两种不同分化程度的胶质瘤细胞系CHG-5和SHG-44细胞骨架系统(胞质骨架和核骨架)的定位及分布特征进行了比较研究，并观察了NDGA诱导分化过程中的细胞骨架蛋白的变化及其与恶性表型逆转的关系。结果显示，两种细胞微管蛋白的定位和分布具有共性，而微丝、中间丝及核骨架的结构和排布方式有所不同，并与瘤细胞的分化程度相关，提示细胞骨架蛋白的分布与构型上的差异构成了CHG-5和SHG-44细胞生物学特性差异的结构基础，并在判定胶 质瘤细胞分化程度和恶性行为上有重要价值。NDGA可改变胶质瘤细 胞骨架蛋白表达与分布，且这种作用与NDGA诱导分化和抑制增殖效 应相一致，提示改善胞质骨架及核骨架构型是NDGA促进瘤细胞分化 作用的重要途径之一。 2．应用基因工程技术分别构建了反义GFAP逆转录病毒载体和携带绿色 荧光蛋白（GFP）的正义 GFAP CDNA真核表达载体，并分别将其成功 导入两种胶质瘤细胞。RT－PCR、ISH和 ICC结果显示，反义 GFAP CDNA 封闭CHG－5细胞内源性GFAP表达后，GFAP mRNA及其蛋白表达减 弱甚至缺失，瘤细胞异型性增加，突起缩短，细胞增殖速度加快。与之 相反，正义 GFAP cDNA转染 SHG－44细胞后，GFAP mRNA及其蛋白、表达增强，瘤细胞形态趋向成熟，突起增多变细，细胞增殖速度显著降 低，群体倍增时间延长，细胞骨架蛋白表达增加。它们对细胞周期也有 截然不同的干预作用。提示GFAP基因功能状态可能是决定胶质瘤细胞 分化程度及恶性行为的重要环节。这一结果在恶性胶质瘤治疗策略上可 能有重要意义。 3．本研究首次探讨了NDGA对反义GFAP CDNA存在下的GFAP基因表 达的诱导作用。结果显示，反义 GFAP CDNA封闭的 CHG－5细胞经 NDGA （10 u M）作用后，GFAP mRNA及其蛋白重新高表达，且在 时间和强度上与NDGA诱导细胞分化、抑制细胞增殖及增加细胞骨架 蛋白含量等作用呈正相关。这些结果提示，上调GFAP基因及其蛋白表 达是NDGA诱导胶质瘤细胞恶性逆转作用的主要靶点。 4．利用活细胞分子探针一GFP基因标记了中国人胶质瘤细胞系SHG－44并 获表达及传代，为体外和在体进一步动态研究胶质瘤细胞生长、分化及 侵袭过程提供了新方法和新材料。 上述研究结果对于深入认识细胞骨架和 GFAP基因在胶质瘤生物－学特性和 NDGA诱导分化机制中的作用具有重要意义。更多还原

【Abstract】 Malignant gliomas are characterized by rapid growth and high invasiveness, which result in the difficulties in treatment. Recently, it has been found that cytoskeleton components and their relationship might be closely related to biologic characteristics of tumors. Our previous studies have shown that nordihydroguaiaretic acid (NDGA) possessed a marked differentiation inducement effect on human glioma cells, which outstandingly presented as an increase in intermediate filaments, up-regulation of GFAP expression, as well as changes of GFAP gene methylation pattern. However, the effects of NDGA on cytoskeleton system and the exact roles of GFAP gene in NDGA-induced differentiation remain obscure.In the present study, cytoskeletal components in malignant glioma cells and the effects of NDGA on them were observed. Meanwhile, antisense GFAP cDNA retrovirus expression vector pLXSN-asGFAP and sense GFAP cDNA fused with green fluorescent protein gene mammalian expression vector pIRGFP-GFAP were constructed. The effects of eliminating or reinforcing endogenous GFAP expression on the biological features were studied. Moreover, reversion of malignant phenotype in GFAP-deficient CHG-5 cells, which induced by NDGA simultaneously, was investigated in this experimental cell system. Our aim was to explore the possible roles of cytoskeleton proteins and GFAP gene in glioma cell proliferation, differentiation and NDGA-induced differentiation. The main results and conclusions are as follows:1. The localization and distribution of cytoskeletal components, including cytoplasmic cytoskeleton and nuclear cytoskeleton, in two glioma cells and itsrelationship with malignant grade were detected using immunofluorescence cytochemistry combined with confocal laser scanning microscopy and whole-mount embedded, selective extraction combined together high voltage transmission electron microscopy. The result showed that the localization and distribution of -tubulin, a subunit of microtubules, was identical in two glioma cell lines CHG-5 and SHG-44. In contrast, the structure and arrangement pattern of microfilaments, intermediate filaments as well as nuclear matrix in cell line CHG-5 were distinct from those of SHG-44, and these were also related to their degrees of differentiation and certain malignant phenotype. These results suggested that the difference of organization of cytoplasmic and nuclear cytoskeleton might provide inherent constructive foundation, and, might also provide an accessory evaluation in degrees of differentiation and malignant grade of glioma cells. It was also found that the skeleton protein expression and distribution could be altered by NDGA, along with its effects of growth inhibition and differentiation. This result suggested that enhancement of cytoskeletal protein synthesis and improvement of cytoplasmic and nuclear cytoskeleton organization might be one of the mechanisms of NDGA-induced differentiation in human glioma cells.2. Using gene recombination technique, antisense GFAP cDNA retrovirus expression vector pLXSN-asGFAP and sense GFAP cDNA fused with green fluorescent protein gene mammalian expression vector pIRGFP-GFAP were constructed respectively. The results of RT-PCR, in situ hybridization and immunocytochemistry showed that the expression of GFAP mRNA and its protein was reduced or even eliminated amongst CHG-5 cells infected with antisense GFAP retrovirus. GFAP-negative CHG-5 cells had marked cellular atypia and enhanced proliferative potential. In contrast with above, ithe expression of GFAP mRNA and its protein was markedly increased in SHG-44 cells after stably transfected with an expression vector containing GFAP cDNAin the sense orientation. The profound morphological changes in these cells, including exhibited stellate, abundant and thin cytoplasmic process formation, reduction of atypia occurred. And cell proliferation rate was markedly reduced. The contents of cytoskeletal proteins were also significantly increased. Cell cycle analysis results also revealed the differ更多还原