INTERMEDIATE FILAMENT FOR HUMAN ASTROCYTOMAS

这项建议的目标是确定不同的表型 恶性人星形胶质细胞的特性，并利用这些特性， 星形细胞瘤诊断分类的生化标志物。的 该项目的工作假设是， 正常星形胶质细胞发育的早期阶段在 恶性星形胶质细胞，因此可以用于识别恶性星形胶质细胞， 表型。 初步研究表明， 中间丝（IF）细胞骨架占据独特时间小生境， 星形胶质细胞发育 一种这样的分化标志物，IF相关的 蛋白（IFAP）-300kD，似乎是共同的所有人类星形细胞瘤，而 角蛋白IF蛋白的产生似乎代表了最恶性的肿瘤。 星形细胞瘤 在这方面，本修订提案的具体目标 1）将IFAP-300 kD的表达定义为一个假定的标志物， 恶性人星形胶质细胞; 2）生物化学表征IFAP-3 OOkD 从人星形细胞瘤细胞; 3）产生替代抗体， IFAP-3 OOkD在固定的人脑星形细胞瘤中的表达 4）研究角蛋白在星形细胞瘤中的表达， 肿瘤恶性指数; 5）用一种新的方法调查肿瘤类型谱， 现有的大鼠星形胶质细胞IF-related单克隆抗体库 蛋白质，以鉴定恶性表型的其他标志物; 6） 以产生针对获得的IF相关蛋白的单克隆抗体， 直接来自人类星形细胞瘤。 表达模式的基础 各种蛋白质标记物将直接在人体中测定 星形细胞瘤组织标本免疫荧光（和免疫过氧化物酶） 使用先前开发的单克隆抗体进行显微镜检查。 标本 来自正常人CNS（用于对照）以及来自人CNS变性 将包括疾病（对于反应性星形胶质细胞）。 的多肽 标记物的组成将通过SDS-PAGE和2-D PAGE/免疫印迹分析。 将对IFAP-3 OOkD进行色谱纯化 从星形细胞瘤中分离并通过氨基酸分析、肽 作图和N-末端序列分析。纯化的IFAP-3 OOkD将 也可以变性并用于产生新的抗体， 可以对标本进行调查。 部分生化特性的 角蛋白交叉反应性物质可能是必要的， 相对于已知的角蛋白多肽的鉴定。 组织 标本将与其他图书馆进行额外调查 免疫荧光显微镜和PAGE/免疫印迹法测定单克隆抗体 分析.最后，通过PAGE比较正常和恶性星形胶质细胞 细胞IF蛋白质谱，恶性表型所特有的蛋白质将 纯化用于生产单克隆抗体，然后将其 筛选以确定星形细胞瘤的其他标志物。

The goals of this proposal are to identify distinctive phenotypic characteristics of malignant human astrocytes and to utilize these biochemical markers for diagnostic classification of astrocytomas. The working hypothesis of the project is that differentiation-specific markers for early stages of normal astrocyte development are re-expressed in malignant astrocytes and can therefore serve to identify malignant phenotypes. Preliminary studies have shown that proteins of the intermediate filament (IF) cytoskeleton occupy unique temporal niches in astrocyte development. One such differentiation marker, IF-associated protein (IFAP)-3OOkD, appears to be common to all human astrocytomas, while keratin IF protein production appears to typify the most malignant grade of astrocytomas. In this context, the Specific Aims of this revised proposal are: 1) to define the expression of IFAP-3OOkD as a putative marker of malignant human astrocytes; 2) to biochemically characterize IFAP-3OOkD from human astrocytoma cells; 3) to produce alternative antibodies to IFAP-3OOkD in order to study its expression using fixed human astrocytoma specimens; 4) to investigate keratin expression in human astrocytomas as an index of tumor malignancy; 5) to survey the spectrum of tumor types with a library of existing monoclonal antibodies to rat astrocyte IF-related proteins in order to identify other markers of the malignant phenotype; 6) to produce monoclonal antibodies against IF-related proteins obtained directly from human astrocytomas. The basis of the expression patterns for the various protein markers will be determined directly in human astrocytoma tissue specimens by immunofluorescence (and immunoperoxidase) microscopy using previously developed monoclonal antibodies. Specimens from normal human CNS (for control) as well as from human CNS degenerative diseases (for reactive astrocytes) will be included. The polypeptide composition of the markers will be determined by SDS-PAGE and 2-D PAGE/inmmunoblot analysis. IFAP-3OOkD will be chromatographically purified from astrocytomas and characterized by amino acid analysis, peptide mapping, and N-terminal sequence analysis. The purified IFAP-3OOkD will also be denatured and used to produce new antibodies so that aldehyde-fixed specimens can be surveyed. Partial biochemical characterization of the keratin cross-reactive species may be necessary as a means of identification relative to the known keratin polypeptides. The tissue specimens will be additionally surveyed with the library of other monoclonal antibodies by immunofluorescence microscopy and PAGE/immunoblot analysis. Finally, after PAGE comparison of normal and malignant astrocytic cell IF protein profiles, proteins unique to the malignant phenotype will be purified for the production of monoclonal antibodies which will then be screened to identify other markers of astrocytomas.