INTERMEDIATE FILAMENT FOR HUMAN ASTROCYTOMAS

这项建议的目标是确定不同的表型 恶性人脑星形胶质细胞的特性及其利用 星形细胞瘤诊断分型的生化标志物。这个 该项目的工作假设是分化特异性标记 对于正常星形胶质细胞发育的早期阶段， 恶性星形胶质细胞，因此可以用来识别恶性 表型。初步研究表明，细胞内的蛋白质 中间丝(IF)细胞骨架占据着独特的时间生态位 星形胶质细胞发育。一个这样的分化标记，IF-相关 蛋白(IFAP)-30OkD似乎在所有人类星形细胞瘤中都很常见，而 角蛋白，如果蛋白生产似乎是典型的最恶性级别 星形细胞瘤。在这方面，这项订正提案的具体目标是 1)将IFAP-30okD的表达定义为可能的标记物 恶性人脑星形胶质细胞；2)IFAP-30okD的生化特性 来自人类星形细胞瘤细胞；3)生产替代抗体 利用固定型人脑星形细胞瘤研究IFAP-30okD的表达 4)研究角蛋白在人脑星形细胞瘤中的表达。 肿瘤恶性程度指数；5)测量肿瘤类型的谱。 现有抗大鼠星形胶质细胞IF相关单抗文库 蛋白质，以确定恶性表型的其他标记；6) 制备抗IF相关蛋白的单抗 直接来自人类星形细胞瘤。的表达模式的基础 各种蛋白质标记物将直接在人体内进行测定 免疫荧光(和免疫过氧化物酶)法检测星形细胞瘤组织标本 使用先前开发的单抗的显微镜检查。标本 来自正常人中枢神经系统(用于对照)以及来自人类中枢神经系统退行性变 疾病(对于反应性星形胶质细胞)将被包括在内。多肽 标记的组成将通过SDS-PAGE和2-D来确定 PAGE/免疫印迹分析。IFAP-30OKD将被层析提纯 来自星形细胞瘤，通过氨基酸分析鉴定，多肽 作图和N-末端序列分析。纯化的IFAP-30OKD将 也可以变性并用来产生新的抗体，这样醛就固定了 可以对标本进行调查。部分生化特性的研究 角蛋白交叉反应物种可能是必要的一种手段 与已知角蛋白多肽相关的鉴定。组织 标本将与其他文库一起进行额外的调查 免疫荧光显微镜和PAGE/免疫印迹法检测单抗 分析。最后，对正常和恶性星形细胞瘤的PAGE进行比较 如果细胞有蛋白质图谱，恶性表型特有的蛋白质将 被提纯以生产单抗，然后将其 筛选出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.