IMMORTALIZED CELL LINES AND NEURAL TRANSPLANTATION

该项目主要涉及开发用于 用于神经移植的连续细胞系的派生 作为原代细胞和组织的替代品，以及用于研究 神经细胞生物学的研究。目前的努力包括开发突变体 SV40大T抗原的截短型，直接细胞法 递送改变细胞周期的蛋白质，以及顺序递送 将基因传递给细胞系。SV40大T抗原的突变形式，它 缺乏P53和DNA结合活性，已被克隆来检测这些 SV40大T抗原永生化所需的特性 中枢神经系统神经元。这种名为T155的突变癌基因能够 克服T64-7b细胞周期停滞并永生化原代细胞 当被脂质体转导时，中脑神经元。T155似乎 在很大程度上干扰了分化表型的表达 比野生型SV40大T抗原小。主要 T155永生化培养的中脑细胞表达分化 神经元标记物(例如神经丝)和胶质标记物(例如GFAP)， 而用野生型SV40大T细胞永生化的细胞很少表达 成熟神经元或神经胶质细胞特有的标记物。当T155-不朽 细胞在长期培养中保持，它们会产生不同的 分化的形态提示成熟的神经元和神经胶质细胞。 由于这种癌基因的体积很小，它可能会传递 它通过与其他载体多肽形成融合蛋白而进入细胞。 干预细胞周期控制的替代方法，使用直接 蛋白质因子的传递和反义方法正在研究中 单独和与SV40大T突变体联合使用。从这些 我们希望通过实验来改进生产永生的方法 细胞系，特别是关于永生化技术用于 神经元。

This project mainly involves the development of techniques for the derivation of continuous cell lines, for use in neural transplantation as an alternative to primary cells and tissues, as well as for studies of neural cell biology. Current efforts include development of mutant truncated forms of SV40 large T antigen, methods for direct cellular delivery of proteins which modify the cell cycle, and sequential delivery of genes to cell lines. A mutant form of SV40 large T antigen, which lacks p53 and DNA binding activity, has been cloned to examine those properties of SV40 large T antigen which are required for immortalizing CNS neurons. This mutant oncogene, called T155, is capable of overcoming cell cycle arrest in T64-7b cells and immortalizing primary mecencephalic neurons when transduced by lipofection. T155 appears to interfere with the expression of differentiated phenotypes to a much smaller degree than wild-type SV40 large T antigen. Primary mesencephalic cell cultures immortalized with T155 express differentiated neuronal markers (e.g., neurofilament) and glial markers (e.g. GFAP), whereas cells immortalized with wild-type SV40 large T rarely express markers characteristic of mature neurons or glia. When T155-immortalized cells are maintained in long-term cultures, they develop variable differentiated morphologies suggestive of mature neurons and glia. Because of the small size of this oncogene, it may be possible to deliver it to cells by forming fusion proteins with other carrier polypeptides. Alternative means of intervening in cell cycle control, using direct delivery of protein factors and antisense approaches, are being studied alone and in combination with SV40 large T mutants. From these experiments we hope to improve the methods for producing immortalized cell lines, especially regarding immortalization techniques for use in neurons.