CYTOLOGICAL STUDIES OF DEVELOPING AND MATURE NEURONS

所提议的工作是《公约》核心目标之一的关键部分， 治疗瘫痪的迈阿密项目;彻底探索使用 雪旺细胞（SC）移植，努力对 神经组织对损伤和疾病的反应。 我们认为 SC影响再生反应的显著能力， 外周和中枢神经组织都表明有可能用于 人神经损伤，特别是脊髓损伤。 我们勾勒出一个 一系列复杂的组织培养实验，旨在获得更完整的 了解SC的基础生物学，尤其是人类SC的基础生物学。 我们相信，研究人类SC生物学的主要努力是 这是必要的，因为它的许多基本特征似乎不同 从啮齿动物组织的研究中得到的结论。 我们首先建议研究大鼠SC功能的机制， 由基底层（BL）组装控制，因为我们相信这方面的 SC功能调节对于理解其能力至关重要 以获得中枢神经组织的全部功能。 我们将 使用髓鞘形成组织培养系统来研究 髓鞘特异性蛋白的表达和BL组装，并将探索 细胞粘附分子L1在信号传导中的作用， 影响早期SC发展。 我们寻求进一步确定， SC表面整合素参与SC功能的调节， SC的细胞骨架是否参与信号传导， 允许SC功能随着BL的组装而前进。 在安排了一个可靠的可行的人类外周神经来源后， 我们建议使用组织培养的方法来改善程序， 从成人外周神经中分离SC，并研究其在神经元中的表达。 调节人SC的细胞、可溶性和基质因子 增殖 提出了实验来确定的能力， 人SC产生细胞外基质（ECM）组分，以及 确定ECM沉积是否调节SC鞘化， 髓鞘形成的人SC，因为它在大鼠。 我们会研究 人类SC为神经嵴提供营养支持的能力- 感觉神经元。 为了不限制我们对人类的观察 SC功能的组织培养研究，我们已经开发了一种方法， 将人SC引入引导通道， 免疫缺陷的啮齿动物外周神经，并将使用该系统， 研究体内人SC增殖的调控，以及 人SC促进外周轴突再生的能力。 最后，我们建议引入一种重组逆转录病毒， β-半乳糖苷酶和碱性成纤维细胞生长因子的序列 并随后比较受感染细胞支持 神经元健康和诱导神经突生长，以及确定如何 这种遗传变化改变了SC承担其基本任务的能力， 轴突鞘化、BL组装和髓鞘形成的功能。 SC生物学的这些基础研究被认为是任何 考虑将该细胞用于人体自体移植 神经损伤患者。 这种细胞影响 轴突再生以及修复脱髓鞘给予这项工作 相当紧迫。

The work proposed is a critical portion of one of the central goals of The Miami Project to Cure Paralysis; to thoroughly explore the use of Schwann cell (SC) transplantation in efforts to favorably influence the response of neural tissue to injury and disease. We believe that the remarkable capability of the SC to influence regenerative responses in both peripheral and central neural tissue indicates potential for use in human neural injury, particularly spinal cord injury. We outline a series of complex tissue culture experiments aimed at gaining a fuller understanding of the basic biology of the SC, especially of the human SC. We believe a major effort to study the biology of the human SC is required, for many of its basic characteristics appear to be different from those that have been elucidated from the study of rodent tissue. We first propose to study the mechanisms by which rat SC function is controlled by basal lamina (BL) assembly for we believe this aspect of the regulation of SC function is critical to understanding its ability to attain full functional capacity in central neural tissues. We will use a myelinating tissue culture system to study the linkage between expression of myelin-specific proteins and BL assembly, and will explore the role of the cell adhesion molecule L1 in the signalling which influences early SC development. We seek further to determine whether SC surface integrins are involved in the regulation of SC function and whether the cytoskeleton of the SC is involved in the signalling which allows SC function to go forward as BL is assembled. Having arranged for a reliable source of viable human peripheral nerve, we propose to use tissue culture methods to improve procedures for separating SCs from human adult peripheral nerve, and to study the cellular, soluble and matrix factors which regulate human SC proliferation. Experiments are proposed to determine the capacity of the human SC to produce extracellular matrix (ECM) components and to determine whether ECM deposition regulates SC ensheathment and myelination by the human SC, as it does in the rat. We will study the ability of the human SC to provide trophic support for neural crest- derived sensory neurons. In order not to limit our observations on human SC function to tissue culture studies, we have developed a method of introducing human SCs into guidance channels placed to bridge gaps in the immune-deficient rodent peripheral nerve, and will use this system to study the regulation of human SC proliferation in vivo, as well as the ability of human SCs to promote peripheral axonal regeneration. Finally, we propose to introduce a recombinant retrovirus which contains sequences for both beta-galactosidase and basic fibroblast growth factor and to subsequently compare the ability of the infected cells to support neuronal health and induce neurite growth, as well as to determine how this genetic change alters the ability of the SC in undertaking its basic function of axonal ensheathment, BL assembly, and myelination. These basic studies of SC biology are considered essential to any consideration of use of this cell in autotransplantation in human patients with neural injury. The potential of this cell to influence axonal regeneration as well as to repair demyelination gives this work considerable urgency.