SCHWANN CELL-AXON INTERACTIONS IN VITRO

在周围神经系统的发育和再生过程中， 决定了雪旺氏细胞是否分化成髓鞘形成细胞。 在损伤的情况下，轴突断裂向雪旺细胞发出信号， 增殖，抑制髓鞘基因表达，表达细胞粘附 分子和神经营养因子。我们的长期目标是了解 细胞外信号如何协调雪旺细胞的变化 表型在任何细胞中，细胞外信号都可以激活细胞内的 一系列转录因子，然后调节 最终决定表型的基因在初步研究中， 确定了四个转录控制基因，表达在 雪旺氏细胞在表型发生变化时。近期目标 就是了解这四个因素在调节雪旺氏细胞中的作用 细胞特性。这些基因是根据我们初步的 数据表明，C-JUN和HLH 462（具体目标1和2， 分别）在脱分化过程中发挥作用，而AP- 2和C/EBP（分别为具体目标3和4）涉及 分化为髓鞘形成表型。 为了确定这些基因中每一个的功能作用，雪旺细胞将 以有义和反义方向用cDNA转染，和 表达将由组成型病毒启动子或 合成的诱导型启动子。将产生稳定的细胞系， 研究了它们的有丝分裂率和表型 标记。[3H]胸苷掺入和倍增时间将用作 增殖指数。间接免疫荧光染色，和 将使用Western和北方印迹分析来确定表达 基因的突变会导致与以下两种情况相关的表型性状的显示： 分化或去分化。功能性的支持性证据 如果反义基因的表达阻止了 对有丝分裂原或分化信号的反应（增加 cAMP或由背根神经节延伸的神经突）。此外，作用 C-JUN和AP-2在调节髓磷脂P-O基因转录中的作用将 接受检查。用于这些研究的方法包括瞬时 转染、蛋白质-DNA结合、体外转录和体外 诱变 虽然其他几个基因将相互作用，在控制雪旺氏 细胞表型，我们试图剖析四个因素的贡献 个体基因更深入地了解雪旺细胞 表型的调节可能最终允许实验操作， 这在很多医学领域都有帮助雪旺细胞移植 目前正在研究脊髓的可能治疗用途 肾上腺嗜铬细胞移植治疗帕金森病 疾病，并作为神经退行性疾病中神经营养因子的来源 疾病此外，详细了解分化过程 最终可以证明在设计治疗髓鞘形成障碍的方法中是有用的， 脱髓鞘疾病

During peripheral nervous system development and regeneration, the axon dictates whether a Schwann cell differentiates into a myelin forming cell. In the event of injury, axonal breakdown signals Schwann cells to proliferate, repress myelin gene expression, and express cell adhesion molecules and neurotrophic factors. Our long term goal is to understand how extracellular signals orchestrate changes in the Schwann cell phenotype. In any cell, extracellular signals are known to activate a series of transcription factors which then regulate the expression of genes that ultimately define the phenotype. In preliminary studies we have identified four transcriptional-control genes that are expressed in Schwann cells at the onset of a change in phenotype. The immediate goals are to understand the role of these four factors in regulating Schwann cell properties. These genes were chosen on the basis of our preliminary data, which indicated that C-JUN and HLH462 (specific aim 1 and 2, respectively) play a role in the process of dedifferentiation, whereas AP- 2 and C/EBP (specific aim 3 and 4, respectively) are involved in differentiation to the myelin-forming phenotype. To ascertain a functional role for each of these genes, Schwann cells will be transfected with cDNA in the sense and antisense orientation, and expression will be regulated by either a constitutive, viral promoter or a synthetic, inducible promoter. Stable cell lines will be generated and studied with respect to their mitotic rate and expression of phenotypic markers. [3H] Thymidine incorporation and doubling times will be used as a index of proliferation. Indirect immunofluorescence staining, and Western and Northern blot analysis will be used to determine if expression of a gene leads to the display of phenotypic traits associated with either differentiation or dedifferentiation. Supporting evidence for a functional role of a gene will be provided if expression of the antisense prevents the response to mitogens or differentiating signals (agents that increase cAMP or neurites extended by dorsal root ganglia). In addition, the role of C-JUN and AP-2 in regulating transcription of the myelin P-O gene will be examined. Methods used for these studies include transient transfection, protein-DNA binding, in vitro transcription, and in vitro mutagenesis. Although several other genes will interplay in controlling the Schwann cell phenotype, we are attempting to dissect out the contribution of four individual genes. A greater understanding of how the Schwann cell phenotype is regulated may ultimately allow experimental manipulation, which could be of help in several medical areas. Schwann cell transplants are currently being studied for possible therapeutic use in spinal cord regeneration, in cografts with adrenal chromaffin cell for Parkinson's disease, and as a source of neurotrophic factors in neurodegenerative disease. Furthermore, detailed knowledge of the process of differentiation can ultimately prove useful in designing therapies for dysmyelinating and demyelinating diseases.