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REGULATION OF MYELIN P2 GENE

髓磷脂 P2 基因的调控

基本信息

批准号：
2267841
负责人：
GIHAN I TENNEKOON
金额：
$ 22.03万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-06-01 至 1995-05-31
项目状态：
已结题

项目摘要

The regulation of the processes of myelination and remyelination are poorly understood. Our long-term goals are to better understand these processes. However, the immediate goals are to understand the regulation of a single protein-the P2 protein. We will study the regulation of expression of the human and mouse P2 genes. To accomplish these studies, we have cloned the mouse and human P2 genes and generated a rat Schwann cell line (MT4H1) and will attempt to generate a human Schwann cell line. In Specific Aim #1, we will identify the important proximal regulatory regions of the mouse P2 gene by transfection into rat Schwann cells. In fact, in the "Preliminary Studies," we have identified a TC-rich region which down-regulates P2 expression and a second region that up-regulates P2 expression. Furthermore, we have identified three transcriptional factors which alter P2 expression: c-jun/c-fos, CAAT/enhance binding protein (C/EBP), belonging to the bZip family of transcriptional factors, and HNF-1, aPOU domain protein. How these three transcriptional factors affect P2 expression will be examined in detail. Furthermore, the regions of the promoter that are necessary and sufficient for tissue-specific expression will be determined by transfecting the 5'-deletion constructs into Schwann cells and other cell types, as well as, producing transgenic animals and examining tissue for beta-galactosidase expression. In Specific Aim #2, we will use strategies previously used to generate rat Schwann cell lines to produce human Schwann cell lines. The cell lines obtained will be characterized by immunocytochemical studies, morphological appearance, biochemical properties, and ability to form myelin when co- cultured with dorsal root ganglia neurites. Only those cells most resembling untransfected human Schwann cells will be used for further studies. Although we have cloned the human P2 gene and know the restriction map, we must still sequence the promoter. Once the sequence is known, studies similar to those undertaken for the mouse P2 promoter will be conducted. These studies should help in understanding the similarities and differences in the regulation of the human and mouse myelin P2 genes. Information about this regulation may provide insights into the processes of myelination and demyelination, which then may be used to help in designing possible therapies to aid recovery in diseases resulting from demyelination or dysmyelination.

髓鞘形成和髓鞘再生过程的调节很差明白我们的长期目标是更好地理解这些过程。然而，当前的目标是了解单个 P2蛋白。我们将研究表达的调控，人和小鼠P2基因。为了完成这些研究，我们克隆了小鼠和人P2基因，并产生大鼠雪旺细胞系（MT4 H1），将尝试制造人类许旺细胞系。具体目标#1，我们将确定小鼠P2的重要近端调控区域，基因转染到大鼠雪旺细胞。事实上，在“初步研究，”我们已经确定了一个富含TC的区域，它下调P2 表达的第二区域和上调P2表达的第二区域。此外，我们已经确定了三个转录因子， P2表达：c-jun/c-fos，CAAT/增强结合蛋白（C/EBP），属于转录因子的bZip家族，和HNF-1，aPOU结构域蛋白这三个转录因子如何影响P2的表达，详细检查。此外，启动子的区域，将确定组织特异性表达的必要和充分条件通过将5 ′-缺失构建体转染到许旺细胞和其它细胞中，细胞类型，以及生产转基因动物和检查组织用于β-半乳糖苷酶的表达。在具体目标#2中，我们将使用以前用于生成大鼠的策略，施旺细胞系以产生人施旺细胞系。的细胞系将通过免疫细胞化学研究、形态学外观、生化特性和共同作用时形成髓鞘的能力，用背根神经节神经突培养。只有那些细胞类似于未转染的人雪旺细胞，问题研究虽然我们已经克隆了人类P2基因，限制性酶切图谱，我们还必须对启动子进行测序。一旦序列已知，类似于对小鼠P2启动子进行的研究将进行。这些研究应该有助于理解以及人类和小鼠髓磷脂P2基因调控的差异。有关这一规定的信息可能会提供深入了解的过程髓鞘形成和脱髓鞘，然后可以用来帮助设计可能的治疗方法，以帮助由以下原因引起的疾病的康复：脱髓鞘或髓鞘形成障碍。