NEUROGENESIS AND GLIOGENESIS IN THE DEVELOPING BRAIN

大脑发育中的神经发生和胶质发生

• 批准号: 2579618
• 负责人: E O MAJOR
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2579618
• 关键词: DNA binding protein; HeLa cells; arachidonate; astrocytes; complementary DNA; developmental genetics; developmental neurobiology; eicosanoid metabolism; embryo /fetus tissue /cell culture; gene expression; genetic library; human fetus tissue; interleukin 1; neurogenesis; neurotrophic factors; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; prostaglandin E; prostaglandin F; transcription factor; transfection /expression vector

The NF-1 and AP-1 transcription factors have adjacent binding sites in the regulatory region of a number of genes. Because of the possible involvement of NF-1 with the expression of genes expressed in the CNS/PNS, we have screened cDNA libraries prepared from human neonatal and fetal brain tissue for the possible presence of a brain specific NF-1 factor with an oligonucleotide probe homologous with the DNA binding domain of the NF-1. Two clones from the neonatal brain library and one clone from the fetal brain library were sequenced. The 3 sequenced cDNA clones were homologous with each other, except for a few bases at the 5' end of the two longest clones and a 150-bp insertion in the 3'-region of the shortest clone. The DNA binding region located at the 5'-end of the clones was highly conserved between the brain clones and that reported for the HeLA NF-1 clone. On the other hand, the 3'-region of the c-DNA clones isolated from the brain libraries were highly homologous but differed from that reported from HeLA cells. The 3'-region of the NF-1 molecules were reported to contain the transcriptional activational domain of the molecular. One of the brain cDNA clones was cloned into a T7 RNA polymerase expression vector, and a specific size protein was overproduced on induction of expression with IPTG. An extract prepared from cells overproducing the specific protein was demonstrated to contain specific binding activity. In order to determine if other classes of NF- 1 could be detected in primary fetal brain cells, RT-PCR analysis was performed with poly A-selected RNA from primary fetal cells and HeLa cells. With the use of class-specific primers the expression of at least four classes of the NF-1 protein family could be detected in both the brain and the HeLA cell lines. In the fetal brain cultures, however, one clone, NF-1/AT1 was highly expressed compared with the other 3 classes. Astrocyte cultures were also analyzed for expression of arachidonic acid metabolites in response to cytokine stimulation. In the presence of IL-1 beta, human astrocytes produce prostaglandin E2 and prostaglandin F2 alpha. Other intermediate compounds in the cyclooxygenase pathway are not made unlike similar cultures of rodent astrocytes. Both of these studies highlight the fact that human astrocytes have phenotypic and genotypic differences compared with rodent models of neuro and gliogenesis.

NF-1和AP-1转录因子具有相邻的结合位点， 许多基因的调控区。由于可能 NF-1参与表达基因的表达， CNS/PNS，我们筛选了从人新生儿和新生儿中制备的cDNA文库， 胎儿脑组织中可能存在的脑特异性NF-1 因子与DNA结合同源的寡核苷酸探针 NF-1的结构域。 两个来自新生儿大脑库的克隆和一个 对来自胎脑文库的克隆进行测序。3个测序的cDNA 除了5'端的一些碱基外，克隆彼此同源 在两个最长克隆的3 ′端插入150-bp， 最短的克隆DNA结合区位于5 '端 克隆在大脑克隆之间是高度保守的， HeLA NF-1克隆 另一方面，c-DNA的3 '-区域 从脑文库中分离的克隆是高度同源的， 与HeLA细胞报道的不同。NF-1的3 '-区域 分子被报道含有转录激活因子， 分子的结构域。 将其中一个脑cDNA克隆克隆到 一个T7 RNA聚合酶表达载体，和一个特定大小的蛋白， 在用IPTG诱导表达时过量产生。 制备的提取物 从过量产生特定蛋白质的细胞中， 特异性结合活性。 为了确定是否有其他类别的NF-κ B- RT-PCR分析表明，在原代胎脑细胞中， 用来自原代胎儿细胞和HeLa的多聚腺苷酸选择的RNA进行 细胞通过使用类特异性引物，至少 NF-1蛋白家族的四个类别可以在两个组织中检测到。 脑和HeLa细胞系。 然而，在胎脑培养中， NF-1/AT 1在3个克隆中的表达量最高。 还分析了星形胶质细胞培养物中花生四烯酸的表达 代谢产物的细胞因子刺激。 在IL-1的存在下， β，人星形胶质细胞产生前列腺素E2和前列腺素F2 α的 环加氧酶途径中的其他中间体化合物是 与啮齿动物星形胶质细胞的类似培养物不同。这两 研究强调了人类星形胶质细胞具有表型和 与啮齿类动物模型相比， 神经胶质生成