DIFFERENTIATION AND REGULATION OF GENE EXPRESSION IN GLIAL CELLS

胶质细胞基因表达的分化和调控

• 批准号: 3969055
• 负责人: L VITKOVIC
• 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/3969055
• 关键词: astrocytes; brain neoplasms; cell differentiation; complementary DNA; cytogenetics; gel electrophoresis; gene expression; genetic library; genetic regulation; genetic transcription; glioma; glutamate ammonia ligase; glutamate decarboxylase; glutamate dehydrogenase; glutaminase; hepatic coma /encephalopathy; human tissue; immunofluorescence technique; kidney cell; liver cells; messenger RNA; molecular cloning; neurons; oncoproteins

Our objective is to investigate the regulation of genes encoding proteins important in the function of astrocytes and in neuron-glial interaction. To this end, we have screened human and rat brain expression libraries for cDNAs encoding enzymes of the GABA-glutamate--glutamine cycle. By screening with antibodies, we have isolated cDNA clones for human glutamate dehydrogenase (GDH) and rat glutaminase (GA). Using these cDNAs as probes and cDNAs for glutamine synthetase (GS) and glutamate decarboxylase (GAD), we have compared mRNA levels for these enzymes in various brain region in liver, and in kidney. We have established that GS mRNA is induced in C6 glioma cells by dexamethasone, a glucocorticoid. In a rat model for hepatic encephalopathy, we have shown that brain mRNA levels for GS, GDS, and GA are altered. Using the gene for glial fibrillary acidic protein (GFAP) as a marker, we have shown that immunofluorescence is much less reliable than immunoblots to determine the presence of GFAP in human gliomas. One cell line was developed which shows 20 fold induction of GFAP synthesis after addition of mycophenolic acid. Several nuclear proteins of neurons and astrocytes were identified, two of which significantly increased in amount when astrocytes where cultured under conditions that in vivo cause neurological disorders (0.4 M ethanol or 3.8 mM ammonia). Two abundant plasma membrane proteins present in both neurons and astrocytes but absent in membranes of kidney and liver cells were identified and purified.

我们的目标是研究编码蛋白质的基因的调控 在星形胶质细胞的功能和神经元-胶质细胞相互作用中起重要作用。 为此，我们筛选了人类和大鼠脑表达文库， 编码GABA-谷氨酸-谷氨酰胺循环酶的cDNA。 通过 用抗体筛选，我们分离了人谷氨酸的cDNA克隆 脱氢酶（GDH）和大鼠转氨酶（GA）。 用这些cDNA作为探针 以及谷氨酰胺合成酶（GS）和谷氨酸脱羧酶（GAD）的cDNA， 我们比较了这些酶在不同脑区的mRNA水平， 肝脏和肾脏。 我们已经确定GS mRNA在C6细胞中被诱导， 地塞米松是一种糖皮质激素。 在大鼠模型中， 肝性脑病时，我们已经证明脑内GS，GDS， 和GA被改变。 利用胶质细胞酸性蛋白基因 （GFAP）作为标记物，我们已经表明免疫荧光要少得多， 比免疫印迹法更可靠地确定人GFAP的存在 神经胶质瘤 开发了一种显示20倍GFAP诱导的细胞系 在加入麦考酚酸后合成。 鉴定了神经元和星形胶质细胞的几种核蛋白， 当星形胶质细胞被培养时， 在体内引起神经障碍的条件下（0.4M乙醇 或3.8mM氨）。 两种丰富的质膜蛋白存在于两个 神经元和星形胶质细胞，但在肾和肝细胞膜中缺失 鉴定并纯化。