Postgenomic approaches to diabetic complications

描述(由申请人提供)： 1型糖尿病的微血管并发症发病率高，再加上大血管并发症，死亡率高。最常见的微血管并发症是神经病变。虽然确切的患病率取决于用于识别神经病变的诊断标准，但大多数研究表明，1型或2型糖尿病患者中有50%有神经病变。 一般认为，I型糖尿病患者循环血糖的巨大变化有助于糖尿病神经病变的发展，并且葡萄糖对基因表达的转录调控在糖尿病神经病变的进展中起主要作用。虽然葡萄糖调节肝细胞和胰岛β细胞基因表达的机制已经确定，但对于葡萄糖调节神经元基因表达的分子机制几乎一无所知。这一建议将为研究神经元Gone表达的葡萄糖调节开发模型系统和新技术。这项工作将涉及费尔德曼和乌勒实验室之间的新合作。 三个特定的目标将服务于拟议的研究，该研究将使用两个神经母细胞瘤细胞系以及大鼠背根神经节神经元进行原代培养。在第一个具体目标中，将采用一种新的高通量转基因程序(表面转染和表达协议或STEP)来适应神经母细胞瘤细胞系和原代神经元。在第二个特定目标中，将使用DNA微阵列杂交来识别受葡萄糖转录调控的候选基因。在第三个特定目标中，我们将使用分步转染法研究葡萄糖对这些候选基因的启动子以及其他具有良好特性的启动子元件的调控。 这些研究的完成不仅将确定导致糖尿病神经系统并发症发生的关键基因组机制，而且将为开发药物筛选分析奠定基础，药物筛选分析将对干扰这些机制的药物进行测试至关重要。

DESCRIPTION (provided by applicant): The microvascular complications of type 1 diabetes carry a high morbidity and, when coupled with macrovascular complications, high mortality. The most common microvascular complication is neuropathy. While exact prevalence depends on the diagnostic criteria used to identify neuropathy, most studies suggest that 50% of patients with either type 1 or type 2 diabetes have neuropathy. It is generally assumed that the large variations in circulating glucose evidenced in type I diabetics contribute to the development of diabetic neuropathy and that transcriptional control of gene expression by glucose plays a major role in the progression of diabetic neuropathy. Although mechanisms have been identified for glucose regulation of gene expression in hepatocytes and pancreatic beta cells, virtually nothing is know about the molecular mechanism by which glucose regulates gene expression in neurons. This proposal will develop model systems and new technologies for the study of glucose regulation of neuronal gone expression. This work will involve a new collaboration between the Feldman and Uhler laboratories. Three specific aims will serve to focus the proposed studies which will employ two neuroblastoma cells lines as well as rat dorsal root ganglion neurons in primary culture. In the first specific aim, a new high-throughput transfection procedure (Surface Transfection and Expression Protocol or STEP) will be adapted to the neuroblastoma cell lines and primary neurons. In the second specific aim, candidate genes transcriptionally regulated by glucose will be identified using DNA microarray hybridization. In the third specific aim, the regulation of promoters from these candidate genes as well as other well-characterized promoter elements by glucose will be studied using STEP transfection. The completion of the studies will not only identify critical genomic mechanisms responsible for the development of neurological complications of diabetes, but they will lay the foundation for the development of drug screening assays that will be essential for the testing of drugs to interfere with these mechanisms.