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Study on the mechanisms of alternative mRNA splicing that regulates the regulation of angiogenesis and diabetic complication susceptibility

mRNA选择性剪接调控血管生成及糖尿病并发症易感性的机制研究

基本信息

批准号：
17590241
负责人：
YONEKURA Hideto
金额：
$ 2.3万
依托单位：
Kanazawa Medical University (2006)Kanazawa University (2005)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

In this research, we studied the mechanisms of alternative pre-mRNA splicing/processing by which mRNAs for soluble RAGE and soluble VEGF receptor are produced, and their roles in the regulation of diabetic vascular complications and angiogenesis. Soluble RAGE has a protective activity against AGE-induced vascular cell injury and soluble VEGF receptor acts as a potent anti-angiogenic factor.(1) We isolated the marine equivalent of soluble RAGE by RT-PCR cloning. This study will provide an animal orthologue of soluble RAGE to clarify its roles in health and disease.(2) We investigated the expression of soluble RAGE protein in human organs and tissues by immunohistochemical analysis, and found that soluble RAGE was widely distributed in various organs and tissues including vascular endothelium, neurons, pancreatic β cells, macrophages/monocytes, bile ducts, salivary glands, digestive tracts, renal tubules, prostate, skin, and thyroid.(3) We developed enzyme-linked immunosorbent assay (ELISA) for human soluble RAGE and examined the association of plasma soluble RAGE level with atherosclerosis, and found that it inversely correlated with carotid or femoral atherosclerosis.(4) We established an assay system for RAGE alternative splicing using a human RAGE mini-gene and HEK293T cells. Transfection experiments with various mutant RAGE mini-genes identified cis-acting elements on RAGE pre-mRNA, which regulated the alternative splicing of soluble RAGE mRNA. We also found the involvement of hnRNP-H in the regulation of soluble RAGE mRNA production.(4) We established an assay system for alternative 3'-end processing of VEGF receptor-1 (Flt-1) mRNA using a human Flt-1 mini-gene and primary cultured human vascular endothelial cells, and identified a cis-acting element on Flt-1 pre-mRNA, which regulated the production of soluble Flt-1 mRNA.

在本研究中，我们研究了选择性前体mRNA剪接/加工的机制，通过该机制产生可溶性VEGF和可溶性VEGF受体的mRNA，以及它们在糖尿病血管并发症和血管生成的调节中的作用。可溶性VEGF对AGE诱导的血管细胞损伤具有保护作用，可溶性VEGF受体是一种有效的抗血管生成因子。(1)我们通过RT-PCR克隆分离了海洋等价物的可溶性大肠杆菌。这项研究将提供一个动物直向同源的可溶性维生素C，以阐明其在健康和疾病中的作用。(2)我们通过免疫组织化学分析研究了可溶性β-淀粉样蛋白在人体器官和组织中的表达，发现可溶性β-淀粉样蛋白广泛分布于各种器官和组织，包括血管内皮、神经元、胰腺β细胞、巨噬细胞/单核细胞、胆管、唾液腺、消化道、肾小管、前列腺、皮肤和甲状腺。(3)我们建立了人可溶性胆红素的酶联免疫吸附试验（ELISA），并研究了血浆可溶性胆红素水平与动脉粥样硬化的关系，发现它与颈动脉或股动脉粥样硬化呈负相关。(4)我们建立了一个使用人β-SMA mini-gene和HEK 293 T细胞的β-SMA选择性剪接的检测系统。用各种突变的p53 mini-gene进行转染实验，鉴定了p53 pre-mRNA上的顺式作用元件，其调节可溶性p53 mRNA的选择性剪接。我们还发现hnRNP-H参与调节可溶性mRNA的产生。(4)我们利用人Flt-1基因和原代培养的人血管内皮细胞建立了VEGF受体-1（Flt-1）mRNA 3 '端替代加工的检测系统，并鉴定了Flt-1前mRNA上的顺式作用元件，该元件调节可溶性Flt-1 mRNA的产生。