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Basic research for gene therapy of urolithiasis by the inhibition of stone matrix protein

抑制结石基质蛋白基因治疗尿石症的基础研究

基本信息

批准号：
09470351
负责人：
KOHRI Kenjiro
金额：
$ 8.32万
依托单位：
Nagoya City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09470351/
关键词：
urolithiasis osteopontin gene transfection gene therapy antisense NFкBデコイビスホスホネート

项目摘要

Urinazy stones contain 1-5% protein, and many reports have suggested the importance of proteins in stone formation. We cloned and sequenced the cDNA encoding osteopontin (OPN), important soluble stone protein components of calcium oxalate stone proteins extracted with O.1M EDTA.Our previous study revealed that osteopontin (OPN), a secreted adhesive phosphoglycoprotein, were strongly expressed by renal distal tubular cells in the rat stone- forming kidney. OPN is clearly produced in the kidney, although its function and significance there remain unclear. Moreover, the association of crystals with renal tubular cells is considered a potential factor in the process of renal stone formation. The mechanisms, however, are not yet well understood. As we spaculated that OPN played a role in crystal-cell interaction, we tried to block the production of OPN by using normal rat kidney cell line (NRK-52E) transfected antisense OPN RNA.The Tetracycline-Regulated Expression System was used. Plasmid PTet-OPNas was constructed by cloning mouse OPN/2ar cDNA sequence in an inverted (antisense) orientation. Two clones of antisense transfectants were identified which showed marked reduction in OPN synthesis upon the absence of tetracycline. Intact NRK-52E cells, NRK-52E cells transfected with empty vector, and tetracycline-treated antisense clones, which did not show inhibition of OPN expression, all under identical conditions associated with calcium oxalate (CaOx) crystals. In contrast, the expression of antisense OPN12ar RNA prevents the association of CaOx crystals with >NRK-52E cells. We suggest that OPN plays a crucial role in this process.

尿石含有1-5%的蛋白质，许多报告表明蛋白质在结石形成中的重要性。我们克隆并测序了骨桥蛋白（osteopontin， OPN）的cDNA编码序列，OPN是0.1 m EDTA提取草酸钙石蛋白的重要可溶性石蛋白成分。我们先前的研究发现骨桥蛋白（OPN）是一种分泌的黏附磷酸糖蛋白，在大鼠结石形成肾的远端肾小管细胞中强烈表达。OPN显然是在肾脏中产生的，尽管其在肾脏中的功能和意义尚不清楚。此外，晶体与肾小管细胞的结合被认为是肾结石形成过程中的一个潜在因素。然而，其机制尚不清楚。由于我们推测OPN在晶体-细胞相互作用中发挥作用，我们试图通过使用正常大鼠肾细胞系（NRK-52E）转染反义OPN RNA来阻断OPN的产生。采用四环素调控表达系统。通过倒置（反义）克隆小鼠OPN/2ar cDNA序列构建质粒PTet-OPNas。在缺乏四环素的情况下，两个反义转染克隆的OPN合成明显减少。完整的NRK-52E细胞，空载体转染的NRK-52E细胞，以及四环素处理的反义克隆，没有显示出OPN表达的抑制，都在草酸钙（CaOx）晶体相关的相同条件下。相反，反义OPN12ar RNA的表达阻止了CaOx晶体与>NRK-52E细胞的结合。我们认为OPN在这一过程中起着至关重要的作用。