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Phenotype analysis of perlecan mutated mice in which heparan sulfates are removed by the use of ES cell.

使用 ES 细胞去除硫酸乙酰肝素的基底膜蛋白聚糖突变小鼠的表型分析。

基本信息

批准号：
11671052
负责人：
MORITA Hiroyuki
金额：
$ 2.18万
依托单位：
Showa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

In the process of glomerular filtration, glomerular basement membranes (GBM) serve as a charge barrier to retain negatively-charged macromolecules in the circulation. On the molecular basis, GBM heparan sulfate prteoglycans (HSPG) have been thought of as key components that constitute the barrier. This negative charge theory has long been generally accepted. However, there is no direct evidence at the molecular level which links the collapse of the barrier to the development of proteinuria. Perlecan is a major GBM HSPG. Perlecan encompasses five molecular domains. Exon 3 of the first domain encodes heparan sulfate attachment sites. The present study was designed to evaluate negative charge theory from a different perspective. By the use of embryonic stem (ES) cell technology, the exon 3 was removed without frame shift. As a result, homozygous mouse was produced having mutated perlecan molecule. Our biochemical analysis at the post-transcriptional level demonstrated fibroblast obtained from the homozygote produed perlecan without heparan sulfates having an intact size of the core molecule. Although homozygotes did not spontaneously show massive proteinuria, intra-peritoneal albumin overload resulted in significant increase in urinary protein excretion after 2 weeks. Our immunoelectron microscopic investigation disclosed PEI stained sites in the GBM of homozygotes was not different from those of wild-type controls. The results strongly indicated other GBM HSPG compensate the loss of perlecan heparan sulfate. Agrin, another GBM HSPS, was not a candidate.

在肾小球滤过过程中，肾小球基底膜（GBM）充当电荷屏障，将带负电的大分子保留在循环中。在分子基础上，GBM 硫酸乙酰肝素蛋白聚糖 (HSPG) 被认为是构成屏障的关键成分。这种负电荷理论早已被普遍接受。然而，在分子水平上没有直接证据表明屏障的崩溃与蛋白尿的发生有关。 Perlecan 是一种主要的 GBM HSPG。 Perlecan 包含五个分子结构域。第一个结构域的外显子 3 编码硫酸乙酰肝素附着位点。本研究旨在从不同的角度评估负电荷理论。通过使用胚胎干（ES）细胞技术，外显子3被无移码去除。结果，产生了具有突变基底膜聚糖分子的纯合小鼠。我们在转录后水平的生化分析证明，从不含硫酸乙酰肝素的纯合子产生的基底膜聚糖获得的成纤维细胞具有完整尺寸的核心分子。尽管纯合子并未自发表现出大量蛋白尿，但腹腔内白蛋白超负荷导致2周后尿蛋白排泄显着增加。我们的免疫电子显微镜研究表明纯合子 GBM 中的 PEI 染色位点与野生型对照没有不同。结果强烈表明其他 GBM HSPG 补偿了基底膜聚糖硫酸乙酰肝素的损失。另一位 GBM HSPS Agrin 不是候选人。