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A novel monoclonal antibody recognizing extracellular space of the atherosclerotic lesions in WHHL rabbits.

一种新型单克隆抗体，可识别 WHHL 兔动脉粥样硬化病变的细胞外空间。

基本信息

批准号：
03454495
负责人：
TAKANO Tatsuya
金额：
$ 4.1万
依托单位：
Teikyo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

项目摘要

In order to study the mechanisms of cholesteryl ester accumulation in the extracellular space of the atherosclerotic aorta, we prepared novel monoclonal antibodies against atherosclerotic lesions using a delipidated crude homogenate of atherosclerotic aorta from Watanabe-heritable hyperlipidemic (WHHL) rabbits as a complex mixture of immunogens. By screening the antibodies histochemically, we were able to obtain for the first time monoclonal antibodies which specifically recognized the extracellular matrix with lipid-laden deposits (EMRla/212D) in atherosclerotic lesions. The purified antigenic material is a glycoprotein with a molecular mass of 66 kDa, and the epitope of the antigenic material contains sialic acid as a major element.We have isolated from rabbit liver cDNA library clones coding for the 66-kDa glycoprotein (GP66). The clone spans the sequence coding for the entire GP66 (456 amino acids) and 19 amino acids of a signal peptide. GP66 was deduced to be rabbit vitronectin from its nucleotide sequence, containing an Arg-Gly-Asp cell attachment sequence and showing 76% amino acid sequence homology with human vitronectin. Furthermore, EMRla/212D recognized rabbit vitronectin purified by heparin affinity chromatography. RNA blot hybridization detected one transcript of the same size in normal and WHHL rabbit liver. The levels of plasma GP66 and liver GP66 mRNA were not altered, whereas 9-fold greater accumulation of GP66 was observed in the thoracic aorta of WHHL rabbit. These results suggest that GP66 is rabbit vitronectin, which selectively accumulates in the atherosclerotic aorta with the development of atherosclerosis.

为了研究胆固醇酯在动脉粥样硬化动脉细胞外积聚的机制，我们以Watanabe遗传性高脂血症(WHHL)兔动脉粥样硬化动脉匀浆的脱脂粗匀浆作为免疫原的复杂混合物，制备了新型的抗动脉粥样硬化病变的单抗。通过对抗体的组织化学筛选，我们首次获得了特异性识别动脉粥样硬化病变中含有脂质沉积的细胞外基质(EMR1a/212D)的单抗。纯化的抗原物是一种分子质量为66 kDa的糖蛋白，其表位以唾液酸为主要成分，我们从兔肝中克隆了编码66 kDa糖蛋白(GP66)的cDNA文库。该克隆跨越了编码整个GP66(456个氨基酸)和19个氨基酸的信号肽的序列。根据其核苷酸序列，推测GP66为兔Vitronectin，含有Arg-Gly-Asp细胞附着序列，与人Vitronectin的氨基酸序列同源性为76%。此外，EMR1a/212D可识别肝素亲和层析纯化的兔玻璃连结蛋白。RNA印迹杂交在正常和WHHL兔肝组织中检测到一个大小相同的转录本。WHHL兔血浆和肝脏中GP66的表达水平无明显变化，但胸主动脉中GP66的积聚增加了9倍。这些结果表明，GP66是一种兔玻璃连结蛋白，随着动脉粥样硬化的发展，它选择性地积聚在动脉粥样硬化的主动脉中。