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The Structure and Function of Retinal Pigment Epithelial Cells

视网膜色素上皮细胞的结构和功能

基本信息

批准号：
07457003
负责人：
HIROSAWA Kazushige
金额：
$ 4.48万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

Monoclonal antibodies were raised against chick and bovine retinal pigment epithelial (RPE) cells using mice as immune animal. Among the monoclonal antibodies, S5D8 and RPE7 were analyzed their characters by means of molecular and cellular biological methods.1) ; S5D8 : The antgen protein has molecular weight of 63kDa. The whole length of cDNA is 2510bp, open reading frame being 1599bp with 2 olyadenylation signals. Amino acid residues are 533 and their molecular size being 60.9kDa. The protein has 3 glycosylation sites. It has neither signal peptide nor membrane expanding region. The size of mRNA is about 2.9kbases. The open reading frame was transfected to cultured fibroblasts, the endoplasmic reticula of which were stained with S5D8.2) ; PRE7 : The localization of RPE7 was on the plasma membranes of RPE and Muller cells of neural retina. This result seem to suggest the protein RPE7 is involved in the supporting function of the retina. The protein has molecular weight of 40-45kDa by Western blotting. RPE7 has an open reading frame of 271 amino acid residues, 3 glycosylation sitesand is found to have a membrane expanding hydrophobic region. Homology research of this cDNA sequences revealed that RPE7 protein belongs to a novel type of immunoglobulin superfamily, one of which has been named EMMPRIN.EMMPRIN is involved in the remodeling of extrcellular matrix and plays a role in the metastasis of cancer cells. Metalloproteinase activity was assayd with cultured bovine RPE by zymography. The culture supernatant contained gelatinaseA,the activity of which became higher when anti-RPE7 antibody was added to the culture medium. It is concluded, from this result, that RPE7 plays an important role in the phagocytosis of outer segment and mobilization of RPE at some pathlogical conditions.

以小鼠为免疫动物，分别制备了针对鸡和牛视网膜色素上皮细胞的单克隆抗体。其中S5D8和RPE7单克隆抗体通过分子生物学和细胞生物学方法分析了它们的特性。1);S5D8：抗原蛋白分子量为63kDa。cDNA全长2510bp，开放阅读框1599bp，含2个聚腺苷化信号。氨基酸残基数为533个，分子量为60.9kDa。蛋白质有3个糖基化位点。它既没有信号肽，也没有膜扩张区。mRNA的大小约为2.9kbase。将开放阅读框转染培养成纤维细胞，对其内质网进行S5D8.2染色；PRE7: RPE7定位于RPE和神经视网膜Muller细胞的质膜上。这一结果似乎表明RPE7蛋白参与了视网膜的支持功能。Western blotting测定该蛋白分子量为40-45kDa。RPE7具有271个氨基酸残基和3个糖基化位点的开放阅读框，并具有膜扩张疏水区。对该cDNA序列的同源性研究表明，RPE7蛋白属于一种新型的免疫球蛋白超家族，其中一个已被命名为EMMPRIN。EMMPRIN参与细胞外基质的重塑，并在癌细胞转移中发挥作用。用培养牛RPE酶谱法测定金属蛋白酶活性。培养上清含有明胶酶ea，在培养基中加入抗rpe7抗体后，明胶酶ea活性提高。由此可见，在某些病理条件下，RPE7在外节吞噬和RPE的动员中发挥了重要作用。