Purification, molecular cloning, and expression of a novel growth-promoting factor for retinal pigment epithelial cells, REF-1/TFPI-2.
Purification, molecular cloning, and expression of a novel growth-promoting factor for retinal pigment epithelial cells, REF-1/TFPI-2.
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视网膜色素上皮细胞新型生长促进因子 REF-1/TFPI-2 的纯化、分子克隆和表达。
DOI：
10.1167/iovs.03-0230
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发表时间：
2004
影响因子：
4.4
通讯作者：
Iwata,Takeshi
中科院分区：
文献类型：
--
作者：
Tanaka,Yasuhiko;Utsumi,Jun;Matsui,Mizuo;Sudo,Tetsuo;Nakamura,Noriko;Mutoh,Masato;Kajita,Akemi;Sone,Saburo;Kigasawa,Kazuteru;Shibuya,Masahiko;Reddy,VenkatN;Zhang,Qiang;Iwata,Takeshi
目的.视网膜色素上皮（RPE）细胞在维持视网膜的稳态和控制脉络膜新生血管形成中起重要作用。本研究的目的是确定一个或多个因素，将刺激RPE细胞增殖。为了分离这样的因子，100 L的人成纤维细胞条件培养基进行离子交换，疏水，反相色谱法，然后十二烷基硫酸钠-聚丙烯酰胺凝胶电泳。在人K-1034 RPE细胞系和人原代RPE细胞中检查该因子的生长促进活性。不同的色谱过程分离出一个31 kDa的因子，具有促进RPE细胞生长的特性。这种因子，我们命名为RPE细胞因子（REF）-1，促进RPE细胞的生长，但不促进人脐静脉内皮细胞（HUVECs）的生长。REF-1的氨基端序列和分子克隆的cDNA与TFPI家族的组织因子途径抑制剂（TFPI）-2和丝氨酸蛋白酶抑制剂胎盘蛋白（PP）-5相同。在中国仓鼠卵巢（CHO）细胞中表达REF-1/TFPI-2，证实其对RPE细胞具有促生长活性。在CHO细胞中表达的重组REF-1/TFPI-2的主要成分具有31 kDa的分子量，并在体外对RPE细胞具有生长促进活性，但对人内皮细胞和成纤维细胞没有生长促进活性。REF-1/TFPI-2也具有蛋白酶抑制活性。其他家族因子，TFPI-1，没有促进RPE细胞生长。REF-1/TFPI-2是一种新的RPE细胞生长促进因子，但对内皮细胞和成纤维细胞没有作用。它的特性使其对眼内治疗修复和维持RPE细胞具有潜在的益处。
purpose. Retinal pigment epithelial (RPE) cells are known to play important roles in maintaining the homeostasis of the retina and in controlling choroidal neovascularization. The purpose of this study was to identify a factor or factors that would stimulate RPE cells to proliferate.methods. To isolate such a factor, 100 L of human-fibroblast–conditioned medium underwent ion-exchange, hydrophobic, and reverse-phase chromatographies followed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The growth-promoting activity of the factor was examined in a human K-1034 RPE cell line and human primary RPE cells.results. The different chromatographic processes isolated a 31-kDa factor that had RPE cell growth–promoting properties. This factor, which we have named RPE cell factor (REF)-1, promotes growth of RPE cells but not of human umbilical vein endothelial cells (HUVECs). The amino-terminal sequence and molecular cloned cDNA of REF-1 were identical with those of tissue-factor pathway inhibitor (TFPI)-2, a family of TFPIs, and placental protein (PP)-5, a serine protease inhibitor. The cDNA expression of REF-1/TFPI-2 with pcDL-pSRα vector in Chinese hamster ovary (CHO) cells confirmed the growth-promoting activity for RPE cells. The major component of the recombinant REF-1/TFPI-2 expressed in CHO cells had a molecular mass of 31 kDa and exerted growth-promoting activity in RPE cells but not in human endothelial cells and fibroblasts in vitro. REF-1/TFPI-2 also had protease inhibitory activity. The other family factor, TFPI-1, did not promote RPE cell growth.conclusions. REF-1/TFPI-2 is a novel growth-promoting factor for RPE cells but not for endothelial cells and fibroblasts. Its properties make it potentially beneficial for intraocular therapy for the repair and maintenance of RPE cells.