Kato K, Ishiguro S, Yamamoto H, Yanai N, Obinata M, Tamai M
Department of Ophthalmology, Tohoku University School of Medicine, Tohoku University, Sendai, Japan.
Cell Struct Funct. 1996 Dec;21(6):459-68. doi: 10.1247/csf.21.459.
We established a retinal pigment epithelium-derived cell line from transgenic mouse harboring temperature-sensitive simian virus 40 T-antigen gene (tsSV40T) and examined its characteristics. We enucleated both eyes from a 2-month-old transgenic mouse and removed the retinal pigment epithelial (RPE) cells and neuroretinal cells. After cloning the RPE cells, we obtained a cell line (RPET). RPET cells grew well at 33 degrees C but not at 37 degrees C, expressing on the temperature-sensitive character of tsSV40T, and maintained characters of RPE cells such as T1-tyrosinase production, phagocytosis of rod outer segments, and presence of cytokeratin, microvilli on the cell surface and lysosome-like granules around the Golgi apparatus in the cytoplasm. Conditioned medium (CM) from a culture of neuroretinal cells harboring tsSV40T was essentially required for growth. The factor(s) in CM was heat-and acid labile, but was resistant to trypsin digestion. In the presence of 3% CM, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) and strong effects on RPET cells, whereas insulin, insulin-like growth factor I (IGF-I), and IGF-II had moderate growth effects. Interestingly, none of these growth factors stimulated the RPET cells in the absence of CM. EHS-Matrix had growth effect, whereas laminin, collagen types I and IV, and fibronectin had no marked growth effects on RPET cells. RPET cells were morphologically changed on a laminin-coated dish. They could not spread on the coated dish, and the majority of the cells floated. But when the floating cells were transferred to non-coated dishes, they immediately attached themselves. These results suggest that RPET cells are a good model for for finding novel growth factor(s) and for investigating the mechanism of cell-laminin attachment.
我们从携带温度敏感型猿猴病毒40 T抗原基因（tsSV40T）的转基因小鼠中建立了一种视网膜色素上皮衍生细胞系，并对其特性进行了研究。我们摘除了一只2个月大转基因小鼠的双眼，分离出视网膜色素上皮（RPE）细胞和神经视网膜细胞。克隆RPE细胞后，我们获得了一个细胞系（RPET）。RPET细胞在33℃时生长良好，但在37℃时不能生长，表现出tsSV40T的温度敏感特性，并保持RPE细胞的特征，如产生T1-酪氨酸酶、吞噬视杆细胞外段、存在细胞角蛋白、细胞表面有微绒毛以及细胞质中高尔基体周围有溶酶体样颗粒。来自携带tsSV40T的神经视网膜细胞培养物的条件培养基（CM）是细胞生长所必需的。CM中的因子对热和酸不稳定，但对胰蛋白酶消化有抗性。在存在3% CM的情况下，表皮生长因子（EGF）和碱性成纤维细胞生长因子（bFGF）对RPET细胞有强烈作用，而胰岛素、胰岛素样生长因子I（IGF-I）和IGF-II有中等生长作用。有趣的是，在没有CM的情况下，这些生长因子均不刺激RPET细胞。EHS-基质有生长作用，而层粘连蛋白、I型和IV型胶原以及纤连蛋白对RPET细胞没有明显的生长作用。RPET细胞在涂有层粘连蛋白的培养皿上形态发生改变。它们不能在涂有层粘连蛋白的培养皿上铺展，大多数细胞漂浮。但当将漂浮的细胞转移到未涂覆的培养皿上时，它们会立即附着。这些结果表明，RPET细胞是寻找新型生长因子和研究细胞与层粘连蛋白附着机制的良好模型。
