Molecular analysis of cell growth regulating factor in retinal, capillary endothelial cells

建立了携带温度敏感性猿猴病毒40大T抗原基因的转基因大鼠永生化视网膜毛细血管内皮细胞系（TR-iBRB）和视网膜周细胞系（TR-rPCT）。研究了TR-iBRB和TR-rPCT细胞之间的旁分泌相互作用，以阐明周细胞中的细胞生长调节因子。将TR-iBRB细胞接种在Transwell小室的插入物上，将TR-rPCT细胞接种在插入物下（接触共培养模型）和孔室上（非接触共培养模型）。TR-rPCT细胞的条件培养基也用于观察TR-iBRB细胞的细胞生长。随着TR-rPCT条件培养基浓度的增加，TR-iBRB细胞的生长速度明显减慢。在接触共培养模型中，TR-iBRB细胞的生长在共培养开始后4天被阻滞。上述结果表明，在视网膜毛细血管内皮细胞中，周细胞释放的某些因子参与了细胞生长的调节，TR-iBRB细胞摄取[^<14>C] L-胱氨酸的过程是一个非饱和Na^+依赖的过程，其Michaelis-Menten常数为9.2 μM，表明由XCT和4F 2 hc mRNA组成的xc系统介导了L-胱氨酸的摄取。在100 μM马来酸二乙酯（氧化应激模型药物）处理后，TR-iBRB细胞中XCT mRNA水平和L-胱氨酸摄取活性以时间依赖性方式增强。同时，TR-iBRB细胞中的谷胱甘肽浓度增加。相反，4F 2 hc mRNA水平在24小时内没有变化，并且通过DEM预处理诱导超过24小时。这些发现表明，L-胱氨酸转运激活系统xc的诱导下的氧化应激条件下，在内部血视网膜屏障。

Conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB) and retinal pericyte cell line (TR-rPCT) were established from transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene. The paracrine interaction between TR-iBRB and TR-rPCT cells has been investigated to elucidate the cell growth regulating factor in pericyte. TR-iBRB cells were seeded on the insert of Transwell chamber and TR-rPCT cell were seeded under the insert (contact co-culture model) and on the well chamber (non-contact co-culture model). The conditioned medium for TR-rPCT cells was also used to observe the cell growth of TR-iBRB cells. The growth of TR-iBRB cells was significantly slowed down with increasing concentration of TR-rPCT conditioned medium. In contact co-culture model, the growth of TR-iBRB cells was arrested 4 days after the beginning of the co-culture. These results suggest that some factor released from pericyte cells is involved in regulating the cell growth in retinal capillary endothelial cells.The [^<14>C] L-cystine uptake by TR-iBRB cells appeared to be mediated through a saturable Na^+-independent process with a Michaelis-Menten constant of 9.2 μM, suggesting system xc, which consists of XCT and 4F2hc mRNA, mediated L-cystine uptake. After 100 μM diethyl maleate treatment, which is a model agent for oxidative stress, the XCT mRNA level and L-cystine uptake activity in TR-iBRB cells were enhanced in a time-dependent manner. Concomitantly, the glutathione concentration in TR-iBRB cells was increased. In contrast, the 4F2hc mRNA level was unchanged up to 24 hours and was induced for more than 24 hours by DEM pretreatment. These findings suggest L-cystine transport is activated by induction of system xc under the oxidative stress conditions at the inner blood-retinal barrier.