Development of inner blood-retinal barrier cell lines from transgehic rats harboring temperature-sensitive SV40 large T-antigen gene

用转SV 40大T抗原基因大鼠（tsA 58 Tg大鼠）建立了永生化大鼠视网膜周细胞系（TR-rPCT）和Mailer细胞系（TR-MUL）。TR-rPCT和TR-MUL细胞具有周细胞和Muller细胞标志物并在体内发挥功能。这些细胞系可以与先前建立的条件永生化大鼠视网膜毛细血管内皮细胞系TR-iBRB共培养。共培养研究表明，来自TR-rPCT细胞的可溶性因子抑制PKC-p44/42 MAPK信号转导的激活和cyclin D1、cdk 4和cdk 6的表达，抑制内皮细胞生长。使用TR-iBRB细胞和TR-MUL细胞，L-胱氨酸转运蛋白，系统xc-被表达并在内部血-视网膜屏障处起作用，并且在向视网膜供应L-胱氨酸作为抗氧化剂的前体中起作用。TR-iBRB细胞和TR-MUL细胞表达xCT和4F 2 hc mRNA和蛋白，并且xCT在马来酸二乙酯处理后的氧化应激条件下被诱导。维生素C作为抗氧化剂和/或自由基清除剂，对视网膜中的自由基进行解毒。GLUT 1在TR-iBRB细胞和TR-MUL细胞中表达，并转运脱氢抗坏血酸，维生素C的氧化形式。这些结果表明，GLUT 1在内部血视网膜屏障中起着向视网膜提供维生素C的作用。TR-iBRB细胞表达LAT 1并具有中性氨基酸的转运功能，表明LAT 1在内部血-视网膜屏障处表达并在向视网膜供应中性氨基酸中起作用。这些发现表明，新开发的条件永生化细胞系是有用的体外模型，以研究内血视网膜屏障转运功能。

Conditionally immortalized rat retinal pericyte cell line(TR-rPCT) and Mailer cell line(TR-MUL) were established from transgenic rats harboring the temperature-sensitive SV 40 large T-antigen gene(tsA58 Tg rat). TR-rPCT and TR-MUL cells have pericyte and Muller cell markers and functions in vivo. These cell lines can be applied in co-culture with previously established conditionally immortalized rat retinal capillary endothelial cell line TR-iBRB). The co-culture studies suggest that soluble factors from TR-rPCT cells suppress the activation of PKC-p44/42 MAPK signaling and the expression of cyclin D1,cdk4,and cdk6,inhibit endothelial cell growth. Using TR-iBRB cells and TR-MUL cells, L-cystine transporter, system xc-was expressed and functioned at the inner blood-retinal barrier and plays a role in supplying L-cystine as a precursor of an antioxidant to the retina. TR-iBRB cells and TR-MUL cells expressed xCT and 4F2hc mRNA and protein and xCT was induced under oxidative stress conditions following diethyl maleate treatment. Vitamin C acts as an antioxidant and/or free radical scavenger to detoxify free radicals in the retina. GLUT1 is expressed in TR-iBRB cells and TR-MUL cells and transports dehydroascorbic acid, oxidized form of vitamin C. These results suggest that GLUT1 at the inner blood-retinal barrier plays a role in supplying vitamin C to the retina. TR-iBRB cells express LAT1 and have transport functions of neutral amino aqids, suggesting that LAT1 is expressed at the inner blood-retinal barrier and plays a role in supplying neutral amino acids to the retina. These findings suggest newly developed conditionally immortalized cell lines are useful in vitro models to study the inner blood-retinal barrier transport functions.