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

用温敏性SV 40大T抗原转基因大鼠(tsA58TG大鼠)建立了条件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细胞在体内具有周细胞和Muller细胞标志物和功能。这些细胞系可用于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了PKC-p44/42 MAPK信号通路的激活，抑制了细胞周期蛋白D1、CDK4和CDK6的表达，从而抑制了内皮细胞的生长。利用tr-iBRB细胞和tr-MUL细胞，L-胱氨酸转运蛋白系统XC-在血-视网膜内屏障表达并发挥作用，并作为视网膜抗氧化剂的前体供应L-胱氨酸。经顺丁烯二酸二乙酯处理后，在氧化应激条件下，tr-iBRB细胞和tr-MUL细胞表达Xct和4F2hc mRNA和蛋白，Xct被诱导。维生素C起抗氧化剂和/或自由基清除剂的作用，以消除视网膜中的自由基。Glut1在tr-iBRB细胞和tr-MUL细胞中表达，并运输脱氢抗坏血酸，维生素C的氧化形式。这些结果表明，位于血-视网膜内屏障的GLUT1在向视网膜供应维生素C方面发挥了作用。RT-iBRB细胞表达LAT1，并具有中性氨基酸转运功能，提示LAT1表达于血-视网膜内屏障，起到为视网膜提供中性氨基酸的作用。这些发现表明，新开发的条件永生化细胞系是研究血-视网膜内屏障运输功能的有用的体外模型。

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.