糖基化终末产物诱导大鼠视网膜微血管内皮细胞表型和功能改变

Phenotypic and functional changes of rat retinal microvascular endothelial cells induced by advanced glycation end products

目的 研究糖基化终末产物(AGEs)诱导大鼠视网膜微血管内皮细胞表型和功能的变化及相关机制.方法 SPF级雄性Wistar大鼠20只,7～8周龄,体重260～280 g,分离培养大鼠视网膜内皮细胞,采用免疫荧光染色、流式细胞术和体外形成毛细血管样网络结构的方法进行鉴定.视网膜内皮细胞在AGEs刺激后,用噻唑蓝(MTT)法分析细胞的增殖能力;用膜连蛋白V/碘化丙啶(Annexin V/PI)双染法检测细胞凋亡;采用逆转录聚合酶链反应(RT-PCR)和流式细胞术检测细胞AGEs受体、人蛋白激酶C(PKC)、细胞间黏附分子1(ICAM-1)和诱导型一氧化氮合酶(iNOS)的表达变化.采用t检验进行统计学分析.结果 分离纯化的大鼠视网膜内皮细胞表达血管性血友病因子(vWF)并在人工基膜上形成毛细血管网络结构.AGEs以时间和剂量依赖的方式抑制大鼠视网膜内皮细胞增殖能力,在200 mg/L的AGEs组培养至第5天时细胞增殖能力低于对照组(t=8.9,P＜0.05),7 d和9 d时抑制作用更明显(t值分别为15.7和46.1,均P＜0.01).400 mg/L AGEs组在第3天开始出现细胞增殖减慢(t=12.5,P＜0.05),从第5天开始增殖速度明显低于对照组(t值分别为22.4、41.5和77.7,均P＜0.01).并且AGEs诱导视网膜内皮细胞凋亡.进一步分析发现AGEs上调了大鼠视网膜内皮细胞AGEs受体、PKC、ICAM-1和iNOS的mRNA表达(t值分别为91.8、9.22、16和42,均P＜0.01)和蛋白水平的表达(t值分别为20.2、12.3、7.7和13.9,均P＜0.01).结论 AGEs可能通过上调AGEs受体诱导大鼠视网膜微血管内皮细胞表型和功能的改变.

Objective To investigate whether the advanced glycation end products (AGEs) can induce the apoptosis of rat retinal microvascular endothelial cells and the related mechanisms. Methods Twenty SPF male Wistar rats aged 7 to 8 weeks were used. Microvascular endothelial cells was isolated from rat retinal, and identified by immunofluorescence staining and flow cytometry for vWF, and the tube formation on Matrigel. MTT assay was used to analyze the effect of AGEs on the proliferation of rat retinal microvascular endothelial cells. Flow cytometry was used to detect AGEs-induced apoptosis. Reverse transcription PCR and flow cytometry were used to analyze the expression level of RAGE, PKC, ICAM-1 and iNOS with or without AGEs in culture medium. t-test were used for statistical analysis. Results The isolated and purified rat retinal microvascular endothelial cells express vWF, and can form capillary-like network structure in Matrigel. AGEs can inhibit proliferation of rat retinal endothelial cells in dose- and timedependent ways: the proliferation of endothelial cells in 200 mg/L AGEs group was lower than control at 5 -day(t =8.9, P ＜0.05) ,and more significant at 7- and 9-day (t = 15.7 or 46.1, both P ＜0.01 ). The inhibition to proliferation of endothelial cells by 400 mg/L AGEs was significant at 3-day ( t = 12.5, P ＜0.05 ), and became greatly at 5-, 7- and 9-day ( t = 22.4, 41.5 or 77.7, all P ＜ 0.01 ). AGEs also induced apoptosis of retinal endothelial cells. Furthermore, AGEs can up-regulate the expression of RAGE, PKC,iNOS and ICAM-1 at mRNA level(t = 91.8, 9.22, 16 and 42, both P ＜ 0.01 ) and protein level (t = 20.2,12.3, 7.7 and 13.9, all P ＜0.01 ). Conclusion AGEs might induced phenotypic and functional changes of rat retinal microvascular endothelial cells by up-regulating RAGE.