缺氧对人视网膜色素上皮细胞G蛋白信号转导调节子5表达的影响及其可能机制

Possible influence of hypoxia on the expression of G-protein signaling 5 regulators in human retinal pigment epithelial cells and its mechanism

目的探讨缺氧环境下人视网膜色素上皮（RPE）细胞中G蛋白信号转导调节子5（RGS5）表达的可能机制。方法实验研究。在正常培养的人RPE细胞培养基中加入终浓度为200／μmol／L的CoCl：建立细胞化学缺氧模型。按缺氧时间分为0、1、3、6、12和24h6组，观察RGS5和血管内皮生长因子（VEGF）表达的变化；另选取12和24h作观察点，按处理因素分为缺氧对照组和VEGF抑制剂Bevacizumab处理组，Bevacizumab包括25、100和250肛咖l3种浓度，观察特异性抑制VEGF表达对RGS5表达产生的影响。应用免疫荧光染色法、Western blot和RT-PCR分别检测RGS5及其mRNA和VEGF蛋白及其mRNA的表达。采用Student’s t检验进行统计学分析。结果正常条件下，RGS5主要在人RPE细胞胞浆中表达。与VEGF存在相同表达区域。随着缺氧时间的延长，RPE细胞中RGS5蛋白及其mRNA表达逐渐上调，24h时达高峰（0．932±0．104，t=3．106，P=-0．01l；0．742±0．083，t=2．852，P=-0．017）；VEGF蛋白及其mRNA表达12h达高峰（1．022±0．141，t=4．144，P=0．002；0．491_＋0．063，t=5．707，P〈0．01），24h时有所下降，但仍高于无缺氧的RPE细胞（0．942±0．125，t=3．306，P=0．008；0．425±0．080，t=3．239，P=0．011）。浓度为100和250μg／ml的Bevacizumab有效抑制VEGF蛋白（￡=2．794，P=0．019；t=3．396，P=0．007）和其mRNA（t=2．823，P=0．018；t=9．584，P〈0．01）表达后，RGS5蛋白（t=2．953，P=0．014；t=2．913，P=0．015）和其mRNA（t=3．009，P=-O．013；t=4．243，P=-0．002）表达也随之受到抑制。结论缺氧条件下人RPE细胞RGS5表达上调，并与VEGF表达具有时相关系；抑制VEGF表达后，RGS5表达随之降低。提示RGS5位于VEGF信号通路下游，可能参与缺血缺氧性RPE相关眼病发生的调控。

Objective To observe the possible influence of hypoxia on the expression of regulators of G-protein signaling 5 （RGS5） in human retinal pigment epithelial （RPE） ceils. Methods Experimental study. To establish a hypoxia model, human RPE cells were cultured with 200 μmol/L COC12. RPE cells were sorted for showing the expressions of RGS5 and vascular endothelial growth factor （VEGF） in the following time frames： 0, 1, 3, 6, 12 and 24 hours. RPE cells were grouped according to treatment protocols： the hypoxia and VEGF inhibitor Bevacizumab groups at 12 and 24 hour time points, treated with concentrations of Bevacizumab at 25, 100 and 250 μg/ml. The expression of RGS5 and VEGF and their mRNA was examined by immunofluorescence, Western blot and RT-PCR. All results were statistically evaluated using a student＇s t test. Results RGS5 protein was mainly found in the cytoplasm of cultured RPE cells in normoxic conditions. The expression of RGS5 protein and mRNA increased in hypoxia conditions. Expression for both of them peaked at 24 hours after hypoxia （0.932±0.104, t=3.106, P=0.011; 0.742±0.083, t=2.852, P=0.017）. The highest levels of VEGF protein and mRNA expression occurred at 12 hours （1.022±0.141, t=4.144, P=-0.002; 0.491±0.063, t=5.707, P〈0.01）, and levels were Still higher than normal at 24 hours （0.942±0.125,t=3.306, P=0.O08; 0.425±0.080, t=3.239, P=0.011）. The expression of RGS5 protein and mRNA was inhibited by Bevacizumab in the 100 μg/ml （t=2.953, P=O.014; t=3.009, P=01013） and 250 Ixg/ml （t=2.913, P=0.015; t=4.243, P=O.O02）groups, following the inhibition of VEGF （t=2.794, P=O.019; t=2.823, P=0.018; t=3.396, P=O.O07; t=9.584, P〈O.O1）. Conclusion The expressions of VEGF increase earlier than RGS5 in hypoxia conditions, and Bevacizumab can effectively inhibit the expression levels of RGS5 and VEGF in human RPE cells. This may suggest RGS5 could be located downstream from the VEGF signaling pathway, and play an important role in regulating RPE-related hypoxic-isehemic ophthalmopathy.