缺氧对培养的人视网膜色素上皮细胞中血管生成素-2表达的影响

Effect of hypoxia on expression of angiopoietin-2 in human retinal pigment epithelium

背景脉络膜新生血管（CNV）是导致一些眼底疾病视力丧失的主要原因，而血管生成素2（Ang-2）与血管生成的关系较为密切。目前在缺氧条件下人视网膜色素上皮（RPE）细胞上Ang-2的表达情况与CNV发病的关系研究较少。目的观察缺氧对体外培养的人RPE细胞中Ang．2表达的影响，探讨Ang-2在CNV形成中的可能作用。方法人RPE细胞经培养和传代，取第4～7代细胞以5×10^7个／L密度接种于培养皿，常氧对照组不加CoCl2，缺氧组换以含终浓度为200μmol／LCoCl2的培养基5ml建立RPE细胞化学缺氧模型，分别于加药导致缺氧后0．5、1、2、4、6、12和24h终止缺氧。采用半定量逆转录聚合酶链反应（RT—PCR）法检测缺氧不同时间对体外培养的人RPE细胞Ang-2mRNA表达的影响；采用酶联免疫吸附反应（ELISA）法检测缺氧不同时间对体外培养的人RPE细胞上清液中Ang-2蛋白质量浓度的影响。结果人RPE细胞复苏后存活率达90％，第4代传代细胞中色素很少，细胞形态多为梭形。不同培养条件组人RPE细胞中Ang-2mRNA／β-actin mRNA值的总体比较差异有统计学意义（F=1086．30，P=0．00）；缺氧条件下培养0．5h后人RPE细胞中Ang-2mRNA／β-actin mRNA值开始明显增加，至缺氧培养后4～6h达高峰，与常氧对照组比较，差异均有统计学意义（P〈0．05）；缺氧培养24h后Ang-2mRNA／β-actin mRNA值接近常氧对照组。ELISA分析结果表明，不同培养条件下人RPE细胞培养基上清液中Ang-2蛋白的总体比较差异有统计学意义（F=1034．00，P=0．00），缺氧条件下培养0．5h后人RPE细胞中Ang-2蛋白的质量浓度开始明显升高，6h达到高峰，与常氧对照组相比，差异均有统计学意义（P〈0．05）；缺氧培养24h后Ang-2蛋白的质量浓度接近常氧对照组。结论缺氧可显著上调体外培养的人RPE细胞中Ang-2的表达，Ang-2于缺氧早期呈高表达，提示Ang-2参与了CNV的形成，可能在CNV形成过程的早期阶段发挥作用。

Background Choroidal neovascularization（CNV）has been described as a main reason of visual loss in a lot of ocular diseases. Researches showed that local hypoxia and retinal pigment epithelial（RPE） cells play an important role in the formation of CNV. A closely relationship of angiopoietin-2 （Ang-2） and angiogenesis has been proved. However, whether the expression of Ang-2 in hypoxic cultured human RPE ceils is associated with the pathogenesis of CNV is still below understood. Objective This study was to investigate the effects of hypoxia on expression of Ang-2 in cultured human RPE cells in vitro, and discuss the possible effects of Ang-2 in the formation of CNV. Methods Human RPE cells were cultured and passaged, and 4-7 generation of cells were used in the experiment. The cells were incubated in cultural plate at the density of 5×10^7 cells/L. The culture medium containing 200 μmol/L CoC12 was used to establish the hypoxia model of human RPE cells cultured in vitro for 0. 5,1,2,4,6,12 and 24 hours, and the RPE cells cultured under normoxia were as controls. Reverse transcription polymerase chain reaction（ RT-PCR） was used to detect the expression of Ang-2 mRNA in cultured human RPE cells, and enzyme- linked immunosorbnent assay （ELISA） was used to assay the content of Ang-2 protein in supernatant of cultured human RPE cells. Results The survival rate of human RPE cells was 90% after resuscitation. The fourth generation of ceils showed the fusiform with the less pigment in them. The Ang-2 mRNA/β-actin mRNA value in human RPE cells was significantly different among various groups （F= 1086. 30,P = 0. 00）,The Ang-2 mRNA/β-actin mRNA value in hypoxia cultured for 0.5 hours group began to increase and peaked in hypoxia culture for 4-6 hours group,with the significant differences in comparison with normoxia control group （P〈 0.05 ）. The Ang-2 mRNA/β- actin mRNA value decreased to the baseline level at hypoxia for 24 hours. The ELISA analysis showed that the concentration of Ang-2 protein in supernatant of RPE cells showed significant difference among groups（ F= 1034.00, P= 0.00）. The concentration of Ang-2 protein increased at hypoxia culture for 0.5 hours and peaked at 6 hours, showing significant differences in comparison with the control group （P 〈 0.05 ）. Conclusions Hypoxia could significantly up-regulate the expression of Ang-2 in human RPE ceils cultured in vitro. Ang-2 expresses highly in the early stage of hypoxia,implying that Ang-2 participates in the formation of CNV.