shRNA抑制人RPE细胞中HIF1α表达下调后IGF-1对VEGF表达的影响

Effect of IGF-1 on expressions of VEGF under suppression of HIF-1a by shRNA in cultured human RPE cells

背景增生性玻璃体视网膜疾病（PVD）是一组眼底视网膜血管性疾病，主要由视网膜色素上皮（RPE）细胞增生所致，胰岛素样生长因子-1（IGF-1）和血管内皮生长因子（VEGF）与RPE细胞的异常增生和病理性新生血管生成有关，但其信号机制及功能尚不完全明了。目的探讨利用小发卡环核糖核酸（shRNA）使人RPE细胞缺氧诱导因子1d（HIF-1α）基因沉默后，IGF-1对VEGF表达的影响。方法收集健康男性供体眼球4只，分离、收集、培养RPE细胞，用SABC法行抗人角蛋白免疫细胞化学染色进行鉴定。用体外转录法合成针对HIF-1α LmRNA序列靶点的shRNA，对3～5代RPE细胞的HIF-1α进行干扰后再经50ug／LIGF-1处理24h，采用逆转录聚合酶链反应（RT—PCR）法检测人RPE细胞中HIF-1α及VEGFmRNA的表达，采用Westernblot法检测人RPE细胞中HIF-1α及VEGF蛋白的水平。结果分离培养的细胞呈扁平不规则多角形，97％的细胞对人角蛋白呈阳性反应。50ug／LIGF-1作用后，人RPE细胞HIF-1αmRNA表达量（1．49±0．18）与0txg／LIGF-1组（1．46±0．17）比较差异无统计学意义（t=0．335，P=0．743），而HIF-1α蛋白表达量（1049．86±172．54w0．00±0．00）、VEGFmRNA（0．95±0．15vs 0．35±0．07）及VEGF蛋白（391．98±56．77vs 214．36±37．15）表达量均明显增高，差异均有统计学意义（t=16．098、9．935、6．928，P〈0．05）。shRNA干扰HIF-1α／mRNA表达后，RNAi转染组HIF-1α、VEGFmRNA及其蛋白水平较RNAi空白对照组及RNAi—C转染组明显下降，3个组间各指标的总体比较差异均有统计学意义（F=68．679、89．904、21．770、6．205．P〈0．05）。结论IGF-1可通过促进人RPE细胞中HIF-1α蛋白的累积诱导VEGF的表达，是导致PVD重要的细胞因子之一。

Background Proliferative vitreo-retinal disease （PVD）is one group of ocular complications marked by the enhanced proliferation of various cells included retinal pigment epithelial （RPE） cells. Insulin-like growth factor-1 （IGF-1）and vascular endothelial growth factor（VEGF） are implicated in the aberrant cell proliferation and pathological neovascularization that characterizes PVD,but the signaling mechanism is unclear now. Objective This study was to explore the effect of IGF-1 on VEGF in cultured human RPE cells under the small hairpin loop RNA （shRNA） keeping hypoxia-inducible factor-1 α（ HIF-1α） silencing. Methods Human retinas were isolated from 4 healthy male donors, and the RPE cells were harvested and cultured. The cells were identified using anti-human keratin antibody. The third to fifth generation of human RPE cells were used in the experiment. One target site of HIF-1α mRNA was chosen by certain design principle, and shRNA was designed and synthesized by the target site and transferred into the cells in vitro, and then the cells were cultivated with 50 μ/L IGF-1 for 24 hours. The mRNA and protein expressions of HIF-1α and VEGF were detected by RT-PCR and Western blot respectively. Results Cultured human RPE cells showed the flat irregularly muhangular shape, and 97% cells appeared the positive response for keratin. HIF-1α mRNA expression in human RPE cells was significantly lower in 50μg/L IGF-1 group than the 0μg/L IGF-1 group（ 1.49±0. 18 vs 1.46±0. 17 ） （ t = 0. 335, P = 0. 743 ） , however, the expressing levels of HIF-1α protein（ 1049.86±172. 54 vs 0.00＋0.00） and VEGF mRNA（0.95±0. 15 vs 0. 35±0.07 ）and VEGF protein （391.98±56.77 vs 214.36±37.15）were raised in the 50μg/L IGF-1 group compared with 0 μg/L IGF-1 group （t= 16. 098,9. 935,6. 928,P〈0.05）. After the HIF-1α-specific shRNA was transferred into cultured RPE cells,the expressions of both HIF-1α mRNA and its protein significantly decreased in RPE ceils under 50 μg/L IGF-1 concentration condition （ F = 68. 679,89. 904,P = 0. 000） , moreover, the expression of VEGF mRNA and its protein were significantly lowed（F=21. 770,6. 205,P〈0. 05）. Conclusions IGF-1 promotes the accumulation of HIF-1α protein and induce the expression of VEGF in human RPE cells,which probably play a pivotal role in the development of PVD.