多聚嘧啶序列结合蛋白相关剪接因子对体外培养的视网膜色素上皮细胞磷脂酰肌醇3激酶/丝氨酸-苏氨酸蛋白激酶信号通路的调控作用  

The regulation of PTB-associated splicing factor on phosphatidylinositol 3 kinase/Akt signaling pathway in retinal pigment epithelial cells

作　　者：漆晨[1,2] 东莉洁 乐毅[1,2] 张晓敏 李筱荣 郭如如[1,2] 茹玉莎 苏畅[1,2] 

机构地区：[1]天津医科大学眼科医院 [2]天津医科大学眼科研究所天津医科大学眼视光学院,300384

出　　处：《中华眼底病杂志》2015年第4期363-367,共5页Chinese Journal of Ocular Fundus Diseases

基　　金：国家自然科学基金（31100991）;天津市应用基础与前沿技术研究计划一般项目（1SJCYBJC24900）;天津市应用基础与前沿技术研究计划重点项目（14JCZDJC36200）;天津医科大学青年基金（2013KYQ09）

摘　　要：目的：观察多聚嘧啶序列结合蛋白相关剪接因子（PSF）对体外培养的视网膜色素上皮（RPE）细胞磷脂酰肌醇3激酶（PI3K）/丝氨酸-苏氨酸蛋白激酶（Akt）信号通路的调控作用。方法将体外培养的 RPE 细胞分为 PSF 高表达组、PSF 高表达对照组、PSF 低表达组、PSF 低表达对照组及假转染组。应用脂质体2000将上调 PSF 表达的真核质粒增强型绿色荧光蛋白（pEGFP）-C2-PSF、下调 PSF 表达的真核质粒 pGenesil-PSF-RNAi 以0.25、0.50、1.00μg 的递增量分别转染至 PSF 高表达组及 PSF 低表达组细胞。PSF 高表达对照组细胞转染 pEGFP-C2空载质粒。PSF 低表达对照组细胞转染 pGenesil-scramble-siRNA 质粒。假转染组仅做转染处理,不加入任何表达质粒。采用水溶性四氮唑法检测胰岛素样生长因子1（IGF-1）刺激条件下 PSF 对 RPE 细胞增生的影响。应用脂质体2000将0.50μg 真核质粒pEGFP-C2-PSF、1.00μg 真核质粒 pGenesil-PSF-RNAi 及 pEGFP-C2空载质粒转染细胞分别作为 PSF 高表达组、PSF 低表达组、对照组,采用实时定量聚合酶链反应（PCR）检测 PSF 对 IGF-1诱导的 RPE 细胞内血管内皮生长因子（VEGF）mRNA 表达的影响。采用蛋白免疫印迹法（Western blot）检测 PSF 对 IGF-1诱导的磷酸化 Akt （pAkt）蛋白表达的影响。在 IGF-1刺激后,将 RPE 细胞分为单纯渥曼青霉素（Wortmannin）处理组、单纯 PSF 高表达组、渥曼青霉素联合 PSF 高表达处理组,并以未经渥曼青霉素处理的常规体外培养的 RPE 细胞为对照组,采用实时定量 PCR 检测各组 VEGF 的表达。结果IGF-1刺激后,PSF 高表达组、PSF 高表达对照组及假转染组之间 RPE 细胞增生率比较,差异有统计学意义（F =29.728,P 〈0.05）；PSF 低表达组、PSF 低表达对照组及假转染组之间 RPE 细胞增生率比较,差异有统计学意义（F =14.121,P〈0.05）。PSF 高表达组 RPE 细胞中 VEGF mRNA 表达较对照组�Objective To observe the regulation of PTB-associated splicing factor （PSF）exerts on phosphatidylinositol 3 kinase （PI3K）/Akt signaling pathway in cultured retinal pigment epithelial （RPE） cells.Methods ARPE-1 9 RPE cells were divided into five groups including PSF overexpression （0.25,0.50,1.00 μg of pEGFP-C2-PSF plasmid DNA）,PSF overexpression control （pEGFP-C2 empty vector DNA）,PSF inhibition （0.25,0.50,1.00 μg of pGenesil-PSF-RNAi plasmid DNA）,PSF inhibition control （pGenesil-scramble-siRNA empty vector）and sham transfected group （treated with lipofactamine 2000 reagent,but without adding plasmid DNA）groups.After transfecting with plasmid DNA,the cells were stimulated with insulin-like growth factor-1 （IGF-1 ）.IGF-1-stimulated ARPE-1 9 cells were also treated with Wortmannin and/or PSF over-expression.WST-1 assay was used to detect the proliferation rates,the VEGF mRNA levels were analyzed using real time polymerase chain reaction （PCR ）, the levels of phosphorylation Akt and total Akt expression were measured by western blotting.Results After IGF-1 stimulation, the difference of the cell proliferation rates between PSF overexpression group, PSF overexpression control group and sham transfected group was statistically significant （F = 29.728,P 〈0.05）.The difference of the cell proliferation rates between PSF inhibition group,PSF inhibition control group and sham transfected was also statistically significant （F =14.121,P 〈0.05）.Compared with control group,the VEGF mRNA levels was decreased in PSF overexpression group （P =0.000 3）,but increased in PSF low expression group （P = 0.030 9）.Furthermore,overexpression of PSF could down-regulate the activation of pAkt after IGF-1 stimulation.When combined with Wortmannin treatment,the VEGF mRNA levels in PSF overexpression group was significantly lower than the control group （P 〈0.05）.Conclusions After IGF-1 treatment,PSF plays a role in suppressing the proliferation and VEGF expression in RPE cells

关 键 词：多聚嘧啶区结合蛋白质 视网膜色素上皮/酶学 磷酸肌醇 3-激酶类 p21 活化激酶类 信号传导 

分 类 号：R774.1[医药卫生—眼科]