多聚嘧啶序列结合蛋白相关剪接因子对视网膜血管内皮细胞IGF-1／VEGF信号通路的抑制作用

Inhibition of PTB-associated splicing factor on IGF-1/VEGF signaling pathway in retinal vascular endothelial cells

背景视网膜新生血管（RNV）是多种眼底血管疾病的共同表现，研究证实胰岛素样生长因子（IGF-1）能够刺激血管内皮细胞的增生，从而促进新生血管的形成，而多聚嘧啶序列结合蛋白相关剪接因子（PSF）在IGF-1信号转导的过程中发挥转录抑制的作用。但PSF在RNV形成过程中的作用尚不清楚。目的研究PSF对IGF-1／血管内皮生长因子（VEGF）信号通路的调控作用。方法将猕猴视网膜血管内皮细胞RF／6A进行培养后分为Pegfp-C2-PSF质粒转染组、pGenesil．PSF-RNAi质粒转染组及各自的对照组，分别在细胞中转人真核质粒Pegfp—C2-PSF、pGenesil—PSF—RNAi及相应的空白质粒。各组细胞培养液中分别添加或不添加胰岛素样生长因子1（IGF-1）以刺激细胞生长，采用MTT法检测各组RF／6A细胞的增生率并筛选各组最适PSF剂量，用于进一步的实验。采用逆转录PCR法测定和比较不同PSF转染组间用或不用U0126处理组间RF／6A细胞中VEGFmRNA的表达；采用Westernblot法验证PSF对IGF-1诱导的细胞外调节蛋白激酶（ERK）活化的促进作用。结果IGF-1刺激后Pegfp．C2．PSF质粒转染组以0．50IxgPSF为最适剂量，其RF／6A细胞的增生率为0．220±0．020，细胞中VEGFmRNA相对表达量为0．79±0．07，分别低于其对照组的0．260±0．006和未转染组的1．26±0．19，差异均有统计学意义（P=0．040、0．016）；IGF-1刺激后pGenesil—PSF—RNAi质粒转染组以1．00IxgPSF作为最适剂量，其RF／6A细胞增生率为0．35±0．02，VEGFmRNA相对表达量为2．29＋0．43，分别高于其对照组的0．210-4-0．019和未转染组的1．26±0．19，差异均有统计学意义（P=0．003、0．019）。IGF-1刺激后1、3、6hPegfp-C2-PSF质粒转染组细胞中pERK蛋白表达量均明显低于未转染组，差异均有统计学意义（P=0．017、0．000、0．000）。Pegfp-C2-PSF质粒转染组U0126’处理后细胞中VEGFmRNA的相对表达量为0．93±0．21，明显低于未转染组的1．32±0．08，差异均有统计学意义（P=0．037），同时明显低于Pegfp—C2-PSF质粒转染组无U0126处理的细胞中VEGFmRNA的相对表达量1．23±0．09，差异有统计学意义（P=0．002）。结论PSF可抑制IGF-1诱导的RF／6A细胞中的ERK信号通路的活化，下调VEGF在RF／6A细胞中的表达，进而抑制RF／6A细胞的增生。

Background Retinal neovascularization （RNV） is common findings of many fundus diseases. Research showed that insulin-like growth factor-1 （IGF-1） induces the proliferation of vascular endothelial ceils and therefore promotes the formation of new blood vessel. Polypyrimidine tract-binding protein-associated splicing factor （PSF） is determined to play the inhibitory effect on the gene trancription in IGF-1 signal pathway,but its effect on RNV is still unelucidated. Objective This study was to observe the regulation of PSF on IGF-1/vascular endothelial growth factor（VEGF） signaling pathway in vitro. Methods RF/6A cells were cultured and divided into four groups. Pegfp-C2-PSF plasmid, Pegfp-C2 plasmid, pGenesil-PSF-RNAi plasmid and pGenesil-RNAi plasmid were transfeeted into the cells with the PSF dose of 0. 00, 0. 25,0.50 and 1. 00 μg respectively. The cells were cultured with or without IGF-1. The proliferation rates of the cells were detected by using MTT assay to determine theoptimal PSF dose. Reverse-transceiption PCR （RT-PCR） was employed to assay and compare the relative expression levels of VEGF mRNA in the ceils among different PSF plasmid tranfected groups or between the treated groups with and without U0126,and the levels of phosphorylized extracelluIar signal-regulation kinase （pERK） in the cells were detected by Western blot assay. Results After IGF-1 stimulation,0.50 μg and 1.00 μg PSF appeared to be the optimal dose in the Pegfp-C2-PSF plasmid group and the pGenesil-PSF-RNAi plasmid group, respectively. The cell proliferation rates and VEGF mRNA levels in the cells were （0.22±0. 02） and （0. 79±0.07）,respectively,in the Pegfp-C2-PSF plasmid group,which were significantly lower than 0. 260±0. 006 in the Pegfp-C2 plasmid group and 1.26±0.19 in the untransfected group （P =0. 040,0. 016）. The mean cell proliferative rate was 0. 32±0.03, and the mean expression level of VEGF mRNA was 2.29±0. 43 in the pGenesil-PSF-RNAi plasmid group,showing significant increases in comparison with 0. 210±0. 019 of the pGenesil-RNAi plasmid group and 1.26±0. 19 of the untransfected group （P=0. 003,0. 019）. The expression levels of pERK protein in the cells were evidently reduced in the Pegfp- C2-PSF plasmid group compared with the untransfected group 1 hour,3,6 hours after addition of IGF-I （P= 0. 017, 0. 000,0. 000）. After treated by UO126,the relative expression level of VEGF mRNA in the Pegfp-C2-PSF plasmid group was 0.93±0. 21 ,which was significantly lower than 1.32±0.08 in the untransfected group （P=0. 037） and 1.23±0.09 in the non-U0126 treated ceils of the Pegfp-C2-PSF plasmid group （P=0. 002）. Conclusions PSF inhibits the activation of ERK signal pathway in the IGF-l-stimulated RF/6A cells and down-regulates the expression level of VEGF in RF/6A cells and thus suppresses the growth and proliferation of RF/6A ceils.