白细胞介素17在人视网膜血管内皮细胞增生、迁移及凋亡中的作用

The effects of interleukin 17 on the proliferation, migration and apoptosis of human retinal vascular endothelial cells

目的 观察白细胞介素17（IL-17）在人视网膜血管内皮细胞（HREC）增生、迁移及凋亡中的作用.方法 体外培养HREC,并取对数生长期的细胞进行实验.采用逆转录聚合酶链反应（RT-PCR）、蛋白免疫印迹法（Western blot）检测HREC中IL-17受体（IL-17R）基因和蛋白的表达.以不含IL-17的培养液培养细胞作为对照组.以浓度50、100、200、500 μg/L的1L-17培养液干预HREC,采用细胞计数试剂盒-8（CCK-8）增生分析法检测不同浓度IL-17对HREC细胞增生的作用,细胞划痕法测定不同浓度IL-17对HREC细胞迁移的影响.以浓度200 μg/L的IL-17培养液干预HREC,采用流式细胞技术检测1L-17对HREC细胞凋亡的影响.以1、2 mg/L的IL-17培养液干预HREC,应用RT-PCR检测HREC中碱性成纤维细胞生长因子（bFGF）、含半胱氨酸的天冬氨酸蛋白水解酶-3（Caspase3）、凝血酶敏感蛋白-1（TSP-1）的mRNA表达,Western blot检测细胞中Caspase-3的蛋白表达.结果 HREC中存在IL-17R的基因和蛋白表达.CCK-8增生分析法结果显示,随着IL-17培养液的浓度不断增加,吸光度[A,旧称光密度（OD）]值也呈上升趋势.与对照组比较,200、500 μg/L干预组A值明显升高,差异均有统计学意义（t=-3.235、-6.276,P=0.032、0.000）.细胞划痕法检测结果显示,干预12、24 h时,100 μg/L干预组（t=-3.551、-2.849,P=0.006、0.019）、200μg/L干预组（t=-10.347、-4.519,P=0.000、0.001）、500 μg/L干预组（t=-3.541、-2.607,P=0.008、0.036）HREC迁移距离均明显增加,差异有统计学意义.流式细胞技术检查结果显示,200 μg/L干预组细胞凋亡比例较对照组明显下调,差异有统计学意义（t=5.682,P=0.047）.RT-PCR及琼脂糖电泳结果显示,经IL-17干预后HREC中bFGF的mRNA表达水平呈升高趋势,而Caspase-3和TSP-1的mRNA表达水平呈下调趋势.Western blot检测结果显示,经IL-17干预后HREC中Caspase-3蛋白表达呈下调趋势.结论 IL-17能促进HREC增生、迁移,并抑制HREC凋亡.其机制可能与促进bFGF表达、抑制Caspase-3表达有关.

Objective To address the effect and mechanism of interleukin 17 （IL-17） on the proliferation,migration and apoptosis of human retinal vascular endothelial cells （HREC）.Methods IL-17 receptor （IL-17R） mRNA and protein expression in human retinal vascular endothelial cells （HREC） were quantified by reverse transcription polymerase chain reaction （RT-PCR） and Western blot.Cell proliferation of HREC was examined using CCK-8 assay in the presence of different concentrations of IL-17.Cell migration of HREC was detected using wound scratch assay.Flow cytometry was used to test the effect of IL-17 on the apoptosis of HREC.The effects of IL-17 on HREC expression of basic fibroblast growth factor （bFGF）,Caspase-3 and thrombospondin-1 （TSP-1） were quantified by reverse transcriptase polymerase chain reaction （RT-PCR）.The effect of IL-17 on HREC expression of Caspase-3 was examined using Western blot.Results IL-17 receptor （IL-17R） expressed in HREC as quantified by RT-PCR and Western blot.The proliferation of HREC in the presence of IL-17 was promoted in a dosage-dependent manner （t=-3.235,-6.276 ;P=0.032,0.000）.Wound scratch assay showed a significant increase in the migrated distance of HREC with IL-17 stimulation under the concentration of 100 μg/L（t=3.551,-2.849; P=0.006,0.019）,200 μg/L（t=-10.347,-4.519; P=0.000,0.001） and 500 μg/L （t=-3.541,-2.607; P=0.008,0.036）.The intervention of 200 μg/L IL-17 can effectively inhibit the apoptosis of HREC,compared with the control group using flow cytometry （t=5.682,P=0.047）.RT-PCR results showed that IL-17 can promote the expression of bFGF and inhibit the expression of Caspase-3 and TSP-1.Western blot result also showed that IL-17 can suppress the protein expression of Caspase-3.Conclusion The mechanism of IL-17 promoting proliferation,migration but suppress apoptosis of HREC may via regulating the expression of bFGF and Caspase-3.