Background Insulin can promote the occurrence of myopia.It has been proven that insulin receptor exists in human retinal pigment epithelial (RPE) cells and can promote RPE cells to secrete transforming growth factor-β2(TGF-β2),which is one of the most important myopic signal molecules.Objective This study was to investigate if PI3K/Akt mediates the promotive effects of insulin on proliferation of human RPE cells and secretion of TGF-β2.Methods Human RPE cell line,ARPE-19 cells,were regularly cultured using DMEM containing 10％ fetal bovine serum,and 10× 103 U/ml insulin,LY294002,10× 103 U/ml insulin+LY294002,Wortmanin,10× 103 U/ml insulin+Wortmanin were added into the medium respectively for 48 hours,and the regularly cultured cells served as blank controls.The proliferation value (absorbance,A) of the cells was evaluated by MTS,and the TGF-β2 level in the cell supernatant was detected by ELISA.The relative expression of TGF-β2 mRNA in the cells was assayed using reverse transcription PCR (RT-PCT) 1 hour and 2 hours after the addition of reagents.Results MTS showed that the proliferation value of the cells in the insulin+LY294002 group was 0.75±0.03,which was significantly lower than that in the insulin group (0.98± 0.04).No significant difference was seen in the proliferative value between the insulin+Wortmanin group and the insulin group (0.97±0.07 versus 0.98± 0.04,P＞0.05).ELISA revealed that the content of TGF-β2 in the the cell supernatant was (11.59±2.85) pg/ml and (49.16± 10.94) pg/ml in the insulin + LY294002 group and the insulin + Wortmanin group,respectively,showing a significant decline in comparison with (548.50±35.18) pg/ml in the insulin group (both at P＜0.05).A significant difference was found in the TGF-β2 content between the insulin+LY294002 group and the insulin+Wortmanin group (t =8.131,P =0.000).The RT-PCR showed that 1 hour and 2 hours after addition of the reagents,the expression levels of TGF-β2 mRNA in the cells were lower in both insulin+LY294002 group and insulin+Wortmanin group than those in the insulin group (P＜0.05).The decline range of TGF-β2 mRNA expression level was more significant in the insulin+LY294002 group than that in the insulin+Wortmanin group at 1 hour (t=4.176,P=0.014) rather than at 2 hours (t=0.756,P=0.492).Conclusions Insulin can promote the proliferation of human RPE cells and secretion of TGF-β2 through PI3K/Akt pathway.This may be one of the mechanisms of insulin causes myopia.%背景 胰岛素具有促进近视发生的作用,胰岛素受体已被证实存在于视网膜色素上皮(RPE)细胞上,转化生长因子β2(TGF-β2)是RPE细胞分泌的重要的近视信号因子之一.目的 研究胰岛素是否通过磷脂酰肌醇-3-羟基酶/苏氨酸激酶(PI3K/Akt)通路促进RPE细胞增生及分泌TGF-β2.方法 使用含质量分数10％胎牛血清的DMEM培养基常规培养人RPE细胞株ARPE-19细胞,分别将10×103 U(商品单位)/ml胰岛素、LY294002、10× 103 U/ml胰岛素+LY294002、Wortmanin和10× 103 U/ml胰岛素+Wortmanin20 μmol/L各20μl加入培养基,另设常规培养的细胞作为空白对照.作用后48 h,采用MTS法检测各组RPE细胞的增生情况,采用ELISA法检测各组RPE细胞上清液中TGF-β2质量浓度,以评估细胞分泌TGF-β2的能力;各种药物作用后1h和2h,采用逆转录PCR(RT-PCR)法检测各组细胞中TGF-β2mRNA的相对表达量.结果 MTS结果显示,胰岛素+LY294002组RPE细胞的增生值(A值)明显低于胰岛素组(0.75±0.03 versus0.98±0.04),差异有统计学意义(P＜0.05),而胰岛素+Wortmanin组RPE细胞增生与胰岛素组相比,差异无统计学意义(0.97±0.07 versus 0.98±0.04,P＞0.05).ELISA法检测结果显示,胰岛素+LY294002组和胰岛素+Wortmanin组RPE细胞上清液中TGF-β2质量浓度分别为(11.59±2.85)pg/ml和(49.16±10.94) pg/ml,明显低于胰岛素组的(548.50±35.18) pg/ml,差异均有统计学意义(P＜0.05),且胰岛素+LY294002组细胞上清液中TGF-β2质量浓度的下降值明显高于胰岛素+Wortmanin组,差异有统计学意义(t=8.131,P=0.000).RT-PCR结果显示,药物作用后1h和2h,胰岛素+LY294002组和胰岛素+Wortmanin组细胞中TGF-β2mRNA的相对表达量均明显低于胰岛素组,差异均有统计学意义(P＜0.05),胰岛素+ LY294002组细胞中TGF-β2mRNA相对表达量的下降程度稍大于胰岛素+Wortmanin组,作用后1h差异有统计学意义(t=4.176,P=0.014),但作用后2h差异无统计学意义(t=0.756,P=0.492).结论 胰岛素可以通过PI3 K/Akt途径促进RPE细胞增生,并促进RPE细胞分泌TGF-β2以进行信号传递,可能是胰岛素促进近视发生的机制之一.
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