摘要/Abstract
摘要： 目的 ·建立转化生长因子 -β2（transforming growth factor-β2，TGF-β2）诱导的人视网膜色素上皮细胞（retinal pigment epithelium cell，RPE）上皮 -间质转化（epithelial-mesenchymal transformation，EMT）模型，探讨叶黄素的作用及其机制。方法 ·体外培养 ARPE-19细胞，分为空白组、 TGF-β2组、TGF-β2+叶黄素组、叶黄素组。采用 real-time PCR检测各组细胞 α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）、纤连蛋白（fibronectin，FN）、上皮钙黏素（E-cadherin）mRNA的表达。 Western blotting检测各组细胞中 α-SMA、FN、闭合蛋白（occludin）的蛋白表达。采用免疫荧光检测 α-SMA的表达。同时用 Western blotting检测 TGF/ Smad通路下游 Smad3的磷酸化水平。结果 · TGF-β2+叶黄素组 EMT程度有明显减轻。纤维化指标 α-SMA、FN的 mRNA、蛋白表达下降，与 TGF-β2组相比差异有统计学意义（均 P<0.05）。同时，上皮细胞相关指标 E-cadherin mRNA、occludin蛋白的表达上调（均 P<0.05）。免疫荧光结果显示，叶黄素可明显抑制上皮细胞转化为肌成纤维细胞。此外， TGF-β2诱导 ARPE-19细胞 EMT过程中发生 Smad3磷酸化水平升高现象可被叶黄素干预（P0.001）。结论 ·叶黄素通过调控 TGF-β/Smad信号通路抑制 ARPE-19细胞 EMT过程，具有潜在的抑制视网膜下纤维化的作用。
关键词: 视网膜下纤维化, 叶黄素, 转化生长因子 -&, beta, 2, 上皮 -间质转化, 视网膜色素上皮细胞
Abstract:
Objective · To establish the transforming growth factor-β2 (TGF-β2) induced epithelial-mesenchymal transition (EMT) model of retinal pigment epithelium cells, and investigate the effect and mechanism of lutein on EMT. Methods · ARPE-19 cells were cultured and divided into 4 groups including control group, TGF-β2 group, TGF-β2+lutein group and lutein group. The mRNA levels of α-smooth muscle actin (α-SMA), fibronectin (FN) and E-cadherin were analyzedreal-time PCR. The protein of α-SMA, FN and occludin were assayedWestern blotting. Immunofluorescence was used to detect the change of α-SMA. Meanwhile, Western blotting was performed to detect the levels of pSmad3 in the TGF/Smad signaling pathway. Results · TGF-β2 induced EMT was inhibitedlutein. Lutein decreased the mRNA and protein levels of the mesenchymal markers α-SMA and FN, and increased the of the epithelial markers E-cadherin and occludin (all P<0.05). Immunofluorescence showed that lutein can inhibit the conversion of epithelial cells into myofibroblasts. Lutein significantly downregulated the high of pSmad3 in TGF-β2 treated ARPE-19 cells (P0.001). Conclusion · Lutein inhibits TGF-β2 induced EMTdownregulating the of pSmad3 in TGF-β/ Smad signaling pathway, indicating it may attenuate subretinal fibrosis.
Key words: subretinal fibrosis, lutein, transforming growth factor-&, beta, 2 (TGF-&, beta, 2), epithelial-mesenchymal transformation (EMT), retinal pigment epithelium cell (RPE)
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