Abstract：
Proliferative vitreoretinopathy (PVR) develops after an unsuccessful or complicated recovery from rhegmatogenous retinal detachment (RRD) surgery. Intraocular scar formation with the contribution of epithelial-mesenchymal transition (EMT) in RPE cells is prominent in the pathology of PVR. In the present study, the EMT process was experimentally induced in human retinal pigment epithelium (RPE; ARPE-19) cells, and the effect of atorvastatin on the process was studied. The mRNA and protein levels of mesenchymal markers actin alpha 2 (ACTA2) / alpha-smooth muscle actin (α-SMA) and fibronectin (FN), and epithelial markers occludin (OCLN) and zonula occludens-1 (ZO-1) were measured using quantitative real-time PCR (qRT-PCR) and western blot methods, respectively. In addition, α-SMA and FN were visualized using immunofluorescence staining. Cells were photographed under a phase contrast light microscope. Changes in the functionality of cells following the EMT process were studied using the IncuCyte scratch wound cell migration assay and the collagen cell invasion assay with confocal microscopy. The induction of EMT in ARPE-19 cells increased the expression of mesenchymal markers ACTA2/α-SMA and fibronectin and reduced the expression of epithelial marker OCLN both at mRNA and protein levels. The mRNA levels of ZO-1 were lower after EMT, as well. Increased levels of α-SMA and FN were confirmed by immunofluorescence staining. Atorvastatin further increased the mRNA levels of mesenchymal markers ACTA2 and FN as well as the protein levels of α-SMA and reduced the mRNA levels of epithelial markers OCLN and ZO-1 under the EMT process. EMT promoted wound closure and cell invasion into the 3D collagen matrix when compared to untreated control cells. These data present cellular changes upon the induction of the EMT process in ARPE-19 cells and the propensity of atorvastatin to complement the effect. More studies are needed to confirm the exact influence of the EMT process and atorvastatin treatment on the PVR development after RRD surgery.
摘要：
增生性玻璃体视网膜病变（PVR）在孔源性视网膜脱离（RRD）手术失败或复杂的恢复后发展。在PVR的病理学中，RPE细胞中上皮-间充质转化（EMT）的作用是眼内瘢痕形成。在本研究中,EMT过程是在人视网膜色素上皮（RPE;ARPE-19）细胞中实验诱导的，研究了阿托伐他汀对该工艺的影响。间充质标志物肌动蛋白α2（ACTA2）/α-平滑肌肌动蛋白（α-SMA）和纤连蛋白（FN）的mRNA和蛋白水平，使用定量实时PCR（qRT-PCR）和Westernblot方法测量上皮标记物occludin（OCLN）和zonulaoccludens-1（ZO-1），分别。此外,使用免疫荧光染色观察α-SMA和FN。在相差光学显微镜下对细胞拍照。使用IncuCyte划痕伤口细胞迁移测定和带有共聚焦显微镜的胶原细胞侵袭测定研究了EMT过程后细胞功能的变化。在ARPE-19细胞中诱导EMT增加了间充质标志物ACTA2/α-SMA和纤连蛋白的表达，并在mRNA和蛋白水平上降低了上皮标志物OCLN的表达。EMT后ZO-1mRNA水平降低,也是。免疫荧光染色证实了α-SMA和FN水平的升高。在EMT过程中，阿托伐他汀进一步增加了间充质标志物ACTA2和FN的mRNA水平以及α-SMA的蛋白水平，并降低了上皮标志物OCLN和ZO-1的mRNA水平。与未处理的对照细胞相比,EMT促进伤口闭合和细胞侵入3D胶原基质。这些数据显示了在ARPE-19细胞中诱导EMT过程后的细胞变化以及阿托伐他汀补充该作用的倾向。需要更多的研究来证实EMT过程和阿托伐他汀治疗对RRD手术后PVR发展的确切影响。
