Luo Wentao, Hu Liumei, Li Weiye, Xu Guotong, Xu Linxinyu, Zhang Conghui, Wang Fang
Department of Ophthalmology, Shanghai Tenth People's Hospital, Tongji University, Shanghai, China.
Department of Ophthalmology, Drexel University College of Medicine, Philadelphia, PA, USA.
Graefes Arch Clin Exp Ophthalmol. 2016 May;254(5):881-90. doi: 10.1007/s00417-016-3290-5. Epub 2016 Feb 23.
In proliferative diabetic retinopathy (PDR), Müller glial cells (MGCs) acquire migratory ability and exhibit a fibroblast-like phenotype. These activated MGCs contribute to the formation of epiretinal membrane, which will stretch the retina, and cause retinal detachment and vitreous hemorrhage. Erythropoietin (Epo) is now found effective in ameliorating renal fibrosis by inhibiting epithelial-to-mesenchymal transition of tubular epithelial cells. This study is undertaken to determine whether Epo has an effect in inhibiting MGCs activation to attenuate epiretinal membrane formation in PDR.
MIO-M1 cell line was used in this study. As a pilot test to determine the most efficient treatment time and concentration of Epo, levels of connective tissue growth factor (CTGF) and transforming growth factor-β (TGF-β) were measured by real-time PCR, after treatment with Epo on MGCs cultured in high glucose. MGCs were cultured in high glucose and normal glucose for 2 days, with or without TGF-β as a pro-fibrogenic cytokine. Epo was introduced at the same time. Immunofluorescence targeting α-smooth muscle actin (α-SMA), fibronectin, and glial fibrillary acidic protein (GFAP) was performed to explore the cell phenotype. Matrix metalloproteinase 9 (MMP9) mRNA level was detected by real-time PCR. Protein levels of CTGF and cytoskeletal proteins like α-SMA and fibronectin were measured by enzyme-linked immunosorbent assay (ELISA) and Western blot respectively. Wound-healing assay was applied to evaluate the migratory ability of MGCs, and actin-tracker green was used to draw the structure of F-actin in MGCs.
After being seeded into high-glucose medium containing TGF-β, MGCs expressed a larger amount of MMP9 mRNA as well as α-SMA, fibronectin at protein level. They secreted more CTGF, and their F-actin reorganized in a parallel manner and showed a stronger ability to migrate. In addition, these changes, including mRNA and protein expression, F-actin assembling, and cell migration, could be attenuated significantly by Epo treatment.
High glucose together with TGF-β promote MGCs to exhibit a fibroblast-like phenotype and develop a greater migratory ability. These changes can be inhibited by Epo, which therefore may contribute to the controlling of epiretinal membrane formation.
在增殖性糖尿病视网膜病变（PDR）中， Müller 胶质细胞（MGCs）获得迁移能力并表现出成纤维细胞样表型。这些活化的 MGCs 有助于视网膜前膜的形成，这将拉伸视网膜，并导致视网膜脱离和玻璃体出血。促红细胞生成素（Epo）现在被发现可通过抑制肾小管上皮细胞的上皮 - 间充质转化来有效改善肾纤维化。本研究旨在确定 Epo 是否具有抑制 MGCs 活化以减轻 PDR 中视网膜前膜形成的作用。
本研究使用 MIO - M1 细胞系。作为确定 Epo 最有效治疗时间和浓度的预试验，在用 Epo 处理高糖培养的 MGCs 后，通过实时 PCR 测量结缔组织生长因子（CTGF）和转化生长因子 -β（TGF -β）的水平。MGCs 在高糖和正常糖条件下培养 2 天，有或没有作为促纤维化细胞因子的 TGF -β。同时加入 Epo。进行靶向α - 平滑肌肌动蛋白（α - SMA）、纤连蛋白和胶质纤维酸性蛋白（GFAP）的免疫荧光以探索细胞表型。通过实时 PCR 检测基质金属蛋白酶 9（MMP9）mRNA 水平。分别通过酶联免疫吸附测定（ELISA）和蛋白质印迹法测量 CTGF 和细胞骨架蛋白如α - SMA 和纤连蛋白的蛋白质水平。应用伤口愈合试验评估 MGCs 的迁移能力，并使用肌动蛋白追踪绿色染料描绘 MGCs 中 F - 肌动蛋白的结构。
接种到含有 TGF -β的高糖培养基中后，MGCs 表达大量的 MMP9 mRNA 以及蛋白质水平的α - SMA、纤连蛋白。它们分泌更多的 CTGF，并且它们的 F - 肌动蛋白以平行方式重组并表现出更强的迁移能力。此外，Epo 处理可显著减弱这些变化，包括 mRNA 和蛋白质表达、F - 肌动蛋白组装和细胞迁移。
高糖与 TGF -β共同促进 MGCs 表现出成纤维细胞样表型并发展出更强的迁移能力。这些变化可被 Epo 抑制，因此 Epo 可能有助于控制视网膜前膜的形成。
