Hollborn Margrit, Tenckhoff Solveig, Jahn Karsten, Iandiev Ianors, Biedermann Bernd, Schnurrbusch Ute E K, Limb G Astrid, Reichenbach Andreas, Wolf Sebastian, Wiedemann Peter, Kohen Leon, Bringmann Andreas
Department of Ophthalmology, University Eye Hospital, University of Leipzig, Leipzig, Germany.
Mol Vis. 2005 Jun 10;11:397-413.
To compare the gene expression pattern of control postmortem retinas with retinas from patients with proliferative vitreoretinopathy (PVR), to determine the expression of the heparin binding epidermal growth factor-like growth factor (HB-EGF) by glial cells in fibroproliferative membranes, and to examine whether cells of the human Müller cell line, MIO-M1, respond to HB-EGF with proliferation, migration, and secretion of the vascular endothelial growth factor (VEGF).
To identify genes that were differently expressed in PVR and control retinas, the RNA from the neural retinas of seven postmortem donors and of two patients with PVR were analyzed for differential gene expression, by hybridization of labeled cRNA probes to an Affymetrix human genome microarray set. The results were validated by real time PCR experiments investigating RNA from 6 postmortem retinas and 4 PVR retinas. Epiretinal PVR membranes were immunohistochemically stained for colocalization of HB-EGF and the glial cell marker, glial fibrillary acidic protein (GFAP). The HB-EGF evoked proliferation of cultured Müller cells was investigated by a bromodeoxyuridine immunoassay, chemotaxis was assessed with a migration assay, and the release of VEGF was evaluated by ELISA.
Out of the 12,600 genes and expressed sequence tags investigated, the levels of 80 showed an increased expression, and 21 were expressed at decreased levels, in the retinas of PVR patients compared to the control retinas. The upregulated signals include genes for nuclear and cell cycle related proteins, extracellular secretory proteins, cytosolic signaling proteins, and proteins of the membrane and the extracellular matrix. The genes of the hepatocyte growth factor and of HB-EGF were found to be expressed in PVR retinas but not in control retinas. In epiretinal membranes of patients with PVR, HB-EGF immunoreactivity partially colocalized with GFAP. In cultured Müller cells, HB-EGF stimulated both proliferation and chemotaxis, and the secretion of VEGF, via activation of the extracellular signal regulated kinases 1 and 2 and of the phosphatidylinositol-3 kinase.
The development of PVR is accompanied by complex changes of the gene expression in the neural retina, with an upregulation of genes that support cell proliferation, cell signaling, cell motility, and extracellular matrix remodeling. HB-EGF is one of the factors that are significantly upregulated in PVR retinas. HB-EGF expression in fibroproliferative tissue and its stimulatory effect on glial cell proliferation, chemotaxis, and VEGF secretion suggest that HB-EGF may be a factor mediating glial cell responses during PVR.
比较对照尸检视网膜与增殖性玻璃体视网膜病变（PVR）患者视网膜的基因表达模式，确定纤维增殖膜中神经胶质细胞对肝素结合表皮生长因子样生长因子（HB-EGF）的表达，并研究人 Müller 细胞系 MIO-M1 的细胞是否对 HB-EGF 产生增殖、迁移及分泌血管内皮生长因子（VEGF）的反应。
为鉴定在 PVR 和对照视网膜中差异表达的基因，通过将标记的 cRNA 探针与 Affymetrix 人类基因组微阵列杂交，分析了 7 名尸检供体和 2 例 PVR 患者神经视网膜的 RNA 差异基因表达。通过实时 PCR 实验对 6 个尸检视网膜和 4 个 PVR 视网膜的 RNA 进行检测，验证结果。对视网膜前 PVR 膜进行免疫组织化学染色，以检测 HB-EGF 与神经胶质细胞标志物胶质纤维酸性蛋白（GFAP）的共定位。通过溴脱氧尿苷免疫测定法研究 HB-EGF 诱导培养的 Müller 细胞增殖，用迁移试验评估趋化性，并用 ELISA 评估 VEGF 的释放。
在研究的 12600 个基因和表达序列标签中，与对照视网膜相比，PVR 患者视网膜中有 80 个基因表达水平升高，21 个基因表达水平降低。上调信号包括与核和细胞周期相关蛋白、细胞外分泌蛋白、胞质信号蛋白以及膜和细胞外基质蛋白相关的基因。发现肝细胞生长因子和 HB-EGF 的基因在 PVR 视网膜中表达，但在对照视网膜中不表达。在 PVR 患者的视网膜前膜中，HB-EGF 免疫反应性与 GFAP 部分共定位。在培养的 Müller 细胞中，HB-EGF 通过激活细胞外信号调节激酶 1 和 2 以及磷脂酰肌醇-3 激酶，刺激增殖和趋化性以及 VEGF 的分泌。
PVR 的发展伴随着神经视网膜基因表达的复杂变化，支持细胞增殖、细胞信号传导、细胞运动和细胞外基质重塑的基因上调。HB-EGF 是 PVR 视网膜中显著上调的因子之一。HB-EGF 在纤维增殖组织中的表达及其对神经胶质细胞增殖、趋化性和 VEGF 分泌的刺激作用表明，HB-EGF 可能是 PVR 期间介导神经胶质细胞反应的一个因子。
