MCP-1及其受体在wAMD小鼠模型视网膜中的表达及作用机制

Expression and function of MCP-1 and its receptor in wAMD model mouse

目的探讨单核细胞趋化因子-1(monocyte chemoattractant protein-1,MCP-1)及其受体CCR2在湿性年龄相关性黄斑变性(wet age-related macular degeneration,wAMD)小鼠模型视网膜中的表达及作用机制。方法制备wAMD小鼠模型,摘取眼球制作冷冻切片,使用MCP-1和CCR2单克隆抗体行免疫荧光检测。使用CD11b和CD68单克隆抗体分别与CCR2抗体进行免疫荧光共表达研究以检测CCR2阳性细胞的来源。制备视网膜的总蛋白和总mRNA提取物,经RT-PCR和Western blot检测视网膜中MCP-1及其受体CCR2 mRNA和蛋白的表达。结果正常对照组小鼠视网膜中无明显的MCP-1表达,wAMD小鼠模型中视网膜色素上皮(retinal pigment epithelium,RPE)层下有显著的新生血管网形成,且RPE层连续性被破坏,其中RPE细胞有明显的MCP-1表达上调。正常对照组小鼠视网膜中无明显的CCR2的表达,wAMD模型小鼠的视网膜中CCR2阳性细胞显著增加。与正常对照组小鼠相比,wAMD模型小鼠眼内MCP-1和CCR2的蛋白和mRNA表达明显上调。免疫荧光共表达结果显示CCR2与CD11b有显著的共表达,双阳性细胞集中在RPE层和视网膜外层;而CCR2与CD68无明显的共表达。结论 MCP-1及其受体可能与wAMD小鼠模型视网膜脉络膜新生血管(choroidal neovascularization,CNV)的形成及其炎症机制相关。

Purpose To detect the expression and the function of MCP-1 and its receptor CCR2 in wet age-related macular degenerative （ wAMD ） model mouse retina. Methods C57BL/6J mouse were enrolled into the study. Model mouse of wAMD was induced with laser. Frozen sections were prepared for histopathological tests. Immunofluorescence study for MCP-1 and CCR2 was carried out. Co-expression study for CCR2/ CDllb or CCR2/CD68 was carried out. Total protein and total mRNA from the eyes of both wAMD and wild type mouse were extraeted. The expression of mRNA and protein of MCP-1 and CCR2 in the eyes were determined by reverse transcription-poly- merase ehain reaction （RT-PCR） and Western blots test, respectively. Results In wild type mouse, both MCP-1 and its receptor CCR2 were not detected in the retina. However in wAMD mouse, an obvious up-regulated MCP-1 and CCR2 expression was seen in the retinal pigment epithelium （RPE） cells accompanied with the increased expression of their mRNA and protein. The co-expression study showed that CCR2 co-expressed with CDllb, but not with CD68. Conclusion MCP-1 and its receptor CCR2 may play a role in the wAMD through stimulation of microglia.