抗血管内皮生长因子单克隆抗体bevacizumab对缺氧Müller细胞水通道蛋白4表达的影响

摘要

Objective To observe the effects of bevacizumab on aquaporin 4 (AQP4) expression in human retinal Müller cells in vitro under hypoxia.To explored the mechanism of treating retinal edema with bevacizumab.Methods Human Müller cells were cultured using the enzymatic digestion method.Transmission electron microscopic analysis and immunofluorescence staining identified Müller cells.With semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), the expression of AQP4 mRNA and vascular endothelial growth factor (VEGF) mRNA in Müller cells cultured under different concentration of CoCl2 for different hours were observed.The expression of AQP4 mRNA in Müller cells cultured using CoCl2 pre-cultured with 200 μg/ml bevacizumab was measured.Results More than 95％ of primary cells showed positive reaction to glial fibrillary acidic protein,glutamine synthetase,vimentin and αsmooth muscle actin with immunofluorescence staining.Characteristic 8 - 10 nm intracellular filaments could be seen in the cytoplasm viewed with transmission electron microscopy.The results using RT-PCR showed that CoCl2 increased the AQP4 and VEGF mRNA expression in Müller cells in a dose and time dependent manner (r=0.952,0.954;P＜0.05).The expression of AQP4 mRNA in Müller cells was increased byVEGF (F=12.43,P＜0.05).The expression of AQP4 mRNA was significantly decreased by bevacizumab(F=2 370.37,P＜0.05).Conclusion Bevacizumab can down-regulate the expression of AQP4 mRNA in human Müller cells under hypoxic conditions partially by VEGF path,which may be a mechanism for treating retinal edema with bevacizumab.%目的 观察抗血管内皮生长因子(VEGF)单克隆抗体bevacizumab(商品名Avastin)对氯化钴(CoCl2)诱导的缺氧Mller细胞水通道蛋白4(AQP4)表达的影响,探讨bevacizumab在治疗视网膜水肿中的作用机制.方法 采用酶消化法培养人视网膜Müller细胞,通过电子显微镜、细胞免疫荧光染色进行Müller细胞的鉴定.采用半定量逆转录-聚合酶链反应(RT-PCR)检测不同浓度的CoCl2和VEGF作用不同时间后Müller细胞AQP4和VEGF mRNA的表达.观察200 μg/ml bevacizumab预处理对CoCl2诱导的缺氧人视网膜Müller细胞AQP4 mRNA表达的影响.结果 细胞免疫荧光染色显示,所培养的细胞95％表达波形蛋白、谷氨酰胺合成酶、α平滑肌肌动蛋白和胶质纤维酸性蛋白；电子显微镜下可见特征性的8～10 nm的微丝.证实培养的细胞为Müller细胞.RT-PCR结果显示,CoCl2上调Mller细胞AQP4和VEGF mRNA表达水平,AQP4和VEGF mRNA表达随CoCl2作用时间延长和CoCl2浓度增加的变化趋势均呈成正相关(r=0.952、0.954,P＜0.05).VEGF可上调Müller细胞上AQP4 mRNA的表达(F=12.43,P＜0.05).bevacizumab可抑制CoCl2诱导的Müller细胞AQP4 mRNA表达的上调(F=2 370.37,P＜0.05).结论 bevacizumab可以下调CoCl2诱导的Müller细胞AQP4的表达.这一效应可能是通过抑制VEGF对AQP4的诱导作用所发挥的,推测这是bevacizumab治疗视网膜水肿的机制之一.