CAJ | 학술논문

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.
목적 관찰항혈관내피생장인자(VEGF)단극륭항체bevacizumab(상품명Avastin)대록화고(CoCl2)유도적결양Mller세포수통도단백4(AQP4)표체적영향,탐토bevacizumab재치료시망막수종중적작용궤제.방법 채용매소화법배양인시망막Müller세포,통과전자현미경、세포면역형광염색진행Müller세포적감정.채용반정량역전록-취합매련반응(RT-PCR)검측불동농도적CoCl2화VEGF작용불동시간후Müller세포AQP4화VEGF mRNA적표체.관찰200 μg/ml bevacizumab예처리대CoCl2유도적결양인시망막Müller세포AQP4 mRNA표체적영향.결과 세포면역형광염색현시,소배양적세포95％표체파형단백、곡안선알합성매、α평활기기동단백화효질섬유산성단백；전자현미경하가견특정성적8～10 nm적미사.증실배양적세포위Müller세포.RT-PCR결과현시,CoCl2상조Mller세포AQP4화VEGF mRNA표체수평,AQP4화VEGF mRNA표체수CoCl2작용시간연장화CoCl2농도증가적변화추세균정성정상관(r=0.952、0.954,P＜0.05).VEGF가상조Müller세포상AQP4 mRNA적표체(F=12.43,P＜0.05).bevacizumab가억제CoCl2유도적Müller세포AQP4 mRNA표체적상조(F=2 370.37,P＜0.05).결론 bevacizumab가이하조CoCl2유도적Müller세포AQP4적표체.저일효응가능시통과억제VEGF대AQP4적유도작용소발휘적,추측저시bevacizumab치료시망막수종적궤제지일.
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.