缺氧对视网膜Müller细胞上水通道蛋白-4表达的影响  

The effects of hypoxia on the expression of AQP-4 in Müller cells

作　　者：曾苗[1] 程扬[1] 曾水清[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院眼科,武汉430030

出　　处：《眼科研究》2010年第3期243-247,共5页Chinese Ophthalmic Research

摘　　要：目的利用体外培养的视网膜Müller细胞,研究在缺氧条件下视网膜Müller细胞上水通道蛋白-4(AQP-4)表达的变化。方法采用组织块培养法从新西兰大白兔视网膜中获取Müller细胞,在含20%胎牛血清的DMEM培养液中原代培养,培养的细胞通过胶质纤维酸性蛋白(GFAP)免疫细胞化学染色及透射电子显微镜进行鉴定。取第2代细胞进行实验,将化学缺氧诱导剂CoCl2联合DMEM培养液培养24h的细胞作为缺氧组;将DMEM培养液单独培养24h的细胞作为对照组。采用免疫细胞化学法及半定量RT-PCR法分别检测2组Müller细胞上AQP-4蛋白及AQP-4 mRNA的表达。结果Müller细胞(第2代)上GFAP的阳性率为90%以上,细胞质染色呈棕色。细胞内含特征性的中间丝,细胞表面有微绒毛,细胞质内含丰富的细胞器。CoCl2联合DMEM培养液培养24h后Müller细胞上AQP-4蛋白的表达较对照组明显增加(t=6.74,P<0.05);AQP-4 mRNA的表达亦明显增加(t=21.79,P<0.05)。结论缺氧能增强Müller细胞上AQP-4的表达,进而使视网膜内液体的积聚增加。提示Müller细胞在糖尿病视网膜病变(DR)或增生性视网膜病变的视网膜水肿形成过程中起重要作用。Background Hypoxia is an important cause resulting in many retinal diseases,such as retinal edema,diabetic retinopathy,proliferative retinopathy and so on. Objective This study is to investigate the effects of hypoxia on the expression of AQP-4 in Muller ceils in vitro. Methods Muller cells were isolated from New Zealand white rabbits and primarily cultured in DMEM containing 20% fetal bovine serum by the explant culture method. The cells were identified by immunostaining for the glial fibrillary acidic protein（ GFAP）. Generation 2 of cells was cultivated with the chemical hypoxia inducer, CoCl2 ,for 24 hours in the hypoxic group and only with DMEM in the control group. The expression of the AQP-4 protein in Mtiller cells was detected by immunocytochemistry. The expression of AQP-4 mRNA was determined by semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR）. Results About 90% of Mtiller cells （generation 2） presented a positive immunoreactivity for GFAP, showing a brown staining in the cytoplasm. Cultured cells displayed the presence of intermediate filaments, microvillus and various cellular organs. The Integral absorbanee of the AQP-4 protein in Mtiller cells was markedly increased 24 hours after incubation with CoC12 in comparison with the control group （t = 6.74 ,P 〈 0.05 ）. The expression level of AQP-4 mRNA in MUller cells was significantly enhanced 24 hours after incubation with CoC12 in comparison with the control group （t = 21.79, P 〈 0.05 ）. Conclusion Hypoxia enchances the expression of AQP-4 in Mutiller cells and further increases fluid accumulation in the retina. These results suggest that Mtiller cells play an important role in the formation of retinal edema in diabetic retinopathy or proliferative retinopathy.

关 键 词：水通道蛋白-4 MÜLLER细胞 视网膜水肿 缺氧 

分 类 号：R774.1[医药卫生—眼科]