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目的探讨缺氧条件下促红细胞生成素(EPO)mRNA及蛋白在体外培养的Müller细胞的表达情况。方法胰酶消化新生大鼠视网膜组织制成单细胞悬液,机械震荡、吹打法分离纯化视网膜Müller细胞,反转录-聚合酶链反应(RT-PCR)、免疫细胞化学方法对缺氧条件下视网膜Müller细胞EPO基因和蛋白的表达进行测定。结果成功获得视网膜Müller细胞,传代后95%以上的细胞呈神经胶质纤维酸性蛋白(GFAP)染色阳性。EPO蛋白的阳性染色位于视网膜Müller细胞的细胞浆及突起上。在正常的视网膜Müller细胞中仅见微弱的EPO mRNA和蛋白表达,而缺氧后表达明显上调,且呈时间依赖性。结论Müller细胞在缺氧条件下EPO mRNA和蛋白表达增强,EPO表达水平的升高可能是缺氧性视网膜病变的神经保护因素之一。
Objective To investigate the expression of erythropoietin (EPO) mRNA and protein in cultured Müller cells under hypoxic conditions. Methods Retinal tissue of neonatal rats was digested with trypsin to make a single cell suspension. The retinal Müller cells were isolated and purified by mechanical shock and blow method. Reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry The expression of EPO genes and proteins in the retinal Müller cells was measured. Results The retinal Müller cells were successfully obtained. More than 95% of the cells were positive for glial fibrillary acidic protein (GFAP) staining after passage. The positive staining of EPO protein is located on the cytoplasm and protrusions of retinal Müller cells. In normal retinal Müller cells, only weak expression of EPO mRNA and protein was seen, while the expression was significantly up-regulated after hypoxia in a time-dependent manner. Conclusions Müller cells express enhanced EPO mRNA and protein under hypoxic conditions. The increased expression of EPO may be one of the neuroprotective factors in hypoxic retinopathy.