碳酸酐酶Ⅳ型在兔角膜内皮细胞中的表达
Expression of carbonic anhydrase Ⅳ in rabbit corneal endothelial cells
目的本研究旨在从分子水平证明碳酸酐酶Ⅳ型(CAⅣ)在兔角膜内皮细胞中的表达.方法采用逆转录多聚酶连反应(RT-PCR),应用CAⅣ的特异性引物,从新鲜的和培养的兔角膜内皮细胞的总RNA中扩增CAⅣ,并对RT-PCR产物进行亚克隆和序列分析.应用大鼠抗CAⅣ的多克隆抗体,采用免疫杂交和间接免疫荧光技术,从新鲜的和培养的兔角膜内皮细胞中检测CAⅣ蛋白的表达和分布.结果新鲜的和培养的兔角膜内皮细胞中,RT-PCR分析都得到了预期长度的CAⅣ阳性条带.序列分析进一步证实了CAⅣ的表达.免疫杂交在大约52KD得到了单一条带.在培养的角膜内皮细胞中,间接免疫荧光染色显示有明显的阳性着染.结论碳酸酐酶Ⅳ在兔角膜内皮细胞中表达.它可能促进经内皮细胞的碳酸氢根离子的运输,从而维持角膜的水合状态和透明性.
更多Objective To demonstrate the molecular expression of carbonic anhydrase Ⅳ (CA Ⅳ) in rabbit corneal endothelium.Methods Reverse transcriptase polymerase chain reaction (RT-PCR) was performed using cultured and fresh rabbit corneal endothelial total RNA and specific primers for CA Ⅳ. The RT-PCR product was subcloned and sequenced. Immunoblotting and indirect immunofluorescence staining were performed to detect protein expression and distribution of CA Ⅳ using fresh and cultured rabbit corneal endothelium and rat anti-CA Ⅳ polyclonal antibody. Results RT-PCR screening gave positive bands at the predicted size for CA Ⅳ from fresh and cultured rabbit corneal endothelium. Sequencing further confirmed the identity of CA Ⅳ in corneal endothelium. Immunoblotting analysis showed a single band at 52 kDa for freshly isolated and cultured endothelial cells. Indirect immunofluorescence staining revealed an apparent positive staining in cultured endothelial cells.Conclusion Carbonic anhydrase Ⅳ is expressed in rabbit corneal endothelium, which could contribute to the transendothelial HCO3 flux that is necessary to maintain corneal hydration and transparency.
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