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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 5 2KD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。
Objective This study aimed to demonstrate the expression of carbonic anhydrase type Ⅳ (CA Ⅳ) in rabbit corneal endothelial cells at the molecular level. Methods Reverse transcription - polymerase chain reaction (RT - PCR) was used to amplify CA Ⅳ from total RNA of fresh and cultured rabbit corneal endothelial cells using CA Ⅳ specific primers. Subcloning and sequence analysis of RT PCR products were performed. The anti-CA IV polyclonal antibody was used to detect the expression and distribution of CAVII protein in fresh and cultured rabbit corneal endothelial cells by immunoblotting and indirect immunofluorescence techniques. Results In both fresh and cultured rabbit corneal endothelial cells, RT-PCR analysis yielded expected length CA IV-positive bands. Sequence analysis further confirmed the CA IV expression. Immunoblotting gave a single band at about 52KD. In cultured corneal endothelial cells, indirect immunofluorescence staining showed significant positive staining. Conclusion Carbonic anhydrase Ⅳ is expressed in rabbit corneal endothelial cells. It may promote the transport of bicarbonate ions through endothelial cells to maintain the hydration and transparency of the cornea.