稳定表达UT-A2的FRT细胞系的建立与鉴定

目的：建立稳定表达尿素通道蛋白A2（UT—A2）的FRT细胞系，为寻找UT—A2抑制剂提供细胞模型。方法：通过真核表达质粒-脂质体介导的方法，将UT—A2 cDNA与真核表达载体pUB6／V5连接后的重组质粒转染入FRT细胞，经稳定筛选建立稳定表达UT—A2的FRT细胞系。功能检测实验将细胞分为对照组和稳定转染组，用2mol／L尿素负荷试验检测稳定表达UT—A2的FRT细胞膜的尿素通透性。结果：经BSD筛选21d后得到稳定表达UT—A2的细胞株；Western blotting证实UT—A2蛋白稳定表达；免疫荧光分析结果提示UT—A2的质膜定位。2mol／L尿素试验证实了该细胞系具有明显尿素通透性。结论：在非肾脏上皮细胞获得了稳定质膜定位表达UT—A2的FRT细胞株，该细胞株可用于UT—A2抑制剂的筛选。

Construction and identification of FRT cell line stably expressing UT-A2

AIM: To obtain an FRT cell line that can stably express urea transporter A2(UT-A2) and provide a cell model for screening UT-A2 inhibitors.METHODS: FRT cells stably expressing aquaporins 1(AQP1) and YFP were transfected with the recombinant plasmid pUB6/V5-UT-A2 by eukaryotic expression plasmid-lipoplast mediating pathway.The stable UT-A2-FRT cell line was cloned by selection with BSD and confirmed by Western blotting and immunofluorescence staining.The urea permeability across the plasma membrane was detected by a 2 mol/L urea lysis assay.RESULTS: We have obtained a stable UT-A2-FRT cell line.Western blotting analysis showed that UT-A2 protein was expressed stably in this cell line.The immunofluorescence staining detection indicated UT-A2 expression in the plasma membrane.It was found that there was significant urea permeability in this cell line by 2 mol/L urea lysis assay.CONCLUSION: We constructed an FRT cell line that could stably express UT-A2 in plasma membrane in the non-renal epithelia cell.The cell line will be used to screen UT-A2 inhibitors.