稳定表达UT-A2的FRT细胞系的建立与鉴定  

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

作　　者：郭丽荣[1] 孟艳[1] 赵丹[1] 赵华山[1] 吕斌[1] 杨宝学[1] 赵雪俭[1] 

机构地区：[1]吉林大学基础医学院病理生理教研室,吉林长春130021

出　　处：《中国病理生理杂志》2007年第4期803-806,共4页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30370572)

摘　　要：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2 mol/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21 d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。2 mol/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。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.

关 键 词：尿素通道蛋白 转染 尿素 

分 类 号：R363[医药卫生—病理学]