WNK4基因在M1细胞中瞬时和稳定表达的位置和作用  

The localization and function of WNK4 gene transient and stable expression in M1 cells

作　　者：段绍斌[1] 刘伏友[1] 彭佑铭[1] 李瑛[1] 李军[1] 周晓蓉[1] 刘虹[1] 刘映红[1] Alan Yu 

机构地区：[1]中南大学湘雅二医院肾内科,湖南长沙410011 [2]美国南加州大学Keck医学院肾脏分子生理研究室

出　　处：《中国医师杂志》2007年第10期1326-1329,共4页Journal of Chinese Physician

摘　　要：目的探讨WNK4（With No K=lysine）基因在鼠肾皮质集合管细胞（M1）中的表达位置和生理作用。方法构建PCSPT-mWNK4-kxx质粒,瞬时转染M1细胞株,采用免疫荧光法检测Myc抗原标记的WNK4基因在M1细胞中表达位置。构建PTrepWNK4质粒,转染M1Tet-Off细胞株,采用免疫荧光法检测Myc抗原标记的WNK4基因在稳定转染M1Tet-Off细胞中表达位置,采用免疫沉淀和Western blot检测WNK4蛋白质表达,采用电压计检测WNK4基因过表达对M1单层细胞跨上皮电阻（TER）的作用。结果瞬时转染野生型WNK4基因,其蛋白质主要在M1细胞的胞膜表达,部分在胞浆。在稳定转染的M1Tet-Off细胞株,野生型WNK4基因表达于细胞的胞膜,免疫沉淀和Western blot结果显示Myc抗原标记的野生型WNK4蛋白在M1Tet-Off细胞中表达。电生理研究显示野生型WNK4基因过表达显著降低M1单层细胞TER。结论野生型鼠WNK4基因主要在M1细胞的胞膜表达,部分表达在胞浆。WNK4基因过表达降低单层细胞跨上皮电阻,增加相邻细胞的通透性,为进一步研究WNK4基因在高血压中的分子机制及其在肾脏的生理功能打下基础。Objective The aim of this study is to study the situation and localization of WNK4 gene transient and stable expression in M1 cells, Methods p^CSPT-mWNK4-KXX plasmid was constructed and transient transfected into M1 cells, Immunofluorescence microscopy was performed to detect the localization of Myc-tagged WNK4 gene in M1 cells, p^TrepWNK4 plasmid was also constructed and transfected into M1 Tet-Off cell line. The localization of WNK4 in transfected M1 Tet-Off cells was determined by immunofluorescence microscopy, the WNK4 protein level was measured by immunoprecipitation and Western blot, and the effect of WNK4 gene over expression on M1 monolayers transepithelia electrical resistance （TER） was determined by a millicell-ERS voltohmeter. Results In transient transfection wild WNK4 cells, the protein was predominantly expressed on M1 cell membrane and could be found in cytoplasm. In stable transfection M1 Tet-Off cell line, wild type WNK4 gene expressed on cell membrane and the expression level was regulated by doxycycline. Immunoprecipitation and Western blot results showed that Myc-tagged wild type WNK4 protein expressed in M1 Tet-Off cells. Electrophysiology result showed over expression of wild type WNK4 gene led to an obvious decreased level of TER. Conclusions Wild-type mWNK4 gene was major expressed on M1 cell membrane and could be found in cytoplasm. Over expression of wild type WNK4 gene caused a decreased level of TER and increased paracellular permeability. The findings would be useful to further identify the role of WNK4 gene in hypertension and its physiology function in the kidney.

关 键 词：基因 蛋白质丝氨酸苏氨酸激酶 细胞系 肾皮质 

分 类 号：R[医药卫生]