TMEM16A钙激活氯离子通道在Fisher大鼠甲状腺滤泡上皮细胞的表达及其电生理特性研究  

Expression of TMEM16A as a calcium-activated chloride channel in Fischer rat thyroid follicular epithelial cells and its electrophysiologic properties

作　　者：郝峰[1] 白雪松[2] 鞠晓红[3] 方芳[3] 藏雨轩[1] 朱杭飞 向国艳[1] 张雲乔 袁忠海[1] 

机构地区：[1]吉林医药学院生物化学检验教研室,吉林吉林132013 [2]吉林医药学院营养教研室,吉林吉林132013 [3]吉林医药学院病原教研室,吉林吉林132013

出　　处：《中国病理生理杂志》2014年第9期1633-1639,共7页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81202031);吉林省教育厅基金资助课题(No.2013-351;No.2009-510);吉林省大学生创新创业训练计划856;吉林医药学院大学生科研基金资助课题(吉医学科字[2012]第12号)

摘　　要：目的:探讨跨膜蛋白16A(TMEM16A)钙激活氯离子通道在Fisher大鼠甲状腺滤泡上皮(FRT)细胞的表达及其电生理特性。方法:构建pUB6/V5-TMEM16A真核表达载体。脂质体方法转染TMEM16A至FRT细胞,同时优化脂质体和载体的量和比例,获得最佳转染效率和表达效果,杀稻瘟菌素(blasticidin)进行抗生素筛选,获取稳定表达TMEM16A的FRT细胞株。RT-PCR和免疫荧光检测TMEM16A于FRT细胞的表达情况;倒置荧光显微镜下观察TMEM16A在FRT细胞中的表达和定位;应用全细胞膜片钳技术和卤族元素敏感的荧光蛋白YFPH148Q/I152L检测TMEM16A钙激活氯离子通道的功能。结果:BamHⅠ和XbaⅠ双酶切的琼脂糖凝胶电泳和测序结果表明目的基因TMEM16A成功克隆到真核表达载体pUB6/V5中;RT-PCR和免疫荧光实验结果表明经杀稻瘟菌素筛选后的FRT细胞在mRNA和蛋白水平表达TMEM16A,倒置显微镜下观察结果表明TMEM16A在FRT细胞膜上有表达;应用全细胞膜片钳技术和卤族元素敏感的荧光蛋白YFP-H148Q/I152L证实稳定表达于FRT细胞的TMEM16A具有经典的钙激活氯离子通道特性。结论:FRT细胞可高效表达TMEM16A。TMEM16A是钙激活氯离子通道的分子基础。AIM： To investigate the expression of transmembrane protein 16A（ TMEM16A） in Fischer rat thyroid follicular epithelial（ FRT） cells and its electrophysiologic properties. METHODS： The eukaryotic expression vector of pUB6 /V5-TMEM16 A was constructed and transfected into FRT cells by liposome-mediated transfection. In order to obtain the high efficiency of gene transfection and expression,the quantity and ratio of lipid /DNA complexes were optimized.The FRT cells stably expressing TMEM16 A were gained by the selection with blasticidin and confirmed by the techniques of RT-PCR and immunofluorescence. The expression and location of TMEM16 A in the FRT cells were observed under an inverted fluorescence microscope. TMEM16 A protein was associated with calcium-dependent chloride current,as measured with halide-sensitive fluorescent protein and patch-clamp technique. RESULTS： The results of double digestion and sequencing indicated that TMEM16 A was cloned into pUB6 /V5. The results of RT-PCR and immunofluorescence confirmed that TMEM16 A was expressed in the FRT cells after transfection with TMEM16 A. The classical calcium-activated chloride channel currents were recorded in the FRT cells stably expressing TMEM16 A by the technique of patch-clamp and halidesensitive fluorescent protein YFP-H148 Q /I152 L. CONCLUSION： The protein expression of TMEM16 A in the FRT cells was observed. TMEM16 A is the molecular identity of calcium-activated chloride channels.

关 键 词：跨膜蛋白16A 钙激活氯离子通道 膜片钳术 Fisher大鼠甲状腺滤泡上皮细胞 

分 类 号：R363[医药卫生—病理学]