CAJ | 학술논문

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YFP-H148Q/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。
목적：탐토과막단백16A（TMEM16A）개격활록리자통도재Fisher대서갑상선려포상피（FRT）세포적표체급기전생리특성。방법：구건pUB6/V5-TMEM16A진핵표체재체。지질체방법전염TMEM16A지FRT세포，동시우화지질체화재체적량화비례，획득최가전염효솔화표체효과，살도온균소（ blasticidin ）진행항생소사선，획취은정표체TMEM16A적FRT세포주。 RT-PCR화면역형광검측TMEM16A우FRT세포적표체정황；도치형광현미경하관찰TMEM16A재FRT세포중적표체화정위；응용전세포막편겸기술화서족원소민감적형광단백YFP-H148Q/I152L검측TMEM16A개격활록리자통도적공능。결과：BamHⅠ화XbaⅠ쌍매절적경지당응효전영화측서결과표명목적기인TMEM16A성공극륭도진핵표체재체pUB6/V5중；RT-PCR화면역형광실험결과표명경살도온균소사선후적FRT세포재mRNA화단백수평표체TMEM16A，도치현미경하관찰결과표명TMEM16A재FRT세포막상유표체；응용전세포막편겸기술화서족원소민감적형광단백YFP-H148Q/I152L증실은정표체우FRT세포적TMEM16A구유경전적개격활록리자통도특성。결론：FRT세포가고효표체TMEM16A。 TMEM16A시개격활록리자통도적분자기출。
AIM:To investigate the expression of transmembrane protein 16A(TMEM16A) in Fischer rat thy-roid follicular epithelial ( FRT) cells and its electrophysiologic properties .METHODS: The eukaryotic expression vector of pUB6/V5-TMEM16A was constructed and transfected into FRT cells by liposome-mediated transfection .In order to ob-tain the high efficiency of gene transfection and expression , the quantity and ratio of lipid/DNA complexes were optimized . The FRT cells stably expressing TMEM16A were gained by the selection with blasticidin and confirmed by the techniques of RT-PCR and immunofluorescence .The expression and location of TMEM 16A in the FRT cells were observed under an in-verted fluorescence microscope .TMEM16A 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 se-quencing indicated that TMEM16A was cloned into pUB6/V5.The results of RT-PCR and immunofluorescence confirmed that TMEM16A was expressed in the FRT cells after transfection with TMEM16A.The classical calcium-activated chloride channel currents were recorded in the FRT cells stably expressing TMEM 16A by the technique of patch-clamp and halide-sensitive fluorescent protein YFP-H148Q/I152L.CONCLUSION:The protein expression of TMEM16A in the FRT cells was observed.TMEM16A is the molecular identity of calcium-activated chloride channels .