ANO1稳定转染细胞模型的构建及生理特性

Establishment and Physiological Characteristics of Stable Transfected ANO1 Cell Line Model

为了构建稳定转染钙激活氯离子通道(CaCCs)细胞模型并研究其生理特性,本试验采用脂质体转染和抗生素筛选获取共表达阴离子通道1(ANO1)蛋白和黄色荧光蛋白双突变体(YFP-H148Q/I152L)的Fisher大鼠甲状腺滤泡上皮(FRT)细胞;用倒置荧光显微镜观察ANO1和YFP-H148Q/I152L在FRT细胞的表达情况;用流式细胞仪检测细胞模型纯度;用荧光淬灭动力学试验检测细胞模型功能;用细胞模型或膜片钳技术研究ANO1生理特性,包括转运阴离子的特性、不同激活剂及抑制剂对ANO1的调控作用、ANO1电流受Ca^(2+)调控作用等。结果显示,倒置荧光显微镜下可观察到清晰的荧光信号;荧光淬灭动力学试验证明,此细胞模型可用于ANO1生理特性的研究;ANO1具有转运阴离子的特性;钙激活氯离子通道激活剂可以使ANO1开放且呈剂量依赖关系;钙激活氯离子通道抑制剂可抑制ANO1开放且具有剂量依赖关系;ANO1电流呈现钙依赖的特性;高浓度Ca^(2+)引起Run-down现象。结果表明,本试验成功构建ANO1细胞模型,ANO1具有经典的钙激活氯离子通道的生理特性。

In order to establish and physiologically characterize a stable cell model transfected with calcium activated chloride channel(CaCCs),Fischer rat thyroid(FRT)cells co-expressing anoctamin 1(ANO1)and yellow fluorescent protein double mutant(YFP-H148Q/I152L)were obtained by liposome transfection,antibiotic screening and limited dilution;the expression of ANO1 and YFP-H148Q/I152L in FRT cells was observed by an inverted fluorescence microscopy;the purity of cell model was detected by flow cytometry;the function of cell model was assessed by fluorescence quenching kinetics experiment;the physiological characteristics of ANO1 were investigated by cell model or patch clamp,including the characteristics of transporting anions,regulatory effects of different channel activators and inhibitors on ANO1,and Ca^(2+) regulation of ANO1 current.The results showed that inverted fluorescence microscope revealed clear fluorescence signal in ANO1 and YFP-H148Q/I152L cell models;fluorescence quenching kinetics experiments indicated that ANO1 cell model was useful for examining the physiological characteristics of ANO1;ANO1 displayed the characteristic of transporting anions;CaCC activator opened ANO1 in a dose-dependent manner;CaCC inhibitors inhibited the opening of ANO1 in a dose-dependent manner;ANO1 current was calcium dependent;high concentration of Ca^(2+) caused run-down phenomenon.Thus,the ANO1 cell model established in this study demonstrated the physiological characteristics of classic CaCCs.