TRPM7通道过表达细胞株的建立、鉴定及其功能的初步研究

Construction and identification of TRPM7 channel overexpression cell strain and preliminary functional research of the channel

通过瞬时转染TRPM7-eGFP质粒至人胚胎肾细胞HEK293T细胞建立瞬时受体电位Melastatin的7型通道(TRPM7通道)过表达体系并利用此体系对通道特性进行研究。经荧光显微镜观察,转染率可达30%～40%,RT-PCR及Western印迹结果显示转染后的细胞在相应的基因及蛋白水平上均有明显的表达。膜片钳结果显示转染后全细胞电流可达2 800 pA,pH 7.4至pH 4.0时通道内向电流显著增加,200μmol/L二苯硼酸-2-氨基乙酯(2-APB)显著抑制通道外向电流。结果表明瞬时转染HEK293T细胞的TRPM7过表达体系成功构建,细胞外酸性条件及TRPM7通道抑制剂2-APB对通道电流有不同影响,提示该通道功能可受到多种因素的影响。

To explore the function of TRPM7 （ transient receptor potential melastatin 7） channel, the overexpres- sion system of TRPM7 channel was constructed by transiently transfection of HEK293T （ human embryonic kidney 293T） cell with TRPM7-eGFP plasmid. Through the fluorescence microscope analysis, the transfection efficiency was about 30%- 40%. The corresponding expression of the gene and protein was observed by RT-PCR and western blotting. And the result of patch clamp showed that the whole cell current was about 2 800 pA. Acidic extracellular solution increased the TRPM7 inward currents and 200 μmol/L 2-APB （2-aminoethyl diphenyl borate） inhibited the TRPM7 outward currents. These results demonstrated that the transiently transfected TRPM7-HEK293T cell line was successfully constructed. The acidic pH and the non-specific inhibitor of TRPM7, 2-APB made different changes on TRPM7, which indicates that the function of the channel can be affected by multiple factors.