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目的构建能够表达TRPV1通道用于实验研究的HEK 293T细胞表达体系。方法采用脂质体转染方式将TRPV1质粒转入人胚胎肾HEK 293T细胞,建立TRPV1型通道瞬时异源表达体系,并对转染后的TRPV1表达体系进行了鉴定,同时对该通道功能特性进行了研究。结果经荧光显微镜观察,转染率可达40%~50%;Western Blot研究发现转染后的HEK 293T细胞在与TRPV1通道蛋白相应的位置上具有明显的条带,提示TRPV1通道蛋白在转染后的细胞中有异源性表达;共聚焦显微成像显示,转染后的HEK293T细胞在受到TRPV1通道激动剂刺激后,胞内荧光强度明显变强,提示TRPV1通道介导钙离子功能正常。结论研究结果表明,基于HEK 293T细胞的TRPV1通道瞬时转染的异源表达体系成功构建。
Objective To construct a HEK 293T cell expression system capable of expressing TRPV1 channel for experimental study. Methods The plasmid of TRPV1 was transfected into human embryonic kidney HEK 293T cells by liposome transfection method to establish transient heterologous expression system of TRPV1 channel and to identify the expression system of TRPV1 after transfection. At the same time, Study. Results The results showed that the transfection rate could reach 40% -50% by fluorescence microscopy. Western Blot showed that the HEK 293T cells transfected with HEK 293T cells showed obvious bands at the positions corresponding to the TRPV1 channel protein, suggesting that TRPV1 channel protein transfection The results of confocal microscopy showed that the intracellular fluorescence intensity of HEK293T cells transfected by TRPV1 channel agonist was significantly enhanced, suggesting that TRPV1 channels mediate the normal function of calcium ions. Conclusion The results show that the HEK 293T cell-based heterologous expression system of TRPV1 transient transfection was successfully constructed.