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A new fluorescence method for determination of sodium-glucose cotransporter 2 activity
Author(s): 
Pages: 725-731
Year: Issue:  5
Journal: Journal of Peking University(Health Sciences)

Keyword:  钠-葡萄糖转运体2脱氧葡萄糖荧光流式细胞术;
Abstract: 目的:建立一种新的荧光标记的钠离子-葡萄糖协同转运蛋白2(sodium-glucose cotransporter 2,SGLT2)葡萄糖转运的测定方法.方法:合成人全长SGLT2 cDNA,构建pIRES2-EGFP-SGLT2重组质粒,酶切鉴定并测序验证其构建的正确性.瞬时转染HEK293细胞后,构建SGLT2高表达细胞株;采用激光共聚焦扫描显微镜、流式细胞仪检测其转染效率及转录活性,Western Blot法检测目的蛋白SGLT2表达情况.以荧光标记的脱氧葡萄糖2-NBDG作为示踪剂,应用流式细胞术检测SGLT2对葡萄糖的转运活性.结果:构建的pIRES2-EGFP-SGLT2质粒经酶切鉴定、测序证明质粒构建正确.该质粒转染HEK293细胞后,与未转染组相比,转染组细胞绿色荧光蛋白(green fluorescent protein,GFP)表达明显增加(P<0.01).与空载体pIRES2 -EGFP转染组相比,重组质粒转染组SGLT2蛋白表达明显增加(P<0.05);而未转染组与空载体转染组SGLT2蛋白表达无明显差异.SGLT2转运试验结果显示,转染组细胞对2-NBDG的钠离子依赖性摄取率较未转染组明显升高(P<0.01).结论:成功构建并应用SGLT2真核表达质粒,建立了一种新的荧光示踪的SGLT2葡萄糖转运活性的测定方法.
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