2-NBDG检测H9c2心肌细胞葡萄糖摄取能力的研究

2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxyglucose as fluorescence probe to detect glucose uptake in H9c2 cells

目的评估荧光标记2-脱氧葡萄糖(2-deoxyglucose,2-DG)即2-NBDG(2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2-deoxyglucose,2-NBDG)作为光学探针检测H9c2心肌细胞葡萄糖摄取的能力。方法间接免疫荧光法测定H9c2心肌细胞葡萄糖转运体(GLUT-1、GLUT-4)的表达。用不同浓度2-NBDG和不同孵育时间处理H9c2心肌细胞,探讨其量效-时效关系,荧光酶标仪测定2-NBDG孵育H9c2细胞的最适浓度和时间。以2-NBDG最适浓度孵育H9c2心肌细胞,同时用共聚焦显微镜和流式细胞仪检测细胞内荧光强度差异,观察心肌细胞摄取2-NBDG情况。使用竞争性抑制剂D型葡萄糖(D-Glu)与2-NBDG共同孵育H9c2心肌细胞,观察D-Glu对2-NBDG的竞争性抑制情况。结果间接免疫荧光法显示H9c2心肌细胞高表达GLUT-1、GLUT-4。2-NBDG孵育H9c2心肌细胞的最适浓度和时间分别为100μmol/L和30 min。共聚焦成像和流式细胞仪均显示H9c2心肌细胞能有效摄取2-NBDG,加入D-Glu后使心肌细胞内荧光信号强度分别降低27.0%和46.7%(P<0.01)。运用2-NBDG检测细胞葡萄糖摄取的方法,证实胰岛素能促进H9c2心肌细胞葡萄糖的摄取。结论 2-NBDG能迅速被高表达GLUT-1、GLUT-4的H9c2心肌细胞摄取并滞留于细胞内,且可以被D-Glu竞争性抑制,表明2-NBDG可作为一个方便且敏感的探针,用于检测细胞葡萄糖摄取的能力。

Objective To assess the possibility of a deoxyglucose analog 2-[N-（7-nitrobenz-2-oxa-1,3-diazol-4-yl） amino]-2-deoxyglucose（2-NBDG） as a fluorescence probe to detect the glucose uptake in H9c2 cells.Methods Glucose transporter 1（GLUT1） and glucose transporter 4（GLUT 4） proteins in H9c2 cells were identified by indirect immunofluorescence（IIF） assays.H9c2 cells were incubated with 2-NBDG at a cohort concentration for different periods and the most suitable concentration and exposure period were determined by fluorescence microplate reader.H9c2 cells were incubated with 100 μmol/L 2-NBDG for 30 min with or without D-Glu,a potent competitive inhibitor of 2-NBDG.After the incubation,the 2-NBDG uptake was observed and recorded by confocal laser scanning microscopy（CLSM） and flow cytometry（FCM）.Results Over-expressed GLUT1 and GLUT 4 proteins were discovered in H9c2 cells.The most suitable concentration and exposure period for H9c2 cells incubation were 100 μmol/L 2-NBDG and 30 min.After incubated with 2-NBDG with or without D-Glu,bright green fluorescence of the 2-NBDG in H9c2 cells was observed under CLSM.The intracellular accumulation of 2-NBDG fluorescence was decreased to 27%（CLSM） and 46.7%（FCM）（P〈0.01） in groups of co-incubating with D-Glu.2-NBDG was used to detect cell glucose uptake and confirmed that insulin to promote glucose uptake in H9c2 cells.Conclusion 2-NBDG is quickly transported by GLUT1 and GLUT4 and accumulated in the H9c2 cells,which can be inhibited by D-Glu.2-NBDG may be used as an optical probe for glucose uptake in H9c2 cells.