"Fluorescent glycogen" formation with sensibility for in vivo and in vitro detection
"Fluorescent glycogen" formation with sensibility for in vivo and in vitro detection
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DOI：
10.1007/s10719-007-9075-7
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发表时间：
2008-08-01
影响因子：
3
通讯作者：
Botana, Luis M.
中科院分区：
文献类型：
--
作者：
Carmen Louzao, M.;Espina, Begona;Botana, Luis M.
目前有许多检测复合碳水化合物的方法，特别是糖原。然而，它们中的大多数需要放射性同位素或破坏组织和糖原水解成葡萄糖。本文提出了一种新的方法，将2- nbdg （2-{N-[7-硝基苯-2-氧-1,3-二唑基]氨基}2-脱氧葡萄糖），一种d -葡萄糖荧光衍生物，掺入糖原中。以克隆9号大鼠肝细胞为细胞模型，采用两种方法；(1)用2- NBDG孵育细胞裂解液，滤水器中碳水化合物沉淀，在微孔板阅读器中测量荧光(2)用2- NBDG孵育活肝细胞，用共聚焦显微镜记录荧光图像。两种方法均采用2- NBDG标记糖原。我们通过与特异性单克隆抗糖原抗体获得的标记进行比较，证实了这一事实。此外，触发糖原合成或降解的药物也分别引起荧光的增加或减少。这是一种简单而有效的2- NBDG检测糖原的方法。它可用于记录活细胞和无细胞系统中糖原储存的变化，并为了解这种重要的能量储备在各种生理和病理生理条件下的作用开辟了前景。
There are presently many methods of detecting complex carbohydrates, and particularly glycogen. However most of them require radioisotopes or destruction of the tissue and hydrolysis of glycogen to glucose. Here we present a new method based on the incorporation of 2-NBDG (2-{N-[7-nitrobenz-2-oxa-1,3-diazol4-yl]amino}2-deoxyglucose), a D-glucose fluorescent derivative, into glycogen. Two kinds of approaches were carried out by using Clone 9 rat hepatocytes as a cellular model; ( 1) Incubation of cell lysates with 2- NBDG, carbohydrate precipitation in filters and measurement of fluorescence in a microplate reader (2) Incubation of living hepatocytes with 2- NBDG and recording of fluorescence images by confocal microscopy. 2- NBDG labeled glycogen in both approaches. We confirmed this fact by comparison to the labeling obtained with a specific monoclonal anti-glycogen antibody. Also drugs that trigger glycogen synthesis or degradation induced an increase or decrease of fluorescence, respectively. This is a simple but efficient method of detecting glycogen with 2- NBDG. It could be used to record changes in glycogen stores in living cells and cell-free systems and opens the prospect of understanding the role of this important energy reserve under various physiological and pathophysiological conditions.