荧光共振能量转移技术在检测环磷酸腺苷信号通路对于MIN6细胞胰岛素分泌机制中的作用

Real-time quantitative detection of cAMP signaling pathway in insulin secretion of MIN6 cells based on FRET technology

目的探讨不同浓度的胰升血糖素(Glg)在无糖、低糖、高糖的环境下通过环磷酸腺苷(cAMP)信号通路对胰岛素分泌的直接调控作用及机制,基于荧光共振能量转移(FRET)的生物传感器技术可作为一种用来在活细胞上实时定量检测第二信使cAMP水平的方法。方法体外培养MIN6细胞,在葡萄糖0、2.8、16.7 mmol/L下予Glg 0、100、500、1000ng/L干预处理,ELISA检测不同处理下MIN6胞内cAMP量和胰岛素释放量;电转染法将质粒ICUE3转染进入MIN6细胞,用FRET技术检测细胞内cAMP水平。结果 ELISA与FRET技术检测的cAMP含量结果基本相同,印证了FRET检测cAMP的可行性;无糖、低糖及高糖环境下,cAMP含量及胰岛素的分泌量为1000ng/L组高于500、100、0ng/L组,500ng/L组高于100ng/L和0ng/L组;在低糖及高糖环境下,100ng/L组高于0ng/L组(P<0.05),且葡萄糖浓度越高趋势越明显。Pearson相关性分析结果显示,MIN6细胞内cAMP含量与胰岛素分泌量呈正相关(R2=0.559,P<0.01)。结论基于FRET的生物传感器技术可以作为一种用来在活细胞上实时定量检测第二信使cAMP水平的方法。Glg可能以浓度梯度的形式通过上调胰岛β细胞胞内cAMP浓度来促进Ins分泌,有一定的葡萄糖依赖性。

Objective To explore the direct regulation effects and mechanism on insulin secretion under different concentrations of glucagon（Glg）through cAMP signal pathway and to evaluate the feasibility of FRET technology used in detecting the cAMP levels in live cells.Methods Cultured isletβcell-MIN6 cells with glucose-free（0 mmol/L）,low-glucose（2.8 mmol/L）and high-glucose（16.7 mmol/L）were respectively intervened with Glg 0,100,500 and 1000 ng/L.The levels of intracellular insulin and cAMP were detected by ELISA.After transfection with ICUE3 plasmid,the levels of cAMP in MIN6 cells were detected using FRET method real-timely and quantitatively.Results The levels of cAMP detected by routine ELISA method were in accordance with those detected by FRET technology.In the conditions of glucose-free,low-glucose and high-glucose,the intracellular cAMP levels and insulin secretions of Glg1000 ng/L groups were higher than those in Glg 500 ng/L,100 ng/L and 0 ng/L groups,with the same trend in Glg 500 ng/L groups than in Glg 100 ng/L and 0 ng/L groups（P〈0.05）.This trend became obvious in higher glucose groups.The levels of cAMP had a positive correlation with insulin secretion in MIN6 cells under different glucose concentrations（R^2=0.559,P〈 0.01）.Conclusion Glg may stimulate insulin secret by increasing cAMP levels in MIN6 cells in the way of concentration gradient and glucose dependent.FRET can be used as a method for detecting the second message cAMP levels in live cells.