荧光比率法测量平滑肌细胞内的钙动力学变化

目的建立一种荧光比率方法来测量细胞内钙离子的动力学变化,并研究血管平滑肌细胞钙动力学变化在重症休克血管反应性降低中的作用.方法复制SD大鼠失血性休克模型,分离肠系膜细动脉血管平滑肌(ASMC),使用荧光探针Fluo-3/AM、FuraRed双标记比率方法结合激光扫描共聚焦显微成像技术测定单个平滑肌细胞钙动力学变化;评估荧光比率法在动态测定胞内钙离子浓度变化中的应用及其观察ATP敏感钾通道(KATP)特异性阻滞剂优降糖对血管反应性和钙动力学影响.结果休克后2 h(失代偿期),血管反应性明显降低,NE作用带来的平滑肌细胞内钙离子浓度升高的程度明显减弱;加入优降糖可明显提高NE对平滑肌细胞内钙离子的升高作用,改善细动脉对NE的反应性,带来血管反应性部分恢复.在37℃,Fluo-3/AM、FuraRed浓度为5μnol/L的条件下,肠系膜细动脉平滑肌细胞负载40 min左右即可获得良好的标记效果.结论优降糖特异性的阻滞ATP敏感钾通道的开放,增多细胞外钙内流并提高血管反应性;使用荧光比率法可以使胞内钙离子浓度的测量不受荧光探针浓度、细胞大小、激光漂白等因素的影响,可以准确、实时、动态测量细胞内钙离子浓度变化.

Detection of kinetic changes for calcium in vascular smooth muscle cells with method of ratio fluorescence

Objective: To establish a ratio fluorescent approach to detect the kinetic changes of calcium in vascular smooth muscle cells and study its role in vascular hyporeactivity in severe hemorrhagic shock.Methods: The hemorrhagic shock was made and mesenteric arteriolar smooth muscle cells (ASMC) were isolated. The kinetic changes of calcium were measured with two highly sensitive Ca2+ fluorescent labeling in conjunction with laser scanning confocal nicroscope (LSCM). The technique of ratio fluorescent approach was evaluated in the application of detecting kinetic changes of calcium. The effect of glibenclamide, a specific inhibitor for ATP sensitive potassium (KATP) channel, on vascular reactivity and the intracellular Ca2+]i were studied. Results: Hemorrhagic shock for 2 h caused an obvious reduction of vascular reactivity and the effect of NE on Ca2+]i were reduced. Glibenclamide could improve the effect of NE on Ca2+]i in ASMC. When ASMC were incubated for 40 min at 37℃ with 5 μmol/L Fluo-3/AM and 5μmol/L FuraRed, the excellent LSCM imaging of intraeellular Ca2+ can be obtained. Conclusions: Glibenclamide, a specific inhibitor for ATP sensitive potassium (KATP) channel can increase intracellular Ca2+]i and improve the vascular reactivity in severe hemorrhagic shock. An approach of ratio fluorescent can be qualified and quantified intracellular Ca2+]i more accurately, since it allows calcium quantitative detecting independent of dye concentration, cell volume,the effect of photobleaching and dye leakage. This paper provided an exactly new method to detect intracellular Ca2+ for real time, continuously and kinetic.