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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℃,Flu-o-3/AM、FuraRed浓度为5μmol/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。
Objective To establish a fluorescent ratio method to measure the intracellular calcium dynamics and to study the role of calcium dynamics in vascular smooth muscle cells in reducing the vascular reactivity in severe shock. Methods The hemorrhagic shock model of SD rats was reproduced and isolated from the mesenteric arteriole smooth muscle (ASMC). Fluorescence probe Fluo-3 / AM and FuraRed dual labeling ratio method were used to determine the calcium dynamics of single smooth muscle cells by confocal laser scanning microscopy To evaluate the application of fluorescence ratio method in the dynamic determination of intracellular calcium concentration and to observe the effects of glyburide-specific blockade agent (GPTK) on vascular reactivity and calcium dynamics. Results After 2 h of shock (decompensation), the vascular reactivity was significantly decreased, and the increase of intracellular calcium concentration in VSMCs induced by NE was significantly attenuated. The addition of glibenclamide significantly increased the intracellular calcium Of the role of raising, to improve the reactivity of fine arteries to NE, resulting in vascular reactivity partially recovered. Under the conditions of 37 ℃, Flu-o-3 / AM and FuraRed concentration of 5μmol / L, the mesenteric arteriole smooth muscle cells were loaded for about 40 min to obtain a good labeling effect. CONCLUSION: GLP can block the opening of ATP-sensitive potassium channel and increase the extracellular calcium influx and increase the reactivity of blood vessels. Fluorescence ratio method can make the measurement of intracellular calcium concentration independent of fluorescence probe concentration, cell size , Laser bleaching and other factors, can accurately, real-time, dynamic measurement of intracellular calcium concentration changes.