荧光定量法测量平滑肌细胞内钙离子浓度的探讨

Fluorescence quantification of intracellular calcium of rat smooth muscle cells

目的研究平滑肌细胞内Ca2+浓度的动力学变化,建立一种定量测量细胞内Ca2+浓度的方法。方法分离肠系膜细动脉血管平滑肌(ASMC)细胞,用荧光探针Indo-1和激光扫描共聚焦显微成像技术测定单个平滑肌细胞Ca2+浓度的动力学变化;首先在37 ℃环境下标定Ca2+ 探针indo-1的解离常数Kd值,根据荧光-浓度换算公式将测量的荧光强度值换算成Ca2+ 浓度值。结果激光扫描共聚焦显微成像技术测量的细胞荧光图像分析显示,平滑肌细胞[Ca2+]i 在地塞米松的刺激下显著上升,有时会出现自发钙波的现象,并且细胞内出现钙超载的现象(细胞荧光图像呈现白色)。通过测量得到的Kd值结合荧光强度-浓度换算公式可准确地测量细胞内[Ca2+]i 。结论荧光定量法结合共聚焦显微成像技术可以简易、快捷地监测细胞内钙离子的动力学变化,不失为一种定量测量细胞内钙离子的方法。

Objective To explore the kinetic changes of calcium in rat smooth muscle cells and establish a method for quantification of intracellular calcium. Methods Rat mesenteric arteriolar smooth muscle cells (ASMCs) were isolated and the kinetic changes of calcium were measured using highly sensitive Ca 2+ fluorescent probe indo-1 with laser scanning confocal microscopy (LSCM). The dissociation constant values (Kd) of the fluorescent probe indo-1 was measured at 37 ℃, and accor- ding to the conversion formula from fluorescence intensity to concentration, the concentration of Ca 2+ was calculated. Results Analysis of the fluorescent images using LSCM showed that i in the ASMCs were significantly elevated in response to stimulation with dexamethasone, and spontaneous calcium waves as well as intracellular calcium overloading were observed occa- sionally. Conclusion Fluorescence quantification with LSCM is applicable for detecting the kinetic changes of intracellular i .