荧光定量法测量平滑肌细胞内钙离子浓度的探讨

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。

Fluorescence quantification of intracellular calcium of rat smooth muscle cells

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 Ca²⁺ 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 according to the conversion formula from fluorescence intensity to concentration, the concentration of Ca²⁺ was calculated. Results Analysis of the fluorescent images using LSCM showed thatCa²⁺]_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 occasionally. Conclusion Fluorescence quantification with LSCM is applicable for detecting the kinetic changes of intracellularCa²⁺]_i.