南方医科大学学报

南方医科大学学报 ›› 2004, Vol. 24 ›› Issue (04): 450-452.

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荧光定量法测量平滑肌细胞内钙离子浓度的探讨

王水云1, 晏芳2, 彭贺新1, 郑昌东3, 柏承文1   

  1. 1. 深圳市人民医院急诊科, 广东, 深圳, 518020;
    2. 第一军医大学组织胚胎学教研室, 广东, 广州, 510515;
    3. 深圳市福田人民医院, 广东, 深圳, 518020
  • 出版日期:2004-04-20 发布日期:2004-04-20
  • 基金资助:
    收稿日期:2003-8-13。
    作者简介:王水云(1951-),男,浙江龙游人,深圳市人民医院急诊科主任,本科,主要从事休克微循环及急症医学研究

Fluorescence quantification of intracellular calcium of rat smooth muscle cells

WANG Shui-yun1, YAN Fang2, PENG He-xin1, ZHENG Chang-dong3, BAI Cheng-wen1   

  1. 1. 深圳市人民医院急诊科, 广东, 深圳, 518020;
    2. 第一军医大学组织胚胎学教研室, 广东, 广州, 510515;
    3. 深圳市福田人民医院, 广东, 深圳, 518020
  • Online:2004-04-20 Published:2004-04-20

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摘要： 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。

Abstract: 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 that[Ca2+]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 intracellular[Ca2+]i.

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