共聚焦显微镜不同倍数物镜下肠系膜动脉三级分支张力和Ca2＋信号同步变化的比较

Comparison of the synchronous changes of vascular tension and intracellular Ca^(2+) signal in third-order branches of mesenteric arteries under the different objective amplification of confocal microscope

本文旨在建立优化的观察肠系膜动脉三级分支(sMA,直径100~300μm)血管张力和血管平滑肌细胞(vascular smooth muscle cells,VSMCs)内Ca^(2+)信号的同步变化的实验方法。分别采用DMT 360CW激光共聚焦微血管张力测定系统和Nikon C2激光共聚焦显微镜,同时记录Ca^(2+)通道激动剂KCl、内皮素-1(endothelin-1,ET-1)以及Ca^(2+)通道抑制剂钆离子(Gd3+)诱导的去内皮sMA血管张力和VSMCs内Ca^(2+)信号的变化,并对共聚焦显微镜不同物镜(10×、20×、40×)下记录到的Ca^(2+)信号荧光值变化量进行对比分析,探索最佳的实验条件。结果显示,KCl可引起sMA显著收缩,20×、40×物镜下VSMCs内Ca^(2+)信号会同步增强,相比40×物镜,20×物镜下Ca^(2+)信号变化量更大,荧光值更稳定,而10×物镜下VSMCs内Ca^(2+)信号变化不明显;不同浓度的ET-1能够引起sMA浓度依赖性收缩,同样20×物镜下VSMCs内Ca^(2+)信号也呈浓度依赖性同步增强;ET-1预收缩sMA后加入Gd3+显著降低ET-1诱导的血管收缩效应,相应地20×物镜下VSMCs内Ca^(2+)信号也显著降低。以上结果表明,Ca^(2+)通道激动剂或抑制剂引起血管收缩或舒张的同时,VSMCs内Ca^(2+)信号会发生相应的变化,提示本实验方法可同步记录两者的变化,而且共聚焦显微镜20×物镜为最佳的实验条件。与分别应用血管张力检测技术测定血管张力变化和动态细胞荧光成像技术测定VSMCs内Ca^(2+)信号变化的方法相比,同步检测张力和Ca^(2+)信号的变化更简单实用,有效避免了不必要的实验误差。

This study was aimed to establish an optimized method to observe the synchronous changes of vascular tension and intracellular Ca^2＋ signal in the third-order branches of mesenteric arteries （sMA, diameter： 100–300 μm）. The vascular tension and intracellular Ca^2＋ signal changes in response to potassium chloride （KCl）, endothelin-1 （ET-1） and Gd3＋ were detected using confocal wire myograph system and confocal laser scanning microscopy imaging technique, respectively. The experimental results were analyzed to explore the optimal experimental conditions. The results showed that KCl caused contraction in sMA significantly, and the intracellular Ca^2＋ level of vascular smooth muscle cells （VSMCs） was also increased under 20× and 40× objective lens. Compared with those under the 40× objective lens, the Ca^2＋ signal change was larger and the fluorescence value was more stable under the 20× objective lens, whereas the Ca^2＋ signal change was not obvious under the 10× objective lens. ET-1 （1–10 nmol/L） caused concentration dependent contraction in sMA significantly, and the intracellular Ca^2＋ signal of VSMCs was also enhanced in a concentration dependent manner. Additionally, Gd3＋ significantly reduced the contraction of sMA and the intracellular Ca^2＋ signal of VSMCs caused by ET-1. The results suggest that the intracellular Ca^2＋ signal of VSMCs changes with vascular contraction or relaxation caused by the agonists or antagonists of Ca^2＋ channels. We successfully recorded both changes synchronously using confocal wire myograph system and confocal laser scanning microscopy imaging technique at the same time. Based on the analysis of the experimental results, we concluded that 20× objective lens provides the best experimental condition. Compared to combination of vascular tone detection method and real- time cellular fluorescence imaging technique, the present synchronous method is convenient and helpful to reduce experimental error.