测量和分析纤毛运动活动的新方法-生物化学与分子生物学专业论文.docxVIP

中南民 中南民族大学硕士学位论文 测量 测量和分析纤毛运动活动的新方法：钙进入调控机械敏感性 I I II II 摘 要 气管中纤毛的运动活动（CBA）通过清除呼吸道中的痰液和病原体扮演着重要的作 用。因为纤毛运动活动极其复杂以至于不能仅仅通过频率来描述其特征，所以我们用激 光共聚焦显微镜的线扫模式记录纤毛运动活动并且定义了六个参数来反应纤毛运动活 动。当测量纤毛运动的小鼠纤毛细胞时，这六个参数的值都大于零。 接下来我们用 10 微摩尔腺嘌呤核苷三磷酸刺激细胞，同时记录细胞内钙水平和纤 毛运动活动。我们发现细胞内钙水平先增高，紧接着纤毛运动活动增强。在这六个参数 之中，频率、幅度和积分面积显著增高，然而上升时间、下降时间和半数衰变时间明显 下降。这些结果表明六个参数适合评估纤毛运动活动而且升高的细胞内钙水平可能增强 纤毛运动活动。 接下来我们用机械刺激纤毛尖端，并且用我们建立的方法观察各个参数的变化。结 果表明机械刺激介导的细胞内钙水平和纤毛运动活动的变化不仅和腺嘌呤 核苷三磷酸引导的变化相似，而且这种变化可以被10微摩尔的钙离子螯合剂BAPTA-A M处理十分钟阻断掉。此外，在无钙条件下，我们检测到相同的阻断效果，当向浴槽中 加入2毫摩尔的钙离子后可以重新恢复细胞内钙水平的增加和纤毛运动活动的增强。 综上所述，我们提供了一种能够同时测量和分析纤毛运动活动并监控细胞内钙水平 的方法。并且用这个方法证明了当机械刺激纤毛尖端时，细胞外钙离子的进入使细胞内 钙水平升高，转而促发纤毛运动活动的增强。 关键词：纤毛细胞；纤毛运动活动；细胞内钙；机械刺激 ABSTRACT Airway ciliary beat activity (CBA) plays a pivotal role in protecting the body by removing mucus and pathogens from the respiratory tract. Since CBA is complicated and cannot be characterized by merely frequency, we recorded CBA using laser confocal line scanning and defined six parameters for describing CBA. The values of these parameters were all above 0 when measured in beating ciliated cells from mouse tracheae. We subsequently used 10 μM adenosine-5′-triphosphate (ATP) to stimulate ciliated cells and simultaneously recorded intracellular Ca2+ levels and CBA. We found that intracellular Ca2+ levels firstincreased, followed by an increase in CBA. Among the sixparameters, frequency, amplitude, and integrated area significantly increased, whereas rise time, decay time, and full duration at half maximum markedly decreased. The results suggest that these six parameters are appropriate for assessing CBA and that increased intracellular Ca2+ levels might enhance CBA. We next used our established methods to observe changes in mechanically stimulated cilia tips. We found that mechanical stimulation-induced changes in both intracellular Ca2+ levels and CBA were not only similar to those induced by ATP, but were also blocked by treatment with a Ca2+ chelator, BAPTA-AM, (10 μM) for 10 min. Moreo