普通光学显微镜下观测离体气管黏膜纤毛活力

In vitro observation of ciliary activity of the tracheal membrane under a common light microscope

目的:探索一种能在普通光学显微镜下体外观测气管黏膜纤毛活动能力的新方法。方法:健康成年家兔9只。乌拉坦静脉麻醉后切取2块等大的颈段气管瓣,随机分配到麻黄素(Eph)组和DMEM对照组。将待测气管瓣黏膜面向上置于有DMEM培养液(DMEM对照组)或用DMEM培养液配制的0.5%麻黄素溶液(Eph组)的玻璃培养皿中,滴入一滴经1%亚甲兰染色的受试动物自体血细胞作为液流的示踪物,在400倍生物显微镜下贴近黏膜表面观察。通过计算机动态图像分析系统观察并自动测量示踪血细胞移动速度,用增减系数(variation coefficient,VC)反映移动速度的变化。结果:DMEM对照组不同时点的VC比较无统计学差异;Eph组VC随时间的推移有规律地下降。结论:利用示踪物建立的在普通光学显微镜下体外动态观测家兔气管黏膜纤毛活动能力的方法简便、可靠、稳定,可用于体外观察和评价呼吸道纤毛功能的领域。

Objective To establish a new method for in vitro the tracheal membrane in rabbit under a common light microscope observation of ciliary activity of Methods Nine healthy adult rabbits were used. Two equal sized cervical trachea flaps were removed after the rabbits were anesthetized by urethane （4mg/kg）. The removed trachea flap was randomly assigned to the ephedrine （ Eph ） and DMEM control group, respectively. The observed trachea flap was placed in a small flat-bottomed glass container with its membranous side upward in DMEM culture medium solution （ DMEM control group ） or in 0.5 % ephedrine solution prepared with DMEM （ Eph group ）. One drop of 1% methylthioninium stained autologous blood cells was added into the glass container as the tracer, and the trachea flap was observed under a common light microscope （400 x ）. The latter was attached with a digital camera linking to an image manipulation system, the computed dynamic image analyser. The velocity of the tracer cell movement worked as the indicator for ciliary activity and was automatically determined by the digital image manipulation system. The variation coefficient （VC） was used as the indicator of the cell movement velocity. Results There was no significant difference among the VC at different time points in the DMEM control group. VC of the Eph group decreased regularly with the time point. Conclusion A thin layer of flowing fluid was found on the surface of the tracheal mucociliary blanket which is driven by the activity of the mucociliary system. The new method of using the tracer to evaluate the ciliary activity of mammalia tracheal membrane in vitro is reliable and stable. It is practical and valuable in the in vitro observation and evaluation of ciliary activity of the tracheal membrane.