兔气管纤毛蛋白磷酸化的初步研究  

Primary Studies on Phosphorylation Profile of Ciliary Proteins in Trachea of Rabbit

作　　者：刘晨[1,2] 韩德民[1,2] 张罗[1,2] 

机构地区：[1]首都医科大学附属北京同仁医院耳鼻咽喉头颈外科 [2]北京市耳鼻咽喉科研究所 耳鼻咽喉头颈科学教育部重点实验室

出　　处：《首都医科大学学报》2009年第1期54-56,共3页Journal of Capital Medical University

基　　金：国家重点基础研究发展计划前期研究专项(2007CB516706);国家自然科学基金(30572026);教育部新世纪优秀人才支持计划(NCET-06-0185)资助项目~~

摘　　要：目的建立分离提取呼吸道纤毛的方法,为进一步研究纤毛蛋白磷酸化在纤毛运动调控机制中的作用提供方法学基础。方法用Triton X-100分离缓冲液孵育兔气管黏膜上皮,使纤毛上皮细胞的纤毛与细胞体分离,通过离心提取分离的纤毛,将提取的标本行透射电镜观察、Western blotting小鼠检测β微管蛋白(β-tubulin)和纤毛蛋白磷酸化情况。结果透射电镜观察可见提取的标本为浓集的纤毛,纤毛的横断面可见典型的"9+2"结构;②Western blotting检测可见高信号强度的β-tubulin条带;③Western blotting检测可见5条酪氨酸磷酸化的蛋白条带。结论经含有Triton X-100的缓冲液孵育的兔气管黏膜上皮离心后可提取到分离的纤毛标本,适于进行进一步的纤毛蛋白磷酸化研究。Objective To establish a method of cilia isolation from airway epithelium and provide the methods of further studying the role of the protein phosphorylation in the mechanisms of ciliary motility. Methods The rabbit trachea epithelium was ineubated in isolation buffer eontaining Triton X-100 in order to isolate the cilia from the epithelial tissue. Cilia were then collected by eentrifugation. The sample was examined by electron microscopy, and the β-tubulin and the phosphorylated proteins were visualized by Western blotting. Results （1） The eleetron mierograph demonstrated a highly enriched ciliary preparation showing the characteristic features of intact axoneme; （2） Western blotting analysis showed visible bands of β-tubulin; （3） Western blotting analysis detected five tyrosine phosphorylated bands. Conclusion The ciliary preparation ean be obtained by ineubating the rabbit airway epithelium in isolation buffer eontalning Triton X-100 and the following eentrifugation. The preparation ean be used to study the role of the protein phosphorylation in the mechanisms of ciliary motility.

关 键 词：纤毛 分离 磷酸化 

分 类 号：Q471[生物学—生理学]