鸡气管上皮细胞分离培养及传支病毒在其培养特性研究  

Isolation and Culture of Chicken Trachea Epithelium and Culture Characteristics of IBV in Chicken Trachea Epithelium

作　　者：孙裴[1] 张彦明[1] 张浩[1] 高春生[2] 张为民[1] 王晶钰[1] 郭抗抗[1] 党如意[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100 [2]河南农业大学牧医工程学院,郑州450002

出　　处：《中国农业科学》2008年第3期861-867,共7页Scientia Agricultura Sinica

基　　金：国家自然科学基金项目(30471290)

摘　　要：【目的】探索鸡气管上皮细胞(chicken trachea epithelium,CTE)分离培养技术,并研究传染性支气管炎病毒(infectious bronchitis virus,IBV)在CTE中培养特性。【方法】利用气管灌注冷消化法分离培养CTE,对CTE特异性8/18角蛋白作免疫组化鉴定上皮细胞。培养的CTE分别接种IBVM41株和T株,分别在接种后第12～96小时观察接毒细胞形态学变化,应用电镜技术观察CTE细胞病变和病毒粒子结构,利用RT-PCR技术检测CTE中病毒核酸并对CTE中IBV的血凝性和鸡胚致病性进行检测。【结果】利用灌注冷消化法可以获得形态完整并带有纤毛的CTE,细胞活性高,经过3步纯化后的CTE均一性高、生长快。两株IBV感染的CTE分别在接毒72和84h后产生明显的细胞病变效应(cytopathic effect,CPE),血凝性和致鸡胚病变检测结果呈阳性。【结论】本研究成功分离培养了CTE,可以为CTE相关研究提供技术支持,并为IBV基础研究和应用奠定基础。【Objective】The purpose of this study was to explore the technology for isolation and culture of chicken trachea epithelium （CTE） and also to study culture characteristics of IBV in CTE. 【Method】 By douche, chicken trachea was digested at 4 by Pronase and ℃ then CTE was isolated and cultured. The experiment of immunohistochemistry for detection of No.8/18 keratins of epithelium was done to identify CTE. The CTE was infected respectively by IBV-M41 strain and IBV-T strain. After infection of 12-96 h, the morphologic changes of CTE were observed, and electron microscopic technique was applied to observe CPE of CTE and virus in the cells. At the same time the method of RT-PCR was used to detect IBV in CTE. Finally, the ability of haemagglutination and pathological changes of chicken embryo caused by IBV were tested and observed. 【Result】 By douche and the method of digestion at 4℃, CTE was isolated and has full morphologic structure, clearly visible cilia and high cytoactive. Through three-step purification CTE got fine uniformity and grew faster. Seventy-two and 84 hours after CTE infected by each IBV strain, CTE appeared obvious cytopathic effect （CPE）, and both of the results of the test of ability of haemagglutination and pathological changes of chicken embryo by IBV were positive. 【Conclusion】 CTE was successfully isolated and cultured, which may provide technology for related CTE research and laid a groundwork for basic research and application of IBV.

关 键 词：鸡气管上皮细胞 分离 培养 IBV 培养特性 

分 类 号：S858.31[农业科学—临床兽医学]