摘要
鸡传染性支气管炎病毒(IBV)具有严格的上皮细胞嗜性。由于缺乏可供IBV复制的传代细胞系,从而阻碍了从细胞水平研究其复制、致病等分子机制。掌握鸡肾上皮细胞等原代细胞的制备、纯化及培养技术显得尤为关键。本项目组通过选择不同日龄的SPF鸡胚或雏鸡进行肾上皮细胞的制备,同时对消化酶、纯化方式及培养条件进行筛选优化,进一步采用具有EGFP标签的r H120-EGFP/5a重组病毒鉴定培养的鸡肾上皮细胞。结果表明:选择1日龄雏鸡,通过胶原酶Ⅰ消化制备的鸡肾上皮细胞的活力最佳;采用多次离心法能有效去除大量单细胞,从而达到纯化上皮细胞的目的;3%胎牛血清足够提供上皮细胞生长所需的营养,并能抑制CEF的生长。试验探索并优化了鸡肾上皮细胞制备及培养的条件,为禽源上皮细胞系的建立和IBV分子致病机制的研究奠定了基础。
Avian infectious bronchitis virus has a strict cell tropism,which can only replicate in epithelial cells of infected tissues. Due to the lack of cell lines that can be used for IBV replication,in vitro investigation of the molecular mechanisms of its replication and pathogenesis are blocked. Therefore,it is very important to master the techniques of preparation,purification and culture of primary cells,such as chicken kidney epithelial cells. This study selected SPF chicken embryos and chickens in different age for preparation of kidney cells. At the same time, in order to improve the purity and viability of the renal epithelial cells,the digestive enzymes,way of purification and concentration of serum were screened. Then r H120-EGFP/5a recombinant virus with EGFP tag was used to identify the cultured renal cells.The results showed that the cell viability of chicken kidney epithelial cells prepared by collagenase Ⅰ from 1-day-age chicks was best. Repeated centrifugation could effectively remove a large number of single cells,which could achieve the purpose of purification of epithelial cells. 3% fetal bovine serum was sufficient to provide the necessary nutrition for the growth of epithelial cells,and could inhibit the growth of CEF. This experiment was made to explore the optimization of culture conditions of chicken kidney epithelial cells,which laid a foundation for the study of IBV molecular pathogenesis and the establishment of avian cell line in the future.
出处
《中国家禽》
北大核心
2016年第10期16-20,共5页
China Poultry
基金
国家自然科学基金项目(31101832
31572524)
江苏省科技支撑计划(BE2012369)
扬州市自然科学基金项目(YZ2014029)
关键词
鸡传染性支气管炎病毒
鸡肾上皮细胞
培养条件
avian infectious bronchitis virus
chicken kidney epithelial cells
cultural condition
