浏阳黑山羊瘤胃上皮细胞周期分布、增殖和凋亡特点  

Characteristics of Ruminal Epithelium Cell Cycle Distribution,Proliferation and Apoptosis of Liuyang Black Goats

作　　者：韩奇鹏 罗玲[1] 揭红东 王凯军[1] 张佩华[1] 周传社[2] 孔志伟[2] 汤少勋[2] 

机构地区：[1]湖南农业大学,动物科学技术学院,畜禽遗传改良湖南省重点实验室,长沙410128 [2]中国科学院,亚热带农业生态研究所,亚热带农业生态过程重点实验室,湖南省畜禽健康养殖工程技术中心,农业部中南动物营养与饲料科学观测实验站,长沙410125

出　　处：《动物营养学报》2016年第7期2269-2277,共9页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：国家自然科学基金面上项目(31372342);国家科技支撑计划课题(2012BAD14B17);中国科学院科技服务网络计划(STS计划)课题(KFJ-EW-STS-071)

摘　　要：本研究旨在建立浏阳黑山羊瘤胃上皮细胞的体外培养模型,并对其周期分布、增殖和凋亡特点进行研究。试验采集60日龄浏阳黑山羊的瘤胃上皮组织,应用0.25%胰蛋白酶＋0.02%乙二胺四乙酸（EDTA）消化法对山羊瘤胃上皮组织进行消化,得到单个的山羊瘤胃上皮原代细胞进行体外培养。通过倒置显微镜对原代和传代培养阶段细胞形态进行观察,采用细胞计数法检测细胞的生长活性,应用细胞免疫组化学方法对传代细胞进行鉴定,并用流式细胞术检测山羊瘤胃上皮传代细胞周期分布情况和凋亡比率。结果显示：1）经0.25%胰蛋白酶＋0.02%EDTA消化获得的山羊瘤胃上皮原代细胞,培养1 d开始贴壁生长,2 d开始生长较快（对数期）,呈典型的＂波峰＂状生长,3～4 d生长最为迅速,7 d生长速度平稳（平台期）。2）经细胞免疫组化学方法的鉴定,细胞胞浆为黄褐色,即细胞角蛋白19呈阳性表达。3）膜联蛋白-V/碘化丙啶联合染色显示,随着培养时间的延长,细胞凋亡比率显著增加（P〈0.01）。结果表明,通过0.25%胰蛋白酶＋0.02%EDTA的消化方法成功得到了浏阳黑山羊瘤胃上皮细胞,可为今后研究反刍动物瘤胃相关机制与功能提供模型。The present study was carried out to establish an in vitro culture model of rumen epithelial cells of Liuyang black goats and investigated cell cycle distribution,proliferation and apoptosis. Ruminal epithelium tissue of 60-day-old Liuyang black goats was collected,and digested with 0.25% trypsin and 0.02% ethylene diamine tetraacetic acid（ EDTA） to obtain individual primary cultured ruminal epithelium cells for in vitro culture. The morphous of ruminal epithelial cells in primary culture and subculture was observed under inverted microscope,the growth activity curve of cells was tested by cell number count assay,the identification of subcultured ruminal epithelium cells was carried out using the method of immunohistochemistry,and the subcultured ruminal epithelium cells cycle distribution and apoptosis ratio were determined through flow cytometry method. The results show ed as follow s： 1） the primary cultured ruminal epithelium cells of goats were obtained by digestion of 0.25% trypsin and 0.02% EDTA,began adherent growth after cultured for 1 d,an exponential growth started and resembled a ＂hill＂in appearance since 2 d,the cell growth rate reached the highest at 3 to4 d,and got stable at 7 d. 2） Identified by immunohistochemistry method,the cytoplasm was brown,which meant that cytokeratin 19（ CK19） was positive expressed. 3） The combination staining of Annexin V and propidium iodide show ed that the apoptosis ratio of cells significantly increased with the prolongation of culture time（ P〈0.01）. In conclusion,the ruminal epithelium cells of Liuyang black goats are obtained successfully by the method of 0.25% trypsin＋ 0.02% EDTA digestion,which provided cell model for further study on mechanism and the function of ruminant rumen.

关 键 词：浏阳黑山羊 瘤胃上皮细胞 周期分布 凋亡 

分 类 号：S826[农业科学—畜牧学]