山羊瘤胃上皮细胞和空肠黏膜上皮细胞原代培养技术研究  

Research on Primary Culture Method for Ruminal Epithelial and Jejunum Epithelial Cells of Goats

作　　者：孙志洪[1,2] 张庆丽[1,3] 贺志雄[1,2] 韩雪峰[1] 谭支良[1] 张恩平[3] 汤少勋[1] 周传社[1] 王敏[1] 

机构地区：[1]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,长沙410125 [2]中国科学院研究生院,北京100049 [3]西北农林科技大学动物科技学院,杨凌712100

出　　处：《动物营养学报》2010年第3期602-610,共9页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：国家自然科学基金(30600436);中国科学院知识创新工程重要方向项目(KSCX2-YW-N-051)联合资助

摘　　要：本研究旨在建立山羊瘤胃上皮细胞(ruminal epithelium cell,REC)和空肠黏膜上皮细胞(jejunum epithelial cell,JEC)的原代培养方法。首先进行胃肠道微生物去除的研究,设计9种不同种类和剂量的抗生素洗涤液,按随机区组原则冲洗采集到的瘤胃上皮和空肠黏膜。根据微生物去除试验结果,选择最佳种类和剂量的抗生素洗涤液对瘤胃上皮和空肠黏膜进行洗涤,然后采用不同的方法对REC和JMC进行分离与培养,根据分离所获的细胞特征以及细胞增殖活性结果确定适合山羊瘤胃上皮和空肠黏膜特性的分离方法。结果表明,为有效去除黏附的微生物,瘤胃上皮需分别经过5倍青链霉素和2倍庆大两性霉素洗涤液的冲洗,而空肠黏膜需分别经过5倍青链霉素和5倍庆大两性霉素洗涤液的冲洗。瘤胃上皮和空肠黏膜分别经2.50%胰蛋白酶+0.02%EDTA和0.20%Ⅳ型胶原酶+0.50mmol/LCaCl2消化后可获得理想的细胞分离效果以及细胞增殖活性。本研究结果提示,根据胃肠道微生物的组成特征以及组织特性选择所确定的抗生素洗涤液以及组织细胞分离方法可用于山羊REC和JEC的原代培养。The present study was carried out to establish a primary culture method for goat ruminal epithelial cell （REC） and jejunum epithelial cell （JEC）. The microbe removing test was carried firstly. According to the randomized block design,9 kinds of antibiotic washing solutions with different types and doses of antibiotic were assigned to wash ruminal epithelium and jejunum mucosa of goat. According to the results of microbe removing test,the antibiotic washing solution with the best efficiency for microbe removing was selected to wash ruminal epithelium and jejunum mucosa. After washing,the REC and JEC were isolated by different methods and then cultured. According the cell isolated characteristics and cell proliferation activity,the fitting isolated methods for the tissue characteristics of ruminal epithelium and jejunum mucosa were confirmed. The results showed as follows： in order to effectively remove the microbes adhering on tissues,ruminal epithelium should be washed by 5 fold penicillin-streptomycin solution and 2 fold gentamicin-amphotericin solution,respectively; and jejunum mucosa should be washed by 5 fold penicillin-streptomycin solution and 5 folds gentamicin-amphotericin solution,respectively. The ideal cell isolation characteristics and cell proliferation activity could be gained when ruminal epithelium and jejunum mucosa were digested by the solution with 2.5% trypsin＋0.02% EDTA and the solution with 0.2% type Ⅳ collagenase＋0.50 mmol/L CaCl2,respectively. The results suggested that the antibiotic washing solution and cell isolation method according to the microbial and histological characteristics of goat’s ruminal epithelium and jejunum mucosa could be used to isolated REC and JEC of goat in primary culture.

关 键 词：反刍动物 原代细胞培养 瘤胃 空肠 山羊 

分 类 号：S827[农业科学—畜牧学]