| 不同培养方法对山羊瘤胃上皮细胞生长及角蛋白18表达量的影响 |
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| 引用本文： | 刘思乐,康劲翮,谭支良,王征.不同培养方法对山羊瘤胃上皮细胞生长及角蛋白18表达量的影响[J].动物营养学报,2016(4):1225-1232. |
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| 作者姓名： | 刘思乐 康劲翮 谭支良 王征 |
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| 作者单位： | 1. 中国科学院亚热带农业生态过程重点实验室，亚热带农业生态研究所，湖南省农业生态过程重点实验室，长沙 410125; 湖南农业大学生物科学技术学院，长沙 410128;2. 中国科学院亚热带农业生态过程重点实验室，亚热带农业生态研究所，湖南省农业生态过程重点实验室，长沙 410125;3. 湖南农业大学生物科学技术学院,长沙,410128 |
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| 基金项目： | 国家自然科学基金项目(31201826)，中央驻湘科研机构技术创新发展专项(2013TF3006) |
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| 摘 要： | 本试验旨在研究不同培养方法对山羊瘤胃上皮细胞生长及角蛋白18(CK18)表达量的影响。采集42日龄山羊的瘤胃上皮组织,分别采用酶消化法和组织块法对其进行体外培养。通过光学显微镜观察原代培养和传代培养阶段的细胞形态,检测第5代山羊瘤胃上皮细胞的生长曲线,并采用细胞免疫荧光法对山羊瘤胃上皮细胞进行鉴定。结果显示:1)经0.25%胰蛋白酶+0.02%乙二胺四乙酸消化获得的原代培养山羊瘤胃上皮细胞于2 d开始贴壁生长,5 d细胞开始明显增多,10 d细胞数量达到最大。2)经组织块法获得的原代培养山羊瘤胃上皮细胞于4 d开始"爬出"组织块,8 d细胞开始明显增多,14 d细胞数量达到最大。3)经免疫荧光染色显示2种方法获得的细胞胞浆内CK18均呈阳性表达且细胞纯度后者明显高于前者。4)组织块法获得的细胞CK18表达量显著高于酶消化法(P0.05)。综合得出,与酶消化法相比,应用组织块法可成功获得纯度更高的山羊瘤胃上皮细胞。
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| 关 键 词： | 山羊 瘤胃 上皮细胞 原代培养 分离 CK18 |
Effects of Different Culture Methods on Growth and Keratin 18 Expression of Ruminal Epithelium Cells of Goats |
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| Abstract: | This research aimed to study the effects of different culture methods on growth and keratin 18 ( CK18) expression of ruminal epithelium cells of goats. The test proceeded with ruminal epithelium cells of goats aged 42 days, and the cells were cultured using the methods of enzyme digestion and tissue mass culture. The morphology of cells at the stages of primary culture and subculture was observed under the optical micro-scope, and the growth curve of the fifth generation of ruminal epithelium cells of goat was tested, meanwhile, ruminal epithelium cells were identified using cell immune fluorescence method. The results showed as follows:1) the primary cultured ruminal epithelium cells of goats obtained by digestion of 0. 25% trypsin and 0. 02%ethylenediamine tetra-acetate began adherent growth on the 2nd day. There was a significant increase in cell number on the 5th day, and the cell number reached a maximum at the 10th day. 2) The primary cultured ru-minal epithelium cells of goats obtained by tissue mass culture began ‘escape’ from the tissue mass on the 4th day. There was a significant increase in cell number on the 8th day, and the cell number reached a maximum at the 14th day. 3) The CK18 in cytoplasm of cells obtained by the 2 methods exhibited a positive reaction by im-mune fluorescence staining, and cell purity was higher in tissue mass culture method. 4) Tissue mass culture method expressed significantly higher CK18 compared with enzyme method ( P<0.05) . In conclusion, ruminal epithelium cells of goats were successfully obtained with high purity using the method of tissue mass culture. |
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| Keywords: | goat rumen epithelium cell primary culture isolation CK18 |
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