作　　者： ; ; ; ; ; ; ;

机构地区： 江西农业大学动物科学技术学院

出　　处： 《江西农业大学学报》 2008年第3期385-389,共5页

摘　　要： 以8～10周龄的雄性黄羽鸡为对象,比较了原位两步胶原酶灌流法、改良的原位两步灌流法及原位一步灌流结合组织块消化法对成年鸡肝细胞的分离效果,同时比较了不同基础培养液对成年鸡肝细胞体外培养的影响。结果表明,采用改良的原位两步灌流法可以获得分离效果很好,均匀一致且活率达90%以上的肝细胞;以Williams’medium E为基础培养液,以1×10^5个/cm^2密度接种,4 h后肝细胞贴壁,随后在含φ=7%新生牛血清的培养液中培养24 h后,肝上皮细胞呈现典型的多角形,核圆透亮,聚集呈岛屿状生长,48 h后增殖旺盛,96 h后基本铺满培养皿底部,出现胆小管样结构,并可维持存活13 d以上。本试验为进一步建立成年鸡肝细胞无血清培养体系,体外研究其功能奠定了基础。 Hepatocytes from 8～10 week-old yellow-feathered male broilers were isolated by two steps in situ collagenase perfusion,modified two steps in situ collagenase perfusion and one step in situ perfusion combined with tissue blocks collagenase digestion,respectively.The separation degree,uniformity and viability of the cells isolated by these three methods were compared.Meanwhile,effects of different mediums on the morphology and growth of the cells were investigated.The results showed that well-dispersed and pure hepatocytes with high viability（〉90%） were obtained by modified two steps in situ collagenase perfusion.The cells attached on the surface of petri dishes（Φ60 mm） 4 h after inoculation（1×10^5 cells/cm^2） using Williams＇ medium E supplemented with 10% new calf serum,10^-6M insulin,10^-6M dexamethasone,10 μg/mL ascorbic acid and the two antibiotics.Hereafter,the medium was changed to Williams＇ medium E supplemented with 7% new calf serum and the two antibiotics and renewed every 24 h.Under this condition,the cells were cultured for more than 13 days keeping their typical parenchymal morphology,with characteristic polygonal shape and formation of bile canaliculi.Other liver cell types were undetectable in daily observations.This preliminary study on the primary culture method of adult chicken hepatocytes established the foundation for further research on the function of adult chicken hepatocytes.

关 键 词： 鸡 肝细胞 分离 原代培养

领　　域： [农业科学] [农业科学]