新生广西巴马小型猪肾成纤维细胞体外培养体系的建立  

Establishment of in vitro culture system for new-born Guangxi Bama mini-pigs kidney fibroblast cells

作　　者：王献伟[1] 卢晟盛[1] 卢克焕[1] 

机构地区：[1]广西大学动物科技学院亚热带农业生物资源保护与利用国家重点实验室,广西南宁530004

出　　处：《中国兽医学报》2011年第10期1526-1532,1536,共8页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30860039);农业部转基因生物新品种培育重大专项资助项目(2009ZX08010-023B);广西自然科学基金重点资助项目(2010GXNSFD013021);广西大学拔尖创新团队建设计划资助项目(2010年)

摘　　要：利用微量液体组织块贴壁法原代培养分离新生广西巴马小型猪肾成纤维细胞,进行常规传代培养、冷冻和复苏;通过接触抑制和解除抑制研究该类细胞的增殖潜力,免疫荧光进行鉴定;并通过脂质体介导对分离到的新生广西巴马小型猪肾成纤维细胞分别进行EGFP-N1和DsRed-N1基因转染。结果成功分离到新生广西巴马小型猪肾成纤维细胞,体外可以大量传代和冷冻,目前已经传至50代以上,生长良好;冷冻复苏和接触抑制解除后仍可以正常培养和传代;细胞免疫荧光检测,波形蛋白表达阳性,角形蛋白表达阴性;转染48h后荧光显微镜下可以观察到绿色荧光和红色荧光,表明瞬时转染新生广西巴马小型猪肾成纤维细胞可以获得成功,其中转染DsRed-N1基因的新生广西巴马小型猪肾成纤维细胞已经传至60代以上。结果表明,本试验成功建立了新生广西巴马小型猪肾成纤维细胞的体外培养体系,在体外能稳定培养和传代,并可以成功获得转基因的新生广西巴马小型猪肾成纤维细胞。Micro-liquid tissue block adhering wall method was used for primary culture and isolation of new-born Bama mini-pigs kidney fibroblasts and routine subculture,freezing,and recovery procedure were performed.Contact inhibition and disinhibition were used to study the proliferative potential of the cells.Immunofluorescence was used to identify the cells.The isolated new-born Bama mini-pigs kidney fibroblasts were used for liposome-mediated EGFP-N1 and DsRed-N1 gene transfection.The results showed that new-born Bama mini-pigs kidney fibroblasts were successfully isolated and could be cultured to a large number of passage cells and frozen.Then they had been cultured for more than 50 passages and all the passage cells grew well.The cells could still be cultured and subcultured after frozen-thawing and disinhibition.Cell immunofluorescence showed that vimentin expression was positive and cytoceratin was negative.At 48 h after transfection,green fluorescence could be observed under fluorescent microscopy,suggesting that it was possible to transiently transfect renal fibroblasts.

关 键 词：广西巴马小型猪 肾成纤维细胞 细胞培养 

分 类 号：S814.8[农业科学—畜牧学]