C57BL/6小鼠胚胎成纤维细胞的分离培养与纯化鉴定  

Isolation,culture,purification and identification of embryonic fibroblasts in C57BL /6 mouse

作　　者：王胜洁[1] 景瑾[1] 杨万路[1] 吴刘成[1,2] 邵义祥[1,2] 

机构地区：[1]南通大学实验动物中心,江苏南通226001 [2]南通大学比较医学研究所,江苏南通226001

出　　处：《黑龙江畜牧兽医》2015年第2期1-4,233,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(30671081);江苏省自然科学基金项目(BK2010279)

摘　　要：为了建立一种易于操作,能够获得高纯度、高活力C57BL/6小鼠胚胎成纤维细胞的分离培养体系,并对分离提取的细胞进行纯化鉴定,试验在无菌环境下取E12.5胎鼠皮肤,剪碎至1 mm2大小,均匀铺于皿中,用含10%胎牛血清的高糖DMEM培养基连续培养4 d,以胰蛋白酶消化-再贴壁法分离纯化目的细胞,分别以NIH3T3、角质形成性细胞作为阳性与阴性对照,采用免疫荧光、Western-blot检测Vimentin及Keratin的表达情况,以鉴定所分离的细胞及其纯度。结果表明:组织块培养24 h即可见成纤维样细胞爬出,培养96 h细胞生长爬满全皿,经胰酶消化、筛网过滤再贴壁接种后能够获得形态均一、活力旺盛、具有成纤维细胞形态学特征的目的细胞;所分离细胞Vimentin表达阳性率为100%,而Keratin表达阳性率为0;与阴性对照角质形成性细胞相比,C57BL/6分离成纤维细胞和NIH3T3细胞均高表达Vimentin,且差异极显著,有统计学意义。说明以E12.5胎鼠皮肤为材料建立成纤维细胞分离培养体系,所分离培养的成纤维细胞具有更高的细胞活力和细胞纯度。To establish an isolation and culture system which can operate easily and obtain high purity and activity embryonic fibroblasts in C57BL/6 （B6） mouse for purifying and identifying the isolated and extracted the cells. The skin of El2.5 feta/ mouse was collected in bacteria - free environment, and then cut into small pieces and spread into a culture dish, continuously cultured for 4 days in high - glucose Dulbecco＇ s Modified Eagle Medium （DMEM） supplemented with 10% fetal bovine serum. The objective cells were isolated and purified by trypsin digestion - adherence method. NIH3T3 ceils and keratinocytes were used as positive and negative controls, respectively, and the expres- sions of vimentin and keratin were detected by immunofluorescence and Western - blot to identify the isolated cells and their purification. The results showed that fibroblast - like cells climbed out using tissue block culture for 24 b, and then grew and covered with the dish using the cul- ture for 96 h. The objective cells were obtained through trypsin digestion, screen filtration, adherence and inoculation, which had uniform form with exuberant vitality and the morphological characteristics of fibroblasts. The positive rate of vimentin expression in the isolated cells was 100, while the positive rate of keratin expression was O. Compared with keratinocytes as negative control, vimentin was highly expressed in the isola- ted fibroblasts from C57BL/6 mouse and the NIH3T3 cells, and the difference was statistically extremely significant. The results indicate that the isolated and cultured fibroblasts have higher cell viability and purity using the isolation and culture system for fibroblasts established based on the skin of El2.5 fetal mouse.

关 键 词：C57BIM6小鼠 E12.5 胚胎成纤维细胞 分离培养 纯化鉴定 

分 类 号：S865.1[农业科学—野生动物驯养] Q786[农业科学—畜牧兽医]