双峰驼胎儿耳缘组织成纤维细胞系的建立及其生物学特性鉴定.PDF

兽类学报,2012,32 (4):340 - 345 Acta Theriologica Sinica 双峰驼胎儿耳缘组织成纤维细胞系的 建立及其生物学特性鉴定  刁睿鑫 张勇 胡俊杰 常卫华 林涛 张志强 徐燕霞 胡少辉 张连梅 赵娜 (甘肃农业大学动物医学院, 兰州 730070 ) 摘要:本研究旨在建立双峰驼胎儿耳缘组织成纤维细胞系。采集双峰驼胎儿耳缘组织,用植块培养法和消化分 离法对其进行原代培养,差速消化和差速贴壁法进行继代培养和细胞纯化,并对培养细胞的形态、 细胞活力、 生长动力学、免疫组织化学、核型、绿色荧光蛋白基因质粒 (GFP )转染表达以及微生物污染等生物学特性进 行了分析。结果表明,原代和传代细胞形态正常,群体倍增时间 (PDT) 为47 2 h,细胞生长较快;不同代数的 染色体众数为2n = 74 ;免疫组织化学鉴定波形蛋白抗体呈阳性,角蛋白抗体呈阴性;外源质粒能在该细胞内整 合和正确表达;微生物污染检测呈阴性。 因此该研究已成功建立了双峰驼胎儿耳缘组织成纤维细胞系,在细胞 水平上保存了双峰驼这一国家重要种质资源,为基因组文库和体细胞克隆等研究提供了理想的生物材料。 关键词:双峰驼;耳缘组织;成纤维细胞系;生物学特性 中图分类号:Q24 文献标识码:A 文章编号:1000 - 1050 (2012)04 - 0340 - 06 Establishment and characteristics of a Bactrian camel fetal ear marginal fibroblast cell line  DIAO Ruixin, ZHANG Yong , HU Junjie, CHANG Weihua, LIN Tao, ZHANG Zhiqiang,XU Yanxia,HU Shaohui,ZHANG Lianmei,ZHAO Na (College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070 , China) Abstract :The experiment was conducted to establish a Bactrian camel (Camelus bactrianus)fetal ear marginal fibroblast cell line. The Bactrian camel fetal ear marginal tissue was cultured with explants or trypsinization,which then was subcul- tured and purified through trypsin digestion and differential anchoring velocity. Analysis on cell morphology,cell viability, growth dynamics,immunocytochemistry,karyotype,transfection and expression of green fluorescent protein marker plas- mid (GFP),and microbial contamination were carried out. The results showed that the population doubling time (PDT ) of the cells was 47 2 h ;the majority chromosome number was 74 ;the immunocytochemistry was