南阳牛成纤维细胞的体外培养及生物学特性研究

Culture and Biological Characteristics of Nanyang Bovine Fibroblasts

建立了南阳牛成纤维细胞系,旨在为制备转基因克隆牛提供核供体细胞。以南阳牛耳缘组织作为试验材料,通过组织块法培养分离南阳牛成纤维细胞,并进行生物学特性分析。结果表明,含有15%FBS的DMEM/F12培养基较适合原代牛成纤维细胞的体外培养。分析了细胞冻存前和复苏后的活率,分别为95.9%和90.2%,差异不显著;生长曲线表明细胞生长正常。南阳牛成纤维细胞传至第6代时,细胞核型正常。流式细胞仪检测结果显示,G0-G1期的F4和F8代细胞占细胞总数的64.35%和64.53%,F11代细胞占细胞总数的81.90%。在S期,F4代细胞占细胞总数的30.69%,F11代细胞占细胞总数的16.37%。结果表明,所培养的南阳牛成纤维细胞可作为体细胞核移植的供体细胞进行转基因克隆牛方面的研究。

The purpose of this study was to preprare transgenic donor cells for somatic cell nuclear transfer by establishing bovine fibroblasts,based on culturing Nanyang fibrocystic successfully and detecting its biological character.The results demonstrated that DMEM/F12 containing 15% FBS is suitable for primary cell cultivation.Meanwhile,Nanyang fibrocystic was detected regularly by growth curve.Cell activity difference between cell freezing and cell reviving is not significant.Cell karyotype is normal when subcultured for six passages.Flow cytometric test results showed that G0-G1 phase of F4 and F8 generation cell increased by 17% to 20%.Compared to the S phase,F4 and F8 generation cell decrease 1% to 4% than that of F11 generation cells.The results indicated that the fibroblast cells obtained may be competent for bovine transgenic cloning.