摘要
[目的]建立永生化绵羊瘤胃成纤维细胞系,为基础研究和动物病毒的研究提供稳定的体外细胞模型。[方法]采用组织块贴壁法培养绵羊瘤胃成纤维细胞(ovine ruminal fibroblasts cells,ORFCs),利用PEI试剂将带有hTERT基因的pCI-neo-hTERT质粒转染ORFCs,经G418筛选得到阳性克隆细胞。采用RT-PCR、Western Blot检测F15和F35代阳性克隆细胞的hTERT基因的转录和表达情况;利用血球计数法绘制原代ORFCs和F35代阳性克隆细胞的生长曲线比较其增殖能力。[结果]将hTERT基因成功转入ORFCs并稳定表达;阳性克隆细胞的增殖能力显著高于原代ORFCs。[结论]该试验成功建立了永生化绵羊瘤胃成纤维细胞系。
[Objective]To establish immortalized ovine ruminal fibroblast cell line and to provide stable in vitro cell model for basic research and animal virus research.[Method]Ovine ruminal fibroblast cells(ORFCs)were cultured by using tissue fragment clinging method.pCI-neo-hTERT plasmid with hTERT gene was transferred into ORFCs using PEI reagents.The positive clone cells were screened by G418.The transcription and expression level of hTERT gene in 15th and 35th generation positive clone cells were detected by RT-PCR and Western Blot.The growth curves of primary cells and 35th generation positive clone cells were drawn with blood cell count method to assess the proliferation ability.[Result]The hTERT gene was successfully transferred into ORFCs and stably expressed.The proliferation ability of positive clone cells was significantly higher than that of primary ORFCs.[Conclusion]Immortalized ovine ruminal fibroblast cell line is successfully established.
作者
赵霏霏
杨丹
辛雅明
杨银凤
ZHAO Fei-fei;YANG Dan;XIN Ya-ming;YANG Yin-feng(College of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot010018,China;Inner Mongolia Key Laboratory of Basic Veterinary Medicine,Hohhot010018,China)
出处
《畜牧与饲料科学》
2021年第3期1-6,共6页
Animal Husbandry and Feed Science
基金
国家自然科学基金项目(32060780)。
关键词
绵羊瘤胃成纤维细胞系
永生化
人源端粒酶逆转录酶
ovine ruminal fibroblast cell line
immortalization
human telomerase reverse transcriptase
