东方田鼠胚胎成纤维细胞永生化细胞系的建立  

Establishment of an Immortalized Microtus fortis Embryonic Fibroblast Cell Line

作　　者：成钢[1] 罗赛群[1] 盖楠[1] 熊德慧[1] 李荣[1] 胡维新[1] 

机构地区：[1]中南大学生物科学与技术学院分子生物学研究中心,长沙410078

出　　处：《中国实验动物学报》2010年第4期292-295,I0003,共5页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家自然科学基金项目(30400256);湖南省研究生科研创新项目(No.2340-74236000001)资助

摘　　要：目的建立东方田鼠胚胎成纤维永生化细胞系,为全面研究东方田鼠抗日本血吸虫机制以及开展不同动物成纤维细胞间比较研究奠定基础和提供细胞实验材料。方法运用脂质体介导的基因转染法将pSV3neo质粒导入第3代东方田鼠胚胎成纤维细胞,经G418筛选抗性克隆并扩大培养,建立永生化细胞系;用PCR检测细胞株中SV40T基因的整合,RT-PCR鉴定SV40T基因在转染细胞中的表达;绘制东方田鼠胚胎成纤维永生化细胞生长曲线。结果阳性细胞克隆已扩大培养并稳定传代50代,经鉴定SV40T抗原已整合到东方田鼠胚胎成纤维细胞中且稳定表达。结论成功建立东方田鼠胚胎成纤维永生化细胞系。Objective To establish an immortalized Microtus fortis embryonic fibroblast （MfEF） cell line in order to lay the foundation for further studies on Microtus fortis against Schistosma japonicum infection and for comparative study of fibroblasts from different animals. Methods A mammalian expression vector （ pSV3 neo） containing the SV40 large T antigen was used to transfect the 3rd passage MfEF cells using Lipofectamine^TM 2000. Colonies were screened by G418 and expanded into immortalized cell lines. PCR was used to detect the integration of the large T antigen with genome in the Microtus fortis embryonic fibroblasts. Expression of SV40 large T antigen gene in the expanded cells was identified by RTPCR. Results Stable growth and serial propagation were observed in the immortalized Microtus fortis embryonic fibroblast line. The mRNA of SV40 T antigen gene was expressed in cells of the immortalized cell line. Conclusion An immortalized cell line of Microtus fortis embryonic fibroblasts has been successfully established.

关 键 词：东方田鼠 胚胎成纤维细胞 永生化 SV40T抗原 

分 类 号：Q25[生物学—细胞生物学]