nm23-H1转染Tca8113细胞后稳定表达细胞株的建立  

Establishment of a cell line of Tca8113 with stable expression of nm23-H1 by gene transfection

作　　者：郅克谦[1] 陈伟辉[2] 温玉明[3] 

机构地区：[1]西安交通大学口腔医院颌面外科,710004 [2]福建医科大学附属协和医院颌面外科 [3]四川大学华西口腔医院颌面外科

出　　处：《实用口腔医学杂志》2005年第6期762-766,共5页Journal of Practical Stomatology

基　　金：国家自然科学基金(编号39870746);福建自然科学基金重点项目(编号C0220002)资助项目

摘　　要：目的:建立稳定高表达nm23-H1的细胞株。方法:将nm23-H1真核表达质粒转化大肠杆菌,制备感受态细菌。提取质粒,酶切琼脂糖电泳对质粒进行鉴定。脂质体介导将质粒转染人舌鳞癌细胞株Tca8113,G418持续筛选5周,对获得的细胞株进行免疫组化、Western-blott和流式细胞仪鉴定。结果:酶切琼脂糖电泳证实插入nm23-H1片段长度为986bp,质粒片段长度为6550bp,nm23-H1的cDNA被插入在pCMV-Bam-Neo中的BamH1区域。免疫组化、Western-blott和流式细胞仪鉴定表明nm23-H1高表达。结论:成功建立稳定高表达nm23-H1的细胞株。Objective：To establish a tongue cancer celt line that stably overexpresses nm23-H1. Methods：The reconstructed plasmid, pCMV-BamH1-Neo-nm23-H1 , was transfected into bacteria and amplified. The plasmid was identified by electrophoresis on a 10 g/L agarose gel and stained with ethidium bromide anti then transfected to the cell line of Tea8113 by lipofectin strategy and was selected by G418 for 5 weeks. The stable expression of nm23-H1 was identified by immunofluorescence,Western-blotting and flow cytometer. Results：Restriction endonuclease Barn H1 examination showed that 986 bp of nm23-H1 was in pCMV-Bam-Neo vector. 5 weeks after transfection positive clones were obtained and the transfected cells were proliferated to confluent. Immunohistochemical examination, Western blot and flow cytometry showed that nm23-H1 expression was increased in the transfected cells. Conclusion： A tongue cancer cell line expressing nm23-H1 is established.

关 键 词：NM23-H1 质粒 转染 细胞株 

分 类 号：R739.8[医药卫生—肿瘤]