摘要
目的建立稳定共表达荧光素酶基因和人端粒酶逆转录酶(hTERT)的小鼠黑色素瘤B16细胞系,并通过尾静脉注射的方式建立小鼠肿瘤肺转移模型。方法利用DNA重组技术将hTERT基因和荧光素酶基因Luc定向插入到真核表达载体,构建真核表达质粒pIRES-neo-hTERT和pIRES-hyg3-Luc,利用阳离子脂质体LipofectamineTM2000共转染小鼠黑色素瘤B16细胞,经G418及潮霉素B加压筛选出稳定转染的细胞株。应用Western blot法及免疫荧光法检测hTERT和Luc基因在B16细胞中的表达;将稳定共表达hTERT和Luc的B16-hTERT/Luc细胞株通过尾静脉注射的方式接种雄性C57BL/6小鼠建立肿瘤肺转移模型,并通过活体成像技术检测小鼠肺部肿瘤的生长。结果建立了稳定共表达hTERT和Luc的小鼠黑色素瘤B16单克隆细胞株B16-hTERT/Luc,经检测hTERT基因和荧光素酶基因Luc在单克隆细胞株中的表达分别为84%和98%。通过尾静脉注射的方式成功建立了小鼠肿瘤肺转移模型,应用活体成像技术能方便地检测到B16-hTERT/Luc肿瘤在小鼠体内的生长情况。结论成功建立了可用于活体成像技术检测的稳定表达hTERT的小鼠黑色素瘤肺转移模型。
Objective To construct the eukaryotic expression vectors of human telomerase reverse transcriptase （hTERT） and luciferase （Luc） gene, and stably transfect B16 cell line with them, and inoculate mice via caudal vein to construct pulmonary metastasis models. Methods The hTERT and Luc cDNA fragments were inserted into eukaryotic expression vectors to obtain the recombinant plasmids plRES-neo-hTERT and plRES-hyg3-Luc. The plasmids were then transfected into B16 cells by LipofectamineTM 2000. Afte the stably transfected cell line was screened by G418 and hygromycin B, the transcription and expression of the hTERT and Luc genes were identified by Western blotting and immunofluorescence staining. The stably transfected cells were inoculated via caudal vein into male C56BL/6 mice to construct pulmonary metas- tasis models. An in vivo imaging system was used to monitor the growth and metastasis of the tumors. Results A stably transfected cell line was established and the express rates of hTERT and Luc gene in the stable cell line were 84% and 98%, respectively, Pulmonary metastasis models were successfully established and the conditions of the lung metastasis could be detected easily using in vivo imaging system. Conclusion The pulmonary metastasis models with stably expressed hTERT for in vivo imaging system in mice has been successfully established.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2013年第1期89-92,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(30840094)
关键词
荧光素酶
HTERT
活体成像
移植瘤
luciferase
hTERT
in vivo imaging system
transplanted tumors
