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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 establish murine melanoma B16 cell line stably co-expressing luciferase gene and human telomerase reverse transcriptase (hTERT), and to establish mouse lung metastasis model through tail vein injection. Methods The recombinant plasmid pIRES-neo-hTERT and pIRES-hyg3-Luc were constructed by inserting the hTERT gene and Luc gene into the eukaryotic expression vector by DNA recombination technology. The cotransfected mice were transfected with lipofectamineTM2000 Melanoma B16 cells were selected by G418 and hygromycin B for stable transfected cell lines. Western blot and immunofluorescence were used to detect the expression of hTERT and Luc genes in B16 cells. Male C57BL / 6 mice were inoculated with B16-hTERT / Luc cell line stably expressing hTERT and Luc through tail vein to establish tumors Lung metastasis model, and detect the growth of mouse lung tumor by live imaging technology. Results The murine melanoma B16 monoclonal cell line B16-hTERT / Luc stably co-expressing hTERT and Luc was established. The expression of hTERT gene and luciferase gene Luc was 84% and 98% respectively in the monoclonal cell line, . Mouse lung metastasis model was successfully established through tail vein injection. The growth of B16-hTERT / Luc tumor in mice could be detected easily by using live imaging technique. Conclusion Mouse hTERT metastasis model of mouse melanoma stably expressing hTERT was established successfully.