摘要
目的:构建稳定过表达及稳定干扰Nodal的黑色素瘤细胞B16细胞株,并鉴定其EMT表型,用于研究Nodal诱导黑色素瘤EMT的现象及机理。方法:将过表达小鼠Nodal基因的质粒pL-tdTomatomNodal,及携带有干扰Nodal基因序列的shRNA质粒pGFP-V-RS-Nodal,分别转染B16细胞。通过抗性筛选富集,阳性克隆挑选及扩大培养,获得稳定转染细胞株B16/dT-mNodal及B16/sh-Nodal。通过实时荧光定量PCR和Western blot技术检测胞内Nodal的过表达及敲除情况和EMT标记物的表达情况。结果:两株细胞均构建成功,B16/dT-mNodal细胞株发出强烈红色荧光,胞内Nodal水平上调明显,并呈现间质细胞特性;B16/sh-Nodal细胞株发出强烈绿色荧光,胞内Nodal水平下调明显,并呈现上皮细胞特性。结论:成功构建稳定过表达Nodal及稳定干扰Nodal的B16细胞株,并构建Nodal影响B16细胞EMT过程的模型,为研究Nodal在黑色素瘤EMT过程中的作用提供了重要的实验工具。
Objective： To generate two B16 melanoma cell lines, stably overexpress or silence Nodal gene and identified their EMT markers for study of phenotypic changes and molecular mechanisms of Nodal induced EMT in melanoma. Methods： The mouse melanoma B16 cells were transfected with recombinant plasmid pL- tdTomato-mNodal or shRNA plasmid pGFP-V-RS-Nodal respectively. The stable cell lines B16/dT-mNodal and B16/sh-Nodal were obtained after resistance screening and enrichment, positive clone selection and expasion. The intracellular gene and protein levels of Nodal and EMT markers in these two cell lines were detected by qRT- PCR and Western blotting. Results： Both stable cell lines were constructed successfully. B16/dT-mNoda cell line exhibited strong red fluorescence with obvious up-regulated of Nodal mRNA and protein and Showed the characteristics of mesenchymal cell. While B16/sh-Nodal cell line displayed strong green fluorescence, as well as notable down-regulated Nodal mRNA and protein levels and Showed the characteristics of epithelial cell. Conclusion： The Nodal stably overexpressing or silencing cell lines B16/dT-mNodal and B16/sh-Nodal have been constructed successfully, providing important experimental tools for the study of Nodal function in the process of melanoma EMT.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2014年第3期1-8,共8页
China Biotechnology
基金
国家重点基础研究发展计划(2011CB935800)
国家自然科学基金(81272311
81071712)资助项目
