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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 construct the B16 cell line which stably overexpresses and stably interfered with Nodal melanoma cells, and to identify its EMT phenotype and to investigate the mechanism and mechanism of Nodal-induced melanoma EMT. Methods: The plasmid pL-tdTomatomNodal, which overexpressed mouse Nodal gene, and pGFP-V-RS-Nodal, a shRNA plasmid carrying the Nodal gene sequence, were transfected into B16 cells. Through resistant screening enrichment, positive clone selection and expansion culture, stable transfected cell lines B16 / dT-mNodal and B16 / sh-Nodal were obtained. Real-time fluorescence quantitative PCR and Western blot were used to detect the overexpression and knockout of Nodal and the expression of EMT marker. Results: Both of the two cell lines were successfully constructed. The B16 / dT-mNodal cell line developed strong red fluorescence, and the intracellular Nodal level was significantly up-regulated and showed stromal cell characteristics. The B16 / sh-Nodal cell line emitted intense green fluorescence and intracellular Nodal Significant down-regulation and epithelial cell characterization. CONCLUSION: B16 cell line stably overexpressing Nodal and interfering with Nodal has been successfully constructed and a model of Nodal affecting the EMT process in B16 cells has been established. It provides an important experimental tool for studying the role of Nodal in melanoma EMT.