Snail基因慢病毒载体的构建及其促进结肠癌细胞上皮间质化的研究

Establishment of Snail Gene Lentiviral Vector and Its Effect in Epithelial-Mesenchymal Transition of Colon Cancer Cells

【目的】构建Snail基因慢病毒载体,研究Snail基因的表达与结肠癌细胞上皮间质化的关系。【方法】利用Gateway Technology法构建Snail基因表达质粒和空白对照质粒,行慢病毒包装。成功构建Snail基因慢病毒载体后,在结肠癌细胞系caco2中转染Snail基因质粒及空白载体对照,构建稳转细胞株caco2-Snail和caco2-vc,并在LoVo细胞中瞬时转染上述质粒,对Snail及E-钙黏蛋白(E-cadherin)的表达进行检测,并用划痕、Tanswell实验对细胞的迁移侵袭能力进行研究。【结果】成功构建Snail基因慢病毒载体并经测序证实。在转染Snail的细胞株中有明显的Snail基因及蛋白表达,其上皮标记E-cadherin的表达比转染空载体的对照组细胞明显下调,且其迁移侵袭能力要强于对照组细胞。【结论】Snail基因慢病毒载体在宿主细胞中表达稳定可靠,可以作为研究上皮间质化很好的工具,Snail基因有明显促进结肠癌细胞上皮间质化的作用。

[ Objective ] To establish snail gene lentiviral vector and explore its effect in EMT （epithelial-mesenchymal transition） of caco2 colon cancer cells. [ Methods ] According to the principle of Gateway technology, the snail expressing plasmid was established and imbedded into lentiviral vector. We transfected Lenti-hSnaill-eGFP/Neo and Lenti-eGFP/Neo （vector control） into caco2 colon cancer cells, thus, two stable cells were established ： caco2-snail and caco2-vc. To validate the effect of snail expressing plasmid in other colon cancer cell lines, we also performed transient transfection of snail expressing vector and empty vector in LoVo cells. The expression level of Snail and E-cadherin were detected in these cell lines. Wound-healing assay and transwell chamber assay were performed to evaluate the migration and invasive ability of these cells. [Results] Snail gene lentiviral vector was successfully established and was proved by gene sequencing. Significant gene and protein expression of snail can be detected in snail expressing cells. The levels of E-cadherin expression was obviously down-regulated in snail expressing cells compared with control cells. Snail promoted migration and invasion of colon cancer cells in wound-healing assay and transwell chamber assay. [ Conclusion ] Snail gene lentiviral vector is a stable and reliable tool for investigating EMT, and snail regulated the EMT of colon cancer cells by promoting cellular migration and invasion.