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目的构建人波形蛋白(vimentin)基因的真核表达载体,转染SW780人膀胱癌细胞,检测vimentin上调对SW780细胞侵袭能力的影响。方法 PCR扩增人vimentin基因的cDNA序列,将其插入pcDNA3.1真核表达载体中,酶切鉴定并测序验证重组质粒pcDNA3.1-vimentin后,转染人膀胱癌SW780细胞。Western blot法检测vimentin及基质金属蛋白酶9(MMP-9)的表达,明胶酶谱分析MMP-9活性,TranswellTM实验检测vimentin对膀胱癌细胞侵袭能力的影响。结果酶切鉴定有预期目的片段,测序证实重组质粒pcDNA3.1-vimentin构建成功。Western blot法检测提示pcDNA3.1-vimentin转染SW780细胞后,vimentin与MMP-9表达水平均上调。明胶酶谱表明vimentin能够显著促进SW780细胞MMP-9的分泌。TranswellTM实验提示vimentin能显著增强SW780细胞的侵袭转移能力。结论成功构建了pcDNA3.1-vimentin真核表达载体并将其成功转染膀胱癌SW780细胞。Vimentin上调能够增强SW780细胞的侵袭能力,可能与上调MMP-9的表达及促进MMP-9分泌有关。
Objective To construct an eukaryotic expression vector of human vimentin gene and transfect SW780 human bladder cancer cells to investigate the effect of vimentin up-regulation on invasiveness of SW780 cells. Methods The cDNA of human vimentin gene was amplified by PCR and inserted into pcDNA3.1 eukaryotic expression vector. The recombinant plasmid pcDNA3.1-vimentin was identified by restriction analysis and transfected into human bladder cancer SW780 cells. The expression of vimentin and matrix metalloproteinase 9 (MMP-9) were detected by Western blot. The activity of MMP-9 was analyzed by gelatin zymography. The effect of vimentin on the invasiveness of bladder cancer cells was detected by TranswellTM assay. Results The target fragment was identified by restriction enzyme digestion and sequencing confirmed that the recombinant plasmid pcDNA3.1-vimentin was successfully constructed. The results of Western blot showed that the expression of vimentin and MMP-9 were upregulated after transfection of pcDNA3.1-vimentin in SW780 cells. Gelatin zymography showed that vimentin can significantly promote MMP-9 secretion in SW780 cells. TranswellTM experiments suggest that vimentin can significantly enhance the invasion and metastasis of SW780 cells. Conclusion The eukaryotic expression vector pcDNA3.1-vimentin was successfully constructed and transfected into SW780 bladder cancer cell line successfully. Upregulation of Vimentin can enhance the invasion of SW780 cells, which may be related to up-regulating the expression of MMP-9 and promoting the secretion of MMP-9.