摘要
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 a recombinant pcDNA3, l-vimentin and transfect it into human bladder carcinoma SVM780 cells, and investigate the effect of vimentin on the invasion of SW780 cells. Methods Human vimentin cDNA was amplified by PCR and inserted into vector pcDNA3, 1 （ - ）. After restriction analysis and sequencing, the recombinant plasmid pcDNA3, 1-vimentin was transfected into SW780 cells. The expressions of vimentin and matrix metalloproteinase 9 （MMP-9） were detected using Western blotting, and the secretion of MMP-9 in SW780 cells was tested using gelatin zymography. The invasion of SW780 cells was examined by TranswellTM assay. Results Enzyme digestion and sequencing analysis confirmed the successful cloning of pcDNA3. 1-vimentin. Western blotting demonstrated that both vimentin and MMP-9 were up-regulated in SW780 cells containing pcDNA3. 1-vimentin. Gelatin zymography indicated that vimentin increased MMP-9 secretion of SW780 cells. TranswellTM assay showed vimentin obviously promoted the invasion of SW780 cells. Conclusion Plasmid pcDNA3, 1-vimentin was constructed successfully and expressed in SW780 cells. Up-regulation of vimentin could promote the invasion of SW780 cells. Up-regulation and over-secretion of MMP-9 may be involved in vimentin-enhanced invasion of SW780 cells.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2014年第7期713-716,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
宁夏自然科学基金(NZ13137)
关键词
波形蛋白
膀胱癌细胞
SW780
侵袭转移
vimentin
bladder carcinoma cells
SW780 cells
invasion and metastasis
