AKR1B10基因过表达载体的构建及其在乳腺癌MCF-7细胞中的表达

Construction of overexpression vector of AKR1B10 gene and expression in breast cancer MCF-7 cells

目的:构建AKR1B10基因真核表达载体pcDNA3.1(+)-AKR1B10,观察其在乳腺癌MCF-7细胞中的表达,为研究AKR1B10基因与乳腺癌的关系提供实验依据。方法:构建真核表达载体pcDNA3.1(+)-AKR1B10,将载体瞬时转染乳腺癌MCF-7细胞,利用RT-PCR与Western blotting方法检测未转染的MCF-7细胞、转染pcDNA3.1(+)-AKR1B10的MCF-7细胞和转染空质粒pcDNA3.1(+)的MCF-7细胞中AKR1B10基因的表达。结果:成功构建真核表达载体pcDNA3.1(+)-AKR1B10。RT-PCR结果显示,将未转染的MCF-7细胞中AKR1B10基因表达量设定为1,转染质粒的MCF-7细胞中AKR1B10基因表达量为1.79,转染空质粒的MCF-7细胞表达量为0.98,转染表达载体细胞AKR1B10基因表达量与转染空质粒的细胞和未转染的细胞比较,差异有统计学意义(P<0.05);Western blotting结果显示,与未转染的MCF-7细胞和转染空质粒的细胞比较,转染表达载体pcDNA3.1(+)-AKR1B10的MCF-7细胞中AKR1B10基因表达量增多。结论:成功构建真核表达载体pcDNA3.1(+)-AKR1B10,AKR1B10基因在转染表达载体的MCF-7细胞中高表达。

Objective To constructed an eukaryotic expression vector pcDNA3.1（＋）-AKR1B10 and observe its expression in breast cancer MCF-7 cells,and to provide experimental basis for study on the relationship between AKR1B10 gene and breast cancer.Methods The breast cancer MCF-7 cells were transfected transiently with the constructed eukaryotic expression vector pcDNA3.1（＋）-AKR1B10.The expressions of AKR1B10 gene in the untransfected MCF-7 cells,the MCF-7 cells transfected with pcDNA3.1（＋）-AKR1B10 and the MCF-7 cells transfected with pcDNA3.1（＋）（control vector） were detected by RT-PCR and Western blotting methods.Results The eukaryotic expression vector pcDNA3.1（＋）-AKR1B10 was successfully constructed.The RT-PCR results showed that if the AKR1B10 gene expression in the untransfected MCF-7 cells was regarded as 1,the expression values were 1.79 in the cells transfected with pcDNA3.1（＋）-AKR1B10 and 0.98 in the cells tranfected with pcDNA3.1（＋）.The expression of AKR1B10 gene in the cells transfected with pcDNA3.1（＋）-AKR1B10 was significantly increased compared with the cells transfected with pcDNA3.1（＋） and the untransfected cells（P〈0.05）.The Western blotting results showed that the expression of AKR1B10 gene in the cells transfected with pcDNA3.1（＋）-AKR1B10 was significantly increased compared with the cells transfected with pcDNA3.1（＋） and the untransfected cells.Conclusion The eukaryotic expression vector pcDNA3.1-AKR1B10 is successfully constructed.The AKR1B10 gene highly expresses in the MCF-7 cells transfected with the expression vectror.