Department of Orthopedics, Second Affiliated Hospital, Medical College, Zhejiang University, 88 Jie Fang Road, Hangzhou, 310009, Zhejiang, P.R. China.
Cell Mol Biol Lett. 2007 Sep;12(3):407-21. doi: 10.2478/s11658-007-0012-3. Epub 2007 Apr 6.
In our previous study, we demonstrated that azurin could selectively trigger apoptosis in human osteosarcoma cell line U2OS cells. However, the rate of apoptosis (35.8 +/- 3.2%) is not very high, and azurin is too expensive to obtain readily. To solve these problems, we constructed a eukaryotic expression plasmid containing the azurin gene with an influenza virus haemagglutinin 9 peptide HA epitope tag, and transfected the recombinant plasmid pcDNA3.1(+)/azurin into U2OS cells. RT-PCR and Western blot analysis validated the successful transfection and the expression of the azurin-HA protein. Conspicuous apoptosis of the transfected cells was detected by flow cytometry (FCM) and the DNA ladder test. The apoptosis rate reached 64.3 +/- 13.1%. The transcriptional levels of the Bax and p53 genes increased significantly in U2OS cells transfected with pcDNA3.1(+)/azurin, but the Bcl-2 mRNA level decreased. There was no difference in the levels of Bcl-xl mRNA and Survivin mRNA. We propose that the transfection of the recombinant plasmid pcDNA3.1(+)/azurin can significantly induce apoptosis in U2OS cells. This is closely associated with the up-regulation of the transcriptional level of the Bax and p53 genes, and the down-regulation of that of the Bcl-2 gene.
在我们之前的研究中，我们证明了蓝铜蛋白可以选择性地诱导人骨肉瘤细胞系 U2OS 细胞凋亡。然而，凋亡率（35.8±3.2%）并不是非常高，而且蓝铜蛋白太贵，不易获得。为了解决这些问题，我们构建了一个含有蓝铜蛋白基因的真核表达质粒，带有流感病毒血凝素 9 肽 HA 表位标签，并将重组质粒 pcDNA3.1(+)/azurin 转染到 U2OS 细胞中。RT-PCR 和 Western blot 分析验证了转染的成功和 azurin-HA 蛋白的表达。通过流式细胞术（FCM）和 DNA 梯带试验检测到转染细胞的明显凋亡。凋亡率达到 64.3±13.1%。转染 pcDNA3.1(+)/azurin 的 U2OS 细胞中 Bax 和 p53 基因的转录水平显著增加，但 Bcl-2 mRNA 水平下降。Bcl-xl mRNA 和 Survivin mRNA 的水平没有差异。我们提出，转染重组质粒 pcDNA3.1(+)/azurin 可以显著诱导 U2OS 细胞凋亡。这与 Bax 和 p53 基因转录水平的上调以及 Bcl-2 基因转录水平的下调密切相关。
