The construction of the eukaryotic expression plasmid pcDNA3.1/azurin and the increased apoptosis of U2OS cells transfected with it.
The construction of the eukaryotic expression plasmid pcDNA3.1/azurin and the increased apoptosis of U2OS cells transfected with it.
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DOI：
10.2478/s11658-007-0012-3
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发表时间：
2007-09
影响因子：
8.3
通讯作者：
Yang DS
中科院分区：
文献类型：
--
作者：
Ye Z;Peng H;Fang Y;Feng J;Yang DS
在我们以前的研究中，我们证明了天青蛋白可以选择性地触发人骨肉瘤细胞系U2 OS细胞的凋亡。然而，凋亡率（35.8 ± 3.2%）不是很高，并且天青蛋白太昂贵而不易获得。为了解决这些问题，我们构建了带有流感病毒血凝素9肽HA表位标签的azurin基因真核表达质粒，并将重组质粒pcDNA3.1（+）/azurin转染U2 OS细胞。RT-PCR和Western blot分析证实转染成功，并表达了azurin-HA蛋白。流式细胞术（FCM）和DNA梯状条带实验（DNALadder test）检测转染细胞凋亡情况。细胞凋亡率达64.3 ± 13.1%。转染pcDNA3.1（+）/azurin后，U2 OS细胞Bax和p53基因转录水平显著升高，Bcl-2 mRNA水平显著降低。Bcl-xl mRNA和Survivin mRNA的表达无明显差异。结论：pcDNA3.1（+）/azurin重组质粒转染U2 OS细胞后，可明显诱导U2 OS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。
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.
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DOI：
10.1016/j.bbrc.2004.03.048
发表时间：
2004-04-30
影响因子：
3.1
作者：
Feng, J;Zhao, LH;Yu, QQ
通讯作者：
Yu, QQ
DOI：
10.1016/j.bbrc.2003.09.217
发表时间：
2003-12-05
影响因子：
3.1
作者：
Punj, V;Das Gupta, TK;Chakrabarty, AM
通讯作者：
Chakrabarty, AM
影响因子：
20.3
作者：
Tu, YP;Xu, FH;Lichtenstein, A
通讯作者：
Lichtenstein, A
影响因子：
64.5
作者：
WILSON, IA;NIMAN, HL;LERNER, RA
通讯作者：
LERNER, RA
DOI：
10.1073/pnas.94.21.11357
发表时间：
1997-10-14
影响因子：
11.1
作者：
Schlesinger, PH;Gross, A;Korsmeyer, SJ
通讯作者：
Korsmeyer, SJ