A20 overexpression under control of mouse osteocalcin promoter in MC3T3-E1 cells inhibited tumor necrosis factor-alpha-induced apoptosis
A20 overexpression under control of mouse osteocalcin promoter in MC3T3-E1 cells inhibited tumor necrosis factor-alpha-induced apoptosis
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MC3T3-E1细胞中小鼠骨钙素启动子控制下的A20过表达抑制肿瘤坏死因子-α诱导的细胞凋亡
DOI：
10.1111/j.1745-7254.2006.00403.x
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发表时间：
2006
影响因子：
8.2
通讯作者：
Li
中科院分区：
文献类型：
--
作者：
Y. Qin;Zhen;Lu;Jin;Yanlin Hou;Tian;Jiacai Wu;Song;Li
目的：构建小鼠骨钙素基因启动子调控下的A20表达载体（OC-A20），并研究稳定过表达A20蛋白对肿瘤坏死因子（TNF）-α诱导的成骨细胞系MC 3 T3-E1凋亡的抑制作用。建立了稳定转染A20的小鼠MC 3 T3-E1细胞系。逆转录-聚合酶链反应（RT-PCR）检测A20 mRNA的表达，Western blot检测A20蛋白的表达。为了确定A20在成骨细胞中表达的特异性，将OC-A20瞬时转染小鼠成骨细胞系MC 3 T3-E1和小鼠胚胎成纤维细胞系NIH 3 T3。结果：A20在MC 3 T3-E1细胞中呈弱阳性表达，A20在MC 3 T3-E1细胞中呈弱阳性表达，A20在MC 3 T3-E1细胞中呈弱阳性表达。RT-PCR和Western blot检测转染OC-A20的MC 3 T3-E1细胞中A20 mRNA和蛋白的表达。用OC-A20瞬时转染MC 3 T3-E1细胞和NIH 3 T3细胞后，仅在MC 3 T3-E1细胞中检测到A20 mRNA的表达。流式细胞仪检测OC-A20组细胞凋亡率明显低于空载体组（P<0.001）。与OC-A20组相比，pcDNA组TUNEL阳性染色明显增加（P<0.001）。结论：我们构建了成骨细胞特异性表达A20蛋白的真核表达载体，在MC 3 T3-E1细胞中表达A20蛋白，证实A20蛋白对TNF-α诱导的成骨细胞凋亡具有保护作用。
AbstractAim:To construct an A20 expression vector under the control of mouse osteocalcin promoter (OC-A20), and investigate osteoblastic MC3T3-E1 cell line, which stably overexpresses A20 protein prevented tumor necrosis factor (TNF)-alpha-induced apoptosis.Methods:OC-A20 vector was constructed by fusing a fragment of the mouse osteocalcin gene-2 promoter with human A20 complementary DNA. Then the mouse MC3T3-E1 cell line, stably transfected by A20, was established. The expression of A20 mRNA and A20 protein in the cells were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. To determine the specificity of A20 expression in osteoblast, the mouse osteoblastic MC3T3-E1 cell line and mouse embryo fibro-blast NIH3T3 cell line were transiently transfected with OC-A20. The anti-apoptotic role of A20 in MC3T3-E1 cells was determined by Flow cytometric analysis (FACS), terminal dUTP nick endo-labeling (TUNEL) and DNA gel electrophoresis analysis (DNA Ladder), respectively.Results:Weak A20 expression was found in MC3T3-E1 cells with the primers of mouse A20. A20 mRNA and A20 protein expression were identified in MC3T3-E1 cells transfected with OC-A20 using RT-PCR and Western blot analysis. Only A20 mRNA expression was found in MC3T3-E1 cell after MC3T3-E1 cells and NIH3T3 cells were transient transfected with OC-A20. A decrease obviously occurred in the rate of apoptosis in the OC-A20 group compared with the empty vector (pcDNA3) group by FACS (P<0.001). A significant increase in TUNEL positive staining was found in the pcDNA group compared with OC-A20 group (P<0.001). Simultaneously, similar effects were demonstrated in DNA gel electrophoresis analysis.Conclusion:We constructed an osteoblast-specific expression vector that expressed A20 protein in MC3T3-E1 cells and confirmed that A20 protects osteoblast against TNF-alpha-induced apoptosis.
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影响因子：
15.9
作者：
Plotkin, LI;Weinstein, RS;Bellido, T
通讯作者：
Bellido, T
影响因子：
56.9
作者：
Lee, EG;Boone, DL;Ma, A
通讯作者：
Ma, A
A20 在细胞凋亡和坏死细胞死亡中的功能二分法。
DOI：
10.1042/bj20041443
发表时间：
2005
期刊：
The Biochemical journal
影响因子：
--
作者：
Storz,Peter;Döppler,Heike;Ferran,Christiane;Grey,ShaneT;Toker,Alex
通讯作者：
Toker,Alex
影响因子：
20.3
作者：
Daniel, S;Arvelo, MB;Ferran, C
通讯作者：
Ferran, C
影响因子：
15.9
作者：
Weinstein, RS;Jilka, RL;Manolagas, SC
通讯作者：
Manolagas, SC