在小鼠骨钙素启动子控制下，MC3T3-E1细胞中A20的过表达抑制了肿瘤坏死因子-α诱导的细胞凋亡。

A20 overexpression under control of mouse osteocalcin promoter in MC3T3-E1 cells inhibited tumor necrosis factor-alpha-induced apoptosis.

作者信息

Qin Yue-juan, Zhang Zhen-lin, Yu Lu-yang, He Jin-wei, Hou Ya-nan, Liu Tian-jin, Wu Jia-cai, Wu Song-hua, Guo Li-he

机构信息

Center for Preventing and Treating Osteoporosis, Osteoporosis Research Unit, Shanghai Sixth People's Hospital, Shanghai Jiao Tong University, Shanghai 200233, China.

出版信息

Acta Pharmacol Sin. 2006 Sep;27(9):1231-7. doi: 10.1111/j.1745-7254.2006.00403.x.

DOI:10.1111/j.1745-7254.2006.00403.x

PMID:16923345

Abstract

AIM

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 fibroblast 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.

摘要

目的

构建在小鼠骨钙素启动子（OC-A20）控制下的A20表达载体，并研究稳定过表达A20蛋白的成骨细胞MC3T3-E1细胞系对肿瘤坏死因子（TNF）-α诱导的细胞凋亡的影响。

方法

通过将小鼠骨钙素基因-2启动子片段与人A20互补DNA融合构建OC-A20载体。然后建立稳定转染A20的小鼠MC3T3-E1细胞系。分别通过逆转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹分析检测细胞中A20 mRNA和A20蛋白的表达。为了确定A20在成骨细胞中表达的特异性，用OC-A20瞬时转染小鼠成骨细胞MC3T3-E1细胞系和小鼠胚胎成纤维细胞NIH3T3细胞系。分别通过流式细胞术分析（FACS）、末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）和DNA凝胶电泳分析（DNA Ladder）确定A20在MC3T3-E1细胞中的抗凋亡作用。

结果

用小鼠A20引物在MC3T3-E1细胞中发现A20表达较弱。用RT-PCR和蛋白质免疫印迹分析在转染OC-A20的MC3T3-E1细胞中鉴定出A20 mRNA和A20蛋白表达。在MC3T3-E1细胞和NIH3T3细胞用OC-A20瞬时转染后，仅在MC3T3-E1细胞中发现A20 mRNA表达。通过FACS分析，与空载体（pcDNA3）组相比，OC-A20组细胞凋亡率明显降低（P<0.001）。与OC-A20组相比，pcDNA组TUNEL阳性染色显著增加（P<0.001）。同时，DNA凝胶电泳分析也显示出类似的结果。