摘要
该文主要探讨p38 MAPK信号通路在人骨形态发生蛋白-2(BMP-2)诱导人牙囊细胞成骨分化中的作用。从人成骨肉瘤细胞株MG-63中克隆hBMP-2基因,通过基因重组技术构建pcDNA3.1(+)-hBMP-2真核表达质粒,采用脂质体法转染人牙囊细胞,以G418筛选得到能够稳定表达BMP-2的细胞克隆。Quantitative Real-time RT-PCR及Western blot检测BMP-2目的基因和蛋白的表达。以p38 MAPK特异性抑制剂SB203580作用于稳定表达BMP-2的人牙囊细胞,观察MAPKAP激酶-2磷酸化蛋白、碱性磷酸酶活性及成骨相关基因表达水平变化。结果显示,通过基因重组和基因转染获得能够稳定表达BMP-2的人牙囊细胞。SB203580明显降低MAPKAP激酶-2磷酸化蛋白的表达水平。BMP-2水平持续高表达能诱导人牙囊细胞碱性磷酸酶活性升高,同时增强OSX、Runx2、OCN、BSP及OPN等成骨相关基因的表达水平,但BMP-2诱导人牙囊细胞成骨分化的作用被SB203580抑制。这表明,p38 MAPK信号通路在BMP-2诱导人牙囊细胞成骨分化中发挥调控作用。
The aim of this paper is to study whether p38 MAPK signaling pathway is involved in BMP-2-induced osteogenic differentiation of human dental follicle cells （HDFC）. hBMP-2 gene was cloned from human osteosarcoma cell line MG-63. The eukaryotic plasmid pcDNA3.1 （＋）-hBMP-2 was constructed by recombinant DNA technology and transduced into HDFC by liposome-mediated cellular delivery. After selection with G418, the resistant clones which can stably overexpress the exogenous BMP-2 were obtained. The expression levels of BMP-2mRNA and protein were determined by quantitative real-time RT-PCR and Western blot respectively. The effect of p38 MAPK specific inhibitor SB203580 on the expression of phospho-MAPKAP kinase-2 protein was examined. Finally, the effect of SB203580 on BMP-2-induced ALP activity and expression of osteogenesis-related genes were investigated. The results showed that HDFC which can stably overexpress the exogenous BMP-2 were obtained by gene recombination and transfection. SB203580 could significantly decrease the protein level of phospho-MAP-KAP Kinase-2. Overexpression of BMP-2 could increase ALP activity and enhance the expression of osteogenesisrelated genes such as OSX, Runx2, OCN, BSP and OPN. However, the effect of BMP-2-induced osteogenic differentiation was inhibited by SB203580. These findings suggest that p38 MAPK signaling pathway is involved in BMP-2-induced osteogenic differentiation of HDFC.
出处
《中国细胞生物学学报》
CAS
CSCD
北大核心
2013年第6期816-823,共8页
Chinese Journal of Cell Biology
基金
国家自然科学基金(批准号:81100752)
浙江省自然科学基金(批准号:LQ12H14001)
浙江省医药卫生一般研究计划(批准号:2012KYA126)
浙江省中医药科学研究基金计划(批准号:2012ZB101)资助的课题~~
