摘要
正弦电磁场(SEMFs)能够显著促进大鼠成骨细胞(ROBs)成熟分化,但其作用机制未知。本研究旨在阐明BMP-Smad信号通路对SEMFs促进ROBs成熟分化的影响。取新生SD大鼠颅骨,多次酶消化体外分离培养得到ROBs传代培养后,利用50 Hz 1.8 m Ts SEMFs处理0,5,15,30和60min,Western印迹检测BMP-2表达和Smad1/5/8磷酸化水平,免疫荧光染色检测P-Smad1/5/8核转位。加入BMP-Smad信号通路的阻断剂noggin后,50 Hz 1.8 m Ts SEMFs分别处理3 d和6 d(2h/d)后,检测胞内碱性磷酸酶(ALP)活性,磁场处理2 d后(2 h/d),real-time PCR和Western印迹分别检测Ⅰ型胶原(collagen1)和成骨相关转录因子RUNX-2基因和蛋白质表达量。结果发现,50Hz 1.8 m Ts SEMFs处理ROBs后,BMP-2的表达量显著增加,胞内Smad1/5/8快速磷酸化,而对非磷酸化的Smad1/5/8表达无影响。同时,SEMFs处理30 min后引起P-Smad1/5/8发生核转位。50Hz 1.8m Ts SEMFs能够显著促进ALP活性增加,促进成骨相关因子collagen1和RUNX-2基因和蛋白质表达。加入BMP-Smad信号通路的阻断剂noggin后,SEMFs促进ALP活性增加,collagen1和RUNX-2基因和蛋白质表达水平被显著抑制。上述结果说明,50 Hz 1.8 m Ts SEMFs促进成骨细胞成骨性分化依赖于BMP-Smad信号通路。
Sinusoidal electromagnetic fields（ SEMFs） have been considered as a stimulator for rat osteoblasts（ ROBs） differentiation. However,the mechanism of the SEMFs-induced osteogenesis is still unknown. In this study,we examined the role of the BMP-Smad signaling pathway in SEMF-induced differentiation of primary ROBs. Cells were obtained by sequential enzyme digestion from the segregated skull of neonatal SD rats. The sub-cultured ROBs were treated in 50 Hz SEMFs at 1. 8 m Ts for 0,5,15,30,60 and 120 min before measuring for BMP-2,total or phospho-Smad1/5/8 proteins by Western blotting or immunofluorescence staining. Noggin 10 nmol/L was used to block the BMP2-Smad1/5/8signaling. Intracellular alkaline phosphatase（ ALP） activity was detected at 3 and 6 days after cells were treated by SEMFs for 120 min/day. The m RNA and protein expression levels of RUNX-2 and type I collagen were measured at day 2 by real-time PCR and Western blotting. The results showed that SEMFs promoted the expression of BMP-2 and rapidly induced the phosphorylation and nuclear translocation of Smad1/5/8 in ROBs. The treatments also significantly promoted ALP activity and the expression of RUNX-2 and type I collagen. The SEMFs-induced osteoblast gene expression could be blocked by noggin. Collectively,we demonstrated that SEMFs promoted osteoblast differentiation by modulating the BMP2-Smad1/5/8 signaling.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2016年第3期305-311,共7页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金项目(No.81270963和No.81471090)资助~~
