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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) can significantly promote the maturation and differentiation of rat osteoblasts (ROBs), but their mechanism of action is unknown. The purpose of this study was to elucidate the effect of BMP-Smad signaling pathway on the maturation of ROBs by SEMFs. The skull of newborn SD rats were isolated and cultured in vitro for several times. After subculturing ROBs, the cells were treated with 50 Hz 1.8 mS SEMFs for 0, 5, 15, 30 and 60 min. Western blotting was used to detect the expression of BMP-2 and Smad1 / 5 / 8 phosphorylation levels, P-Smad1 / 5/8 nuclear translocation was detected by immunofluorescence staining. After the addition of noggin, a blocker of BMP-Smad signaling pathway, alkaline phosphatase (ALP) activity was detected at 50 Hz and 1.8 mS SEMFs for 3 d and 6 d (2 h / d), respectively. (2 h / d). Real-time PCR and Western blot were used to detect the mRNA and protein expression of collagen type 1 and RUNX-2. The results showed that the expression of BMP-2 was significantly increased after treatment with 50 Hz 1.8 m SEM SEMs, and the phosphorylation of Smad1 / 5/8 was not affected by the rapid phosphorylation of Smad1 / 5/8. Meanwhile, SEMFs treated for 30 min caused nuclear translocation of P-Smad1 / 5/8. 50Hz 1.8m Ts SEMFs can significantly promote the increase of ALP activity and promote the expression of osteoblast-related factors collagen1 and RUNX-2 gene and protein. After addition of noggin, a blocker of BMP-Smad signaling pathway, SEMFs promoted ALP activity and inhibited the expression of collagen1 and RUNX-2 gene and protein. The above results show that 50 Hz 1.8 mTs SEMFs promote osteoblast differentiation depends on the BMP-Smad signaling pathway.