多囊蛋白2对低频脉冲电磁场促进成骨细胞分化成熟的影响

Effect of polycystin2 on differentiation and maturation of osteoblasts promoted by low-frequency pulsed electromagnetic fields

摘要:

已知低频脉冲电磁场(pulsed electromagnetic fields,PEMFs)可以促进体外培养大鼠颅骨成骨细胞(rat calvarial osteoblasts,ROBs)的分化成熟，但ROBs感知PEMFs物理信号并启动成骨性分化的机制至今不明。本研究探究了0.6 mT 50 Hz PEMFs促进ROBs成骨性分化与位于ROBs表面的初级纤毛上的多囊蛋白2(polycystin2,PC2)的关系。首先用免疫荧光染色法研究了PC2是否位于ROBs初级纤毛内，然后通过Western blotting检测了经PEMFs处理不同时间后，ROBs内PC2蛋白表达的变化情况。接着用PC2阻断剂盐酸阿米洛利(amiloride HCl,AMI)预处理ROBs，检测碱性磷酸酶(alkline phosphatase,ALP)活性和PC2蛋白表达受PEMFs处理的影响情况，以及与骨形成相关的Runx-2、Bmp-2、Col-1、Osx的蛋白及基因表达的变化情况。再用RNA干扰法抑制ROBs内PC2的表达后，检测与骨形成相关基因表达情况。结果发现，PC2被定位于ROBs初级纤毛上，PEMFs处理增加了PC2蛋白表达量。PC2被AMI阻断后，PEMFs不再能提高PC2蛋白表达水平及ALP活性，PEMFs对成骨相关蛋白和基因表达的促进作用也被抵消。使用RNA干扰法抑制PC2的表达后，PEMFs也不再能提高骨形成相关基因的表达。结果表明：存在于成骨细胞初级纤毛表面的PC2在感知并传递PEMFs发出的物理信号中扮演着不可或缺的角色，PEMFs促进ROBs成骨性分化依赖于PC2的存在。本研究为阐明低频脉冲电磁场促进骨形成及治疗骨质疏松的机制研究奠定了基础。

Abstract:

It is known that low-frequency pulsed electromagnetic fields (PEMFs) can promote the differentiation and maturation of rat calvarial osteoblasts (ROBs) cultured in vitro.However,the mechanism that how ROBs perceive the physical signals of PEMFs and initiate osteogenic differentiation remains unknown.In this study,we investigated the relationship between the promotion of osteogenic differentiation of ROBs by 0.6 mT 50 Hz PEMFs and the presence of polycystin2(PC2) located on the primary cilia on the surface of ROBs.First,immunofluorescence staining was used to study whether PC2 is located in the primary cilia of ROBs,and then the changes of PC2 protein expression in ROBs upon treatment with PEMFs for different time were detected by Western blotting.Subsequently,we detected the expression of PC2 protein by Western blotting and the effect of PEMFs on the activity of alkaline phosphatase (ALP),as well as the expression of Runx-2,Bmp-2,Col-1 and Osx proteins and genes related to bone formation after pretreating ROBs with amiloride HCl (AMI),a PC2 blocker.Moreover,we detected the expression of genes related to bone formation after inhibiting the expression of PC2 in ROBs using RNA interference.The results showed that PC2 was localized on the primary cilia of ROBs,and PEMFs treatment increased the expression of PC2 protein.When PC2 was blocked by AMI,PEMFs could no longer increase PC2 protein expression and ALP activity,and the promotion effect of PEMFs on osteogenic related protein and gene expression was also offset.After inhibiting the expression of PC2 using RNA interference,PEMFs can no longer increase the expression of genes related to bone formation.The results showed that PC2,located on the surface of primary cilia of osteoblasts,plays an indispensable role in perceiving and transmitting the physical signals from PEMFs,and the promotion of osteogenic differentiation of ROBs by PEMFs depends on the existence of PC2.This study may help to elucidate the mechanism underlying the promotion of bone formation and osteoporosis treatment in low-frequency PEMFs.