摘要
目的本文探讨上皮钠离子通道(ENa C)对破骨细胞功能和活性的影响。方法采用大鼠巨噬细胞集落刺激因子和核转录因子-κB受体活化因子配体诱导大鼠骨髓单核细胞使其分化为破骨细胞。以1.5×104密度接种到12孔板,查随机表分3孔为1组,分为4组:Control组和不同浓度阿米洛利(Ami,ENa C的抑制剂)组。抗酒石酸酸性磷酸酶(TRAP)染色进行阳性破骨细胞鉴定;将破骨细胞和骨片共同培养,测定骨吸收陷窝的数目;用RT-PCR技术分析破骨细胞标志酶基因组织蛋白酶K(CK)的表达。结果不同浓度的Ami处理破骨细胞后,TRAP染色阳性破骨细胞减少,抑制破骨细胞的形成和骨吸收,而且降低破骨细胞特异性基因CK的表达。结论本次实验在细胞水平证明ENa C在破骨细胞上的表达并且调控破骨细胞的分化和骨吸收,说明ENa C可能参与破骨细胞的功能调节,提示破骨细胞可能存在一个与ENa C相关调节的新途径,为骨代谢的研究提供了一个新思路。
Objective To explore the role of epithelial sodium channel(ENa C) in regulating the functional activity of osteoclasts.Methods Multinucleated osteoclasts were obtained by inducing the differentiation of rat bone marrow cells with macrophage colony-stimulating factor(M-CSF) and RANKL. The osteoclasts were exposed to different concentrations of the ENa C inhibitor amiloride, and the expression of ENa C on osteoclasts was examined using immunofluorescence technique. The osteoclasts were identified with tartrate-resistant acid phosphatase(TRAP) staining, and the positive cells were incubated with fresh bovine femoral bone slices and the number of bone absorption pits was counted by computer-aided image processing. RT-PCR was performed to analyze the expression of cathepsin K in the osteoclasts. Results Exposure to different concentrations of amiloride significantly inhibited the expression of ENa C and reduced the number of TRAP-positive osteoclasts. Exposure of the osteoclasts to amiloride also reduced the number of bone resorption pits on bone slices and the expression of osteoclastspecific gene cathepsin K. Conclusions ENa C may participate in the regulation of osteoclast differentiation and bone resorption, suggesting its role in functional regulation of the osteoclasts and a possibly new signaling pathway related with ENa C regulation for modulating bone metabolism.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2016年第8期1148-1152,共5页
Journal of Southern Medical University
基金
广东省科技计划项目(2014A020212241)
关键词
破骨细胞
上皮钠离子通道
阿米洛利
免疫荧光
osteoclast cell
epithelial sodium channel
amiloride
immunofluorescence
