| 引用本文: | 谢艳芳,石文贵,王鸣刚,陈克明.初级纤毛对蛇床子素促进成骨细胞成骨性分化的影响[J].中国现代应用药学,2016,33(7):837-841. |
| XIE Yanfang,SHI Wengui,WANG Minggang,CHEN Keming.Role of Primary Cilium in the Osthole Induced Differentiation and Mineralization of Osteoblasts[J].Chin J Mod Appl Pharm(中国现代应用药学),2016,33(7):837-841. |
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| 摘要: |
| 目的 研究初级纤毛对蛇床子素促进成骨细胞成骨性分化的影响。方法 取新生SD大鼠颅骨，多次酶消化法获得成骨细胞，免疫荧光染色观察成骨细表面初级纤毛，RNA干扰法抑制鞭毛运输系统蛋白(IFT88)的表达，从而抑制成骨细胞初级纤毛的产生。以浓度为1×10-6 mol·L-1蛇床子素分别作用于RNA干扰成骨细胞，3，6 d后检测胞内碱性磷酸酶(ALP)活性；药物处理24 h后，Real-time PCR检测1型胶原(collagen 1，COL-1)、RUNX-2、ALP mRNA的表达量；Western blot检测COL-1、RUNX-2蛋白表达量。结果 超过70%的成骨细胞表面具有长度约为5 μm的初级纤毛，RNA干扰法显著抑制IFT88基因和蛋白的表达，并抑制了初级纤毛的发生。1×10-6 mol·L-1蛇床子素能够显著地促进胞内ALP的活性，成骨性分化相关的因子COL-1、RUNX-2和ALP基因的表达也相应增高，同时COL-1、RUNX-2蛋白表达量显著增加。当成骨细胞初级纤毛干扰后，药物促进ALP活性升高的作用消失，COL-1、Runx-2基因和蛋白的表达量也相应的降低。结论 成骨细胞初级纤毛的去除，能够显著性地抑制蛇床子素促进成骨细胞成骨性分化的能力。 |
| 关键词: 蛇床子素 初级纤毛 成骨细胞 RNA干扰 |
| DOI： |
| 分类号:R966；R932 |
| 基金项目:国家自然科学基金(81270963，81471090) |
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| Role of Primary Cilium in the Osthole Induced Differentiation and Mineralization of Osteoblasts |
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XIE Yanfang1, SHI Wengui2, WANG Minggang1, CHEN Keming3
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1.School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China;2.Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730050, China;3.Institute of Orthopaedics, Lanzhou General Hospital, Lanzhou Command of CPLA, Lanzhou 730050, China
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| Abstract: |
| OBJECTIVE To study the role of primary cilium in the osteogenic differentiation process of rat calvarial osteoblasts induced by osthole. METHODS The neonatal SD rat skull was segregated, and enzyme digestion was used to obtain bone cells. Primary cilium of osteoblasts was detected by immunostaining and cilium was abrogated by knockdown of IFT88. Transfected cells were treated with 1×10-6 mol·L-1 osthole. The alkaline phosphatase (ALP) activity was determined at the 3rd and 6th day. Total RNA was isolated and the gene expression of ALP, collagen-1(COL-1) and RUNX-2 were investigated by real-time PCR. Finally total protein was also isolated and the secretion of COL-1 and RUNX-2 was examined by Western blotting. RESULTS The percentage of cells possessing primary cilia was found to be >70%, and the cilium was about 5 mm in length projecting from cell surface. Knockdown of IFT88 caused an reduction of cells with primary cilia. The ALP activity of the group treated with 1×10-6?mol·L-1 osthole was significantly higher than the control group, and mRNA expression levels of ALP, COL-1 and RUNX-2 after of treatment changed in similar tendency. Similarly, the stimulating effects of osthole on protein expression of COL-1 and RUNX-2. However, osthole-induced promoting effects on ALP activity and other osteogenesis-related markers were suppressed when primary cilia of osteoblasts were abrogated using RNA interference. CONCLUSION The abrogation of primary cilium can dramatically blocked the differentiation and mineralization of osteoblasts induced by osthole. |
| Key words: osthole primary cilium osteoblast RNA interference |
