基于骨细胞研究PTH/TGF-β信号通路在氟骨症骨转换机制中的作用

The mechanism underlying bone lesions induced by fluoride are the key problem to be solved urgently in the pathogenesis of endemic fluorosis. Among them，the high bone turnover was outstanding characteristic of progressive period in skeletal fluorosis. We established a stable method to isolate and identify primary osteocytes in vitro, and observed the stimulant action of fluoride on osteocytes, as well as significantly regulating expression of sost and RANKL related with osteogenesis and osteoclastogenensis in osteocyte. Rats co-exposed to fluoride with PTH or TGF-β inhibitor changed the status of bone turnover and osteoid-osteocyte around lesion, and the marker genes of bone turnover varied in rats co-treated with PTH and fluoride. The microRNA chip assay was used to analyzed the expression profiling of osteoblast and osteoclast exposed to fluoride on same condition. The significantly different expression of microRNAs related with regulation of TGF-β and PTH signaling were found in two kind of cells.These data provided new clues to answer the above question. The plasmid transfection, small interfering RNA, DeltaVision high- and super-resolution imaging systems, molecular biology and coculture system techniques will be carried out in this research. The aim of project is to explore the role of osteocyte in the mechanism of bone turnover induced by fluoride, and the action of PTH involved in this mechanism. The specific pathway was investigated about TGF-β and PTH signaling pathway involved in the mechanism of bone turnover of the skeletal fluorosis and action on coupling between bone resorption and formation. Completion of this project will help to provide more scientific and comprehensive description to the mechanism of active bone turnover and conversion between bone lesions in the progressive period of skeletal fluorosis.

氟诱导的不同骨组织病变机制是氟中毒研究中亟待解决的关键问题。其中骨转换加速是氟骨症病变进展期重要特征。我们建立了稳定的体外骨细胞分离和培养方法，观察到染氟骨细胞的活性增加，以及骨细胞内与成骨和破骨密切相关的Sost和RANKL表达显著改变。投氟联合应用PTH或TGF-β抑制剂改变了氟诱导的大鼠骨转换状态及周围骨样骨细胞，而且氟加PTH促进了骨转换标志性基因的变化。染氟诱导了成骨细胞和破骨细胞内多个调控TGF-β和PTH信号通路的microRNA表达差异，前期工作为回答上面问题提供了线索。本项目拟采用质粒转染、siRNA、超高分辨率显微镜、分子生物学和细胞共培养等先进技术探讨骨细胞在氟诱导骨转换中的地位以及PTH在其中作用；TGF-β和PTH信号通路参与氟骨症骨转换具体机制及其对成骨与破骨偶联的作用。本项目的完成有助于为氟骨症进展期骨转换及不同骨骼损害之间的转换机制提供更科学、更全面的说明。

慢性氟中毒骨相损害复杂多样而令人困惑。成骨、破骨活跃和骨转换加速是形成氟骨症病变多样性的病理基础，也是探究氟骨症发病机制的关键点。本项目考察了骨细胞在氟骨症骨转换中的地位，分析TGFβ1和PTH在氟骨症骨转换机制中的作用，包括它们对成骨细胞和破骨细胞及其相互偶联的影响。体外实验结果表明骨细胞较成骨细胞和破骨细胞耐受氟能力强，一定浓度的氟刺激骨细胞内核因子κB受体活化因子配体（RANKL）表达，但抑制硬化蛋白（SOST）表达，从而发挥刺激成骨和破骨作用。利用MicroRNA 芯片技术筛选出多条信号通路同时参与氟调控的成骨细胞和破骨细胞内机制。TGFβ1参与氟调控早期分化的成骨细胞增殖和凋亡机制，亦通过激活Smad3信号因子促进成骨细胞和破骨细胞的分化和功能。采用细胞共培养方法不仅观察到氟调控骨细胞分泌SOST来影响RANKL和骨保护素（OPG）的表达，激活破骨细胞内RANK和NFATc1信号因子诱导破骨细胞分化；而且氟抑制骨细胞SOST表达促进成骨细胞分化。PTH主要协同氟作用的是骨细胞诱导的成骨细胞分化阶段。在成骨细胞与破骨细胞偶联方面，PTH和TGFβ1主要激活Smad3信号通路升高两种细胞内的EphB4/ephrinB2表达，以增强氟对成骨细胞和破骨细胞之间的偶联信息传递。我们复制氟骨症大鼠中骨转换为骨吸收向骨形成过渡阶段，印证了骨细胞来源的SOST与RANKL参与氟骨症骨转换活跃的机制。TGFβ1刺激骨组织内Smad3信号通路，调控RANKL/OPG和Runx2表达来参与氟诱导的骨转换过程。PTH加速氟诱导的骨转换从破骨向成骨过渡进程，验证了Wnt10b和Smad3信号通路参与了该机制。本项目从骨细胞调控成骨细胞、破骨细胞角度探索氟骨症骨量降低的机制，明确TGFβ1和PTH对氟骨症骨转换调控的具体机制，从而为骨细胞在氟骨症发病机制中的重要地位奠定了理论基础。