基于Ihh/PTHrP信号通路研究过量氟抑制软骨内成骨的分子调控机制

Previous studies have suggested that fluorosis impaired growth plate cartilage in animals and delayed bone growth of children, which suggested that excessive fluoride suppressed endochondral ossification, but the underlying molecular mechanism remains to be elucidated. Our previous studies demonstrated that excessive fluoride significantly inhibited longitudinal growth of cultured fetal rat metatarsal bone, proliferation and differentiation of chondrocytes, while chondrocytes appotosis was increased. Expressions of Ihh, PTHrP mRNA and protein in growth plate of fluorosis rats were significantly increased, which suggested Ihh/PTHrP signaling pathway was involved in the growth plate cartilage impairment of fluorosis rats. The present study was designed to address the roles of Ihh/PTHrP, BMPs, FGFs and Wnt signaling pathways, as well as Sox9, Runx2 transcription factors in the process of fluorosis endochondral ossification. To achieve this, we use animal experiment, ex vivo model of cultured fetal rat metatarsal bones and mesenchymal chondrogenic cell line RCJ3.1C5.18 to explore the roles of Ihh/PTHrP, BMPs, FGFs, Wnt, Sox9 and Runx2. In cultured fetal rat metatarsal bones and RCJ3.1C5.18, signaling pathway inhibitors and RNAi will be used to validate the roles of Ihh/PTHrP, BMPs, FGFs and Wnt signaling pathway in endochondral ossification after excessive fluoride treatment. We hope this study will facilitate to find novel breakthrough in the exploration of the mechanism of skeletal fluorosis.

研究发现氟中毒可损伤动物生长板软骨、延缓儿童骨骼的生长发育，提示过量氟抑制了正常的软骨内成骨，但其发生的分子机制尚不十分清楚。前期我们应用跖骨器官培养发现过量氟显著抑制跖骨的纵向生长，软骨细胞增殖和分化降低，而细胞凋亡增加；氟中毒大鼠生长板Ihh、PTHrP mRNA和蛋白高表达，由此推测Ihh/PTHrP信号轴与氟中毒生长板软骨损伤密切相关。本课题将以Ihh/PTHrP信号轴为切入点，采用动物实验、器官和细胞培养分别研究Ihh/PTHrP信号轴及相关的BMPs、FGFs、Wnt等信号通路和Sox9、Runx2等转录因子在氟中毒软骨内成骨各阶段中的表达并进行综合分析。在体外培养的跖骨和RCJ3.1C5.18软骨细胞中，采用信号通路抑制剂和RNA干扰技术来进一步验证以Ihh/PTHrP为主的各信号通路在过量氟抑制软骨内成骨中的具体调控作用，期望本项目能在氟骨症发病机制的探索中产生新的突破。

地方性氟中毒是我国流行最为广泛、病情最为严重的重点地方病之一。尽管地氟病病因明确，但尚无特效治疗措施，因此阐明地氟病的发病机制是控制乃至消除地氟病的根本途径。研究发现氟中毒可损伤生长板软骨、延缓儿童骨骼的生长发育，提示氟中毒抑制软骨内成骨，但其发生的分子机制尚不十分清楚。基于Ihh/PTHrP信号通路在软骨内成骨过程中的核心作用和课题组前期实验结果，本项目以Ihh/PTHrP信号轴为切入点，从动物模型、体外跖骨器官培养和细胞培养等方面系统探讨氟对生长板软骨细胞增殖、分化、基质矿化、凋亡及血管侵入等软骨内成骨过程的影响，并分析Ihh/PTHrP信号轴和相关的FGFs、mTOR等信号通路以及SOX9、RunX2等转录因子在其中的调控作用。研究发现氟通过下调血清胰岛素样生长因子（IGF-Ⅰ）和以Ihh/PTHrP信号轴为核心的多种局部信号通路共同作用抑制软骨内成骨，进而抑制长骨生长。氟通过抑制PI3K/AKT/mTOR信号通路、上调FGFR3抑制软骨细胞增殖；氟通过上调SOX9、下调RunX2等转录因子的表达抑制软骨基质降解及血管侵入；氟中毒大鼠生长板软骨基质中硫酸乙酰肝素异常增多，激活FGFR3信号通路，抑制Ihh/PTHrP负反馈环进而抑制软骨内成骨。本项目明确了氟中毒对软骨内成骨主要过程的影响，阐明氟以Ihh/PTHrP信号通路为主的调控网络抑制软骨内成骨的分子机制，为氟骨症的发病机制、预防和干预措施提供了实验依据。 本项目共发表标注本项目资助的SCI论文2篇，中文1篇，参加国际学术会议1人次，参加国内学术会议1人次并做分会报告，培养硕士研究生4名。