基于AMPK/Akt/mTOR信号途径研究TSH在破骨细胞分化中的作用

It has been believed that osteoporosis associated with hyperthyroidism due to elevated thyroxine level. But patients with subclinical hyperthyroidism, decline in the level of thyroid stimulating hormone (TSH) alone, are at equal risk of osteoporosis. Research has shown that there is thyroid-stimulating hormone receptor (TSHR) on the osteoclast, but whether TSH regulates osteoclast directly and its mechanisms are still not be stated clearly. In our previous researches, TSH inhibited RAW264.7 osteoclast cell line differentiation and down-regulated gene expression of tartrate-resistant acid phosphatase (TRAP), matrix metallo-proteinase-9 (MMP-9) and cathepsin K, followed by activity of Adenosine Monophosphate Activated Protein Kinase (AMPK) was increased. In Tshr-/- mice with hormone replacement, the T4 level was normalized, but bone resorption was increased and trabecular bone volume was still significantly lower. These results prompt that TSH regulates osteoclast through TSHR. In this study, in combination of in vivo and in vitro, we took three different kinds of animal models and two types of cells as the main study objects, by using gene knockout, RNA interference and other advanced molecular biology techniques, removing the effects of thyroxine, to study the effects of TSH on osteoclast differentiation and bone metabolism from different aspects and viewpoints. The research focused on whether TSH regulate osteoclast differentiation is mediated through AMPK / Akt / mTOR signaling pathway. The research will provide experimental evidence for the role of TSH plays in bone metabolism and new ideas for the prevention and treatment of bone loss caused by thyroid diseases and other causes of osteoporosis.

甲状腺功能亢进症伴发的骨质疏松一直被归因于甲状腺激素水平升高；但亚临床甲亢患者仅有促甲状腺激素（TSH）水平下降，同样易发生骨质疏松。已证明破骨细胞存在TSH受体，目前TSH对破骨细胞是否有直接调节作用及其机制一直不清。我们前期研究发现TSH可抑制小鼠单核/巨噬细胞系RAW264.7细胞分化并下调TRAP、MMP-9 、 Cathepsin K 基因转录，伴随AMPK 活性加强，提示TSH 通过TSHR调节破骨细胞。本项目采取体内与体外实验相结合的策略，以3种动物模型和2类破骨细胞为研究对象，排除甲状腺激素的作用，利用基因敲除、RNA干扰等分子生物学技术，从不同层面、不同角度地论证TSH对破骨细胞分化和骨代谢的作用，并着重研究TSH调控破骨细胞分化的作用是否通过AMPK/Akt/mTOR信号途径介导。为认识TSH对骨代谢的作用提供实验依据；为甲状腺疾病及其他原因所致骨丢失的干预靶点提供依据

甲状腺功能亢进症往往伴随着骨质疏松的发生。以往认为甲状腺激素（T3、T4）水平升高是甲亢性骨质疏松的主要原因，但临床研究发现，亚临床甲亢患者甲状腺激素水平正常，仅仅存在促甲状腺激素（TSH）水平下降时，同样易并发骨质疏松，因此提出TSH可能对骨代谢有调节作用，但还存在争议。本课题分别通过对体内人体甲状腺功能正常男性骨密度与血清促甲状腺激素（TSH）水平的关系、TSHR基因敲除小鼠骨量及骨强度的变化以及体外不同剂量TSH对成骨、破骨细胞影响等研究了TSH对骨代谢的直接影响及其作用机制。.结果发现：（1）随血清 TSH 浓度的升高，甲功正常男性 BMD也随之升高，提示 TSH 对骨量调节有直接影响；（2）与Tshr+/+ 组小鼠相比，Tshr-/- 和Tshr+/- 组小鼠股骨弹性载荷（Fp）、最大载荷(Fb)、弹性模量和最大弯曲应力均显著降低，甲状腺素替代治疗的Tshr-/- 组小鼠仅弹性模量稍有升高；（3） 与Tshr+/+ 组小鼠相比，股骨不脱钙切片显示Tshr-/- 和Tshr+/- 组小鼠股骨骨小梁体积（BV/TV）、类骨质表面（OS/BS）、类骨质宽（O.Th）、成骨细胞表面（Ob.S）等骨形成指标均降低，而骨吸收指标破骨细胞表面（Oc.S）显著增加；甲状腺素替代治疗后Tshr -/- 组小鼠骨形成与骨吸收均有所增加；（4） 小鼠单核巨噬细胞系RAW264.7细胞诱导形成的破骨样细胞细胞膜表面存在TSHR；不同浓度的TSH呈剂量依赖性地抑制破骨细胞分化，下调破骨细胞TRAP、MMP-9和CTSK的mRNA和蛋白表达水平；TSH能够抑制破骨细胞的分化及功能，这可能是TSH抑制破骨细胞介导的骨吸收的部分作用机制；（5）不同浓度 TSH 可剂量依赖性促进成骨细胞增殖，增加分化及调节相关基因ALP、COI1、BMP2、Runx2mRNA 表达。TSH 100mu/ml 时呈时间依赖性上调 ALP、BMP2 mRNA表达；Western-Blot 显示与对照组相比，实验组随着 TSH 浓度增加 Runx2、Osterix 表达呈增高趋势。TSH起到促进骨形成的作用。.课题的科学意义在于通过体内及体外研究证实了TSH抑制破骨细胞介导的骨吸收，同时能够促进成骨细胞的增殖分化，从而对骨代谢具有不依赖于甲状腺激素的直接作用，将为甲状腺功能异常性骨质疏松的治疗提供新的方向。