基于CKIP-1介导的mTOR自噬信号通路探讨原发I型骨质疏松致病机理及益气温经法对其治疗机制的研究

Primary type I osteoporosis,a common orthopedic disease,has been becoming a serious threaten to the health of the middle-aged people and the elders.Due to the lackness of effective specific markers and prognostic factors,we are in great need to discuss the new pathogenesis and potential target for diagnosis and treatment.Previous studies had showed that CKIP-1,the negative regulatory factor of bone formation,and mTOR autophagy signaling pathway might play a key role in the development of type I osteoporosis,but the specific pathogenesis remains unknown.In view of this point,we will build osteoblast and osteoclast cells by over-expression and low-expression of CKIP-1,and animal models in ovariectomized rats,draw support from our unique Reinforcing Qi Warming Meridians prescription and other drugs such as micromolecule moderators of autophagy signaling pathway,the usage of luciferase assay system, co-immunoprecipitation and other techniques,to make further studies of CKIP-1,autophagy,pathogenesis and therapeutic functions of primary type I osteoporosis in cells and animal models.We aim to obtain strong evidences of the promoting roles of autophagy in osteoporosis.CKIP-1 and micromolecule of autophagy signaling pathway will provide new potential targets for the diagnosis and therapies in osteoporosis.The combined intervention of Traditional Chinese Medcine(Reinforcing Qi Warming Meridians prescription)and Western Medicine(target drugs of autophagy)will provide a new idea for the treatment of osteoporosis.

原发I型骨质疏松是严重威胁中老年人健康的骨科常见病，且缺乏有效的特异性诊疗标记物及预后因子，亟需探讨其新的发病机制和可能的诊疗靶点。我们前期研究发现，骨形成的负性调节因子—CKIP-1及自噬的mTOR通路在I型骨质疏松的发生发展中可能起到了关键作用，但其具体的致病机理尚无清晰证据。鉴于此，本项目通过建立CKIP-1过表达和低表达成骨、破骨细胞株，去卵巢大鼠动物模型，借助我们独特的益气温经方药和自噬相关通路小分子调节剂等药物，以及荧光素酶检测系统、免疫共沉淀等技术手段，从细胞和动物水平对CKIP-1经自噬干预原发I型骨质疏松的致病机制和治疗功能进行深入研究，获得自噬对骨质疏松的促进作用的有力证据。CKIP-1及自噬通路相关分子为骨质疏松治疗提供了新的靶点，益气温经方药和自噬靶点药物中西医联合干预也为抗骨质疏松治疗提供了新思路。

骨质疏松症对绝经后妇女健康已构成严重威胁，益气温经法可促进骨形成，用于临床多年，疗效显著。前期研究发现CKIP-1蛋白对骨代谢平衡有重要的调节作用，而益气温经法可以抑制CKIP-1蛋白的表达水平，但是内在机制并不十分明确。细胞自噬对于骨代谢平衡的调控有着深远的影响，CKIP-1是否可能通过影响细胞自噬从而达到干预骨代谢平衡的目的，在国内外尚未见相关报道。本次研究中，我们以蛋白组学为主要实验方法，观察CKIP-1对成骨细胞和破骨细胞的不同影响，及强骨饮的干预效应；通过免疫共沉淀的方式，观察CKIP-1是否会靶向结合自噬信号通路的关键蛋白。我们发现，CKIP-1可促进成骨、破骨细胞中自噬水平，它与P13K/AKT/mTOR信号轴中的关键因子AKT的结合，激化AKT，激活mTOR的上游信号，从而激活P13K/AKT/mTOR信号通路，上调自噬水平。强骨饮在破骨细胞可以促进CKIP-1与AKT的结合激活自噬，而在成骨细胞中发挥着相反的作用。这为我们研究强骨饮抗骨质疏松症的机制提供了新的开拓的思路，也为为防治原发I型骨质疏松的基础、临床和中医药的现代化研究提供新的研究角度。