SIRT6/AMPK轴精细调控MSCs成骨分化的机制研究

Cranial and maxillofacial bone defect restoration is one of the common problems of dental clinical work. It not only can promote MSCs application in bone tissue engineering, but also is beneficial to screen the new therapeutic target of osteoporosis to understand the mechanisms regulating osteogenic and adipogenic differentiation of bone marrow MSCs. In our previous study, we have confirmed that the aging-related gene SIRT6 plays important roles in MSCs osteogenesis and adipogenesis. However, the fine-tuning mechanism is not clear. In this research, we hypothesize that SIRT6/AMPK axis can fine-tune osteogenic differentiation of MSCs at different stages. To evaluate the effect of SIRT6/AMPK on osteogenic different in various stages, MSCs with enhanced or reduced AMPK/GLUT1 function driven by different promoters are developed in vitro, and then the conditional knockout mice are employed to investigate the function of SIRT6 in vivo. We expect to illuminate the mechanism of SIRT6/AMPK axis’s effect on the osteogenic differentiation of MSCs in the early and late stages of MSCs osteogenic differentiation. This study will provide us theoretical basis to regulate MSCs differentiation, and to treat osteoporosis.

颅颌面大块骨缺损修复是口腔临床工作常见的难题之一。了解骨髓MSCs成骨和成脂分化的精细调控机制，不仅可以促进MSCs在骨组织工程中的应用，而且可以寻找到治疗骨质疏松症（OP）的新靶点。在前期研究中，我们已证实衰老相关蛋白SIRT6调控MSCs成骨和成脂分化，但是其精细的调节机制并不明确。本课题提出SIRT6通过AMPK通路在不同阶段精细调控MSCs的成骨分化。为验证以上假说，我们首先在体外不同时期激活或者抑制AMPK通路，验证过表达或沉默SIRT6对LKB1/AMPK通路活性的影响及AMPK通路活性对MSCs成骨向分化的作用；其次，检测SIRT6是否可以通过调控GLUT1来抑制AMPK通路活性，促进成骨细胞的成熟；最后，通过研究SIRT6条件性敲除小鼠，观察在不同阶段敲除SIRT6对小鼠成骨相关组织的影响。本研究将为MSCs定向分化的调控和OP的防治提供新的理论支持。

项目背景：临床问题：颅颌面骨缺损修复是口腔临床工作常见的问题和治疗难点之一，了解调控间充质干细胞（MSCs）成骨和成脂方向分化的关键基因可以更好地促进MSCs成骨分化，有利于MSCs在骨再生医学领域的应用，为骨质疏松病人的临床治疗提供新的靶点。.研究内容：SIRT6 如何通过GLUT1/AMPK调控终末分化细胞如小鼠成牙骨质细胞系OCCM的矿化能力；SIRT6下游蛋白PCSK9对牙周膜细胞（PDLCs）细胞分化调节作用；PCSK9对炎症状态下OCCM矿化能力的调节作用；SIRT6下游蛋白PCSK9对牙周炎的调控作用。.关键数据：SIRT6的过表达抑制终末分化细胞成牙骨质细胞OCCM-30的矿化；GLUT1促进成牙骨质细胞OCCM-30的矿化；SIRT6通过抑制GLUT1的表达抑制成牙骨质细胞OCCM-30的矿化；SIRT6通过抑制GLUT1或者激活AMPK通路抑制成牙骨质细胞OCCM-30的矿化；SIRT6下游蛋白PCSK9过表达促进PDLCs矿化；PCSK9通过直接结合TRAF2蛋白促进PDLCs成骨分化；PCSK9过表达通过NF-kb通路在炎症因子短暂刺激下促进OCCM矿化能力；PCSK9在小鼠牙周炎模型中上调表达；PCSK9敲除降低牙周炎的严重程度；PCSK9敲除促进小鼠牙周内巨噬细胞对致病细菌Pg的吞噬。.科学意义：本研究证明SIRT6通过抑制GLUT1促进OCCM的矿化；本研究证明PCSK9敲除抑制牙周炎的发展，是牙周炎治疗的重要靶点；本研究牙骨质的再生和牙周炎的治疗提供了理论依据。