骨细胞Wnt-Jag1微环境因子调控骨形成新机制及其对骨流失的保护作用研究

Bone reconstruction based on its development will fit tremendously-unmet clinic needs in treatment and rehabilitation of muscular-skeletal disorders. Mediation of self-renewal of mesenchymal stem cells (MSCs) and differentiation of osteoblasts is the key. . To do so, we uncovered that (1) osteocytes, the terminal-stage cells of osteoblast differentiation, are the targeting cells responsible for orchestrating Wnt anabolic action with increased bone formation and unexpected higher resorption without inducing leukemia (PNAS, 2015). The action happens without decreasing the expression of Sost/sclerostin, an inhibitor of Wnt signaling and involuntary bone formation, contradicting to the traditionally recognized knowledge of osteocytic action for osteogenesis. Moreover, osteocytic Wnt does not inhibit bone resorption due to higher level of Wnt-upregulated Opg, because the ratio of RankL/Opg in bones is even with higher higher, unlike osteoblastic Wnt action that increases Opg, to inhibit bone resorption but with induced leukemia. . Also, we identified that (2) Notch signaling pathway inhibits osteoblast differentiation in MSCs, leading to the maintenance of the MSC pool in the bone marrow with its underpinning mechanism; and that Notch mediates temporal and spatial action of Notch on osteoblast differentiation (Nat Med 2008; PLoS Genet, 2012). . Further analysis of this pathway indicates Notch signaling lies downstream of Wnt, which is also confirmed in other three anabolic actions by PTH intermittent injection, PTH receptor conditional knock-out in osteocytes, and bone mechanical stimulation. These effects were reversed by inhibiting Wnt signaling and was recapitulated in vitro in osteocytic cell line and authentic osteocytes of parietal bones. Mechanistically, in vitro studies leading to this application show that Wnt and PTH transcribes Notch ligand Jag1, respectively, and that Jag1-expressing cells inhibit and enhance osteoblast differentiation in MSCs and marrow stromal cells, respectively. . This application aims to test the novel hypothesis that osteocytic Wnt-Jag1 niche controls bone reconstruction. We plan to test that Jag1 is the target of osteocytic Wnt/β-catenin signaling and mediates Notch signaling in MSCs and its derived cell lineages responsible for their self-renewal and differentiation towards osteoblasts to form new bone; and to examine the extent of protection by the niche with dual function of bone formation and resorption in bone loss. . This study will provide fundamental information of drug development of early diagnosis, prevention, and safe therapy and rehabilitation of bone diseases such as bone loss.

基于骨发育的健康骨重新形成是许多疾病治疗和康复极为需要的，这一目标的实现仍存在诸多难题。间充质干细胞（MSC）体内自我更新和成骨分化的调控是关键。申请人的前期工作证明：1）骨细胞是经典Wnt产生骨合成代谢的效应细胞、提高骨生成和吸收。其成骨不通过调节sclerostin实现，不同于过去对骨细胞的认识；也别于成骨细胞经典Wnt抑制骨吸收而增骨。2）率先发现Notch信号抑制MSC分化、保持MSC库的功能和分子机制，及其时空调控成骨分化。3）骨细胞Wnt和其它三种骨合成代谢模型测量结果表明Notch信号介入其中；并在体外检测了这种调控的细胞和分子基础。本研究首次提出骨细胞Wnt-Notch配体Jag1微环境因子调控骨形成假说，旨在明确骨细胞Wnt控制Jag1转录、调控MSC及其系列细胞的Notch信号、实现MSC自我更新和成骨分化；探测具有骨生成和吸收双功能的微环境因子对骨流失的保护作用。

间充质干细胞在骨再生的应用中受到限制，主要原因是对其在体内增殖和成骨分化的调控缺乏必要的了解。申请人率先发现骨细胞是经典Wnt信号产生骨合成代谢的效应细胞，提高骨生成和骨吸收，这一功效不受骨细胞特异表达的sclerostin调节，不同于过去对骨细胞的认识。本研究从全新角度测定“骨细胞Wnt-Jag1 微环境因子调控骨形成”科学假说，探讨发育终端的骨细胞反向调控骨发育的生理机制。取得结果如下：. 建立了原代骨细胞分离培养技术，鉴定了骨细胞Wnt激活小鼠骨细胞具有促进骨髓基质细胞（BMSC）成骨分化的细胞学功能和机制。发现骨细胞Wnt完全阻止失重骨丢失的功能与机制。模拟失重两周可以100%地保护失重骨丢失，没有产生野生型小鼠失重后sclerostin表达上升现象；维持了成骨分化，抑制了失重造成的破骨细胞分化和骨吸收的提高。取得了失重研究中罕有的保护效果。小分子药物分别逆转骨细胞Wnt的失重骨保护和野生型小鼠的失重骨丢失，初步验证了骨细胞Wnt保护失重骨丢失的分子机制。通过模拟失重试验初步查明的骨细胞Wnt产生失重骨保护的细胞和分子基础具有转化应用价值，丰富失重生理学内容。. 鉴定了骨细胞Wnt-Jag1(Notch信号配体)微环境因子具有独立成骨分化功能。系统比较了Notch配体Dll1/3/4和Jag1/Jag2，证明了骨细胞Wnt产生Notch配体对成骨细胞发育的促进作用，明确这一作用与其激活BMSC的Notch信号有关。从而证明骨细胞Wnt-Jag1-Notch信号轴的成骨工作模式，也具有转化应用价值。. 意外地发现：激活骨细胞Notch信号产生成骨分化障碍，骨量大减，破骨细胞增多；小鼠在围产期死亡，死亡原因不明。这与国外的报道差异很大。但是，我们在体外验证了激活原代骨细胞Notch信号抑制BMSC成骨分化的作用。还发现老龄化基因表达增多，这一结果类似于老年骨松病人的情况，亟待立项研究。. 构建了骨细胞过表达Jag1转基因小鼠，尚未完成转基因分析，以及骨表型和对失重骨保护的作用分析。制备了骨细胞Notch信号失活小鼠, 正在分析骨表型及其对失重骨流失的作用。