基于髓枯骨痿理论和Wnt/β-Catenin信号探讨骨碎补对骨质疏松BMSCs成骨、成脂分化及OPG/RANKL表达的调节作用

The balance between of osteoblastogenesis and adipogenesis of bone marrow mesenchymal stem cells (BMSCs) has an important role in pathogenesy of age-related and post-menopause osteoporosis. Wnt/β-Catenin signaling pathway may control the differentiation of mesenchymal stem cells to either osteoblasts or adipocytes. Traditional Chinese medicine believes the kidney generating marrow and dominating bone, and marrow atrophy resulting in Osteoporosis. In this study, age-related rat osteoporosis and post-menopause rat osteoporosis models will be established by selected old rat and ovariectomy respectively. Proceeding from marrow atrophy resulting in osteoporosis and BMSCs regulated by Wnt/β-Catenin signaling pathway, BMSCs will be separated, cultured and investigated from age-related and ovariectomy-induced osteoporotic rat. Firstly, the expression and modifications of Wnt/β-Catenin signaling pathway and OPG/RANKL，the potentiality of the differentiation of BMSCs to either osteoblasts or adiocytes and will be investigated and the correlation will be analysised. Secondly, this study will investigate effect of Rhizome of Drynaria containing serum on osteoblastogenesis and adipogenesis of osteoporotic BMSCs, Simultaneously, detect and analyze the expression and modifications of Wnt/β-Catenin signaling pathway and OPG/RANKL. Finally, BMSCs will be separated, cultured after age-related and ovariectomy-induced osteoporotic rats are treated with Rhizome of Drynaria, the adjustments of Wnt/β-Catenin signaling pathway, expressiong of OPG/RANKL and osteoblastogenesis and adipogenesis of osteoporotic BMSCs will be further investigated and analysised. In conclusion, such researches will show the modification of Wnt/β-Catenin signaling pathway resulting in unblance between osteoblastogenesis and adipogenesis os osteoporotic BMSCs could have an important roles in the pathogenesy of age-related and post-menopause osteoporosis, which may be substantial Celluar and Molecular Biology foundation of Marrow atrophy resulting in Osteoporosi; on the other hand, it will also reveal Rhizome of Drynaria regulates osteoblastogenesis and adipogenesis of bone marrow mesenchymal stem cells derived from osteoporostic rat and adjust the expression of OPG/RANKL to inhibit steoclasticogenesis by modulation of Wnt/β-Catenin signaling pathway, which may be a mechanism of Rhizome of Drynaria to treat osteoporosis.

骨髓间充质干细胞（BMSCs）成骨、成脂分化失衡在年龄相关和绝经后骨质疏松症中扮演了重要角色。本研究基于髓枯骨痿理论，以Wnt/β-Catenin信号管理下的BMSCs为切入点，分析Wnt/β-Catenin信号在骨质疏松大鼠BMSCs中的改变与其成骨、成脂分化失衡和OPG/RANKL表达改变的相关性；观察骨碎补含药血清对体外培养骨质疏松BMSCs以及经骨碎补治疗后对骨质疏松大鼠BMSCs成骨、成脂分化能力的影响，并分析Wnt/β-Catenin信号及OPG/RANKL表达的变化。揭示Wnt/β-Catenin信号管理下的BMSCs成骨、成脂分化失衡可能是骨质疏松发病的一种细胞分子机制，是髓枯骨痿的细胞分子生物学基础所在；揭示骨碎补可能通过补肾荣髓再激活Wnt/β-Catenin信号，从而促进BMSCs成骨分化，抑制成脂分化，改变OPG/RANKL表达间接抑制破骨细胞分化的抗骨质疏松机理。

本项目基于髓枯骨痿理论，探讨Wnt/β-Catenin在老年和绝经后骨质疏松相关的BMSCs成骨/成脂分化失衡和OPG/RANKL表达改变中的作用机制及骨碎补抗骨质疏松的作用机理。发病机制方面：分别构建老年和去卵巢诱导的骨质疏松大鼠模型，并以正常大鼠模型作为对照，分别分离来自各组大鼠的骨髓BMSCs细胞并传代培养，制作生长曲线分析细胞增殖特性，茜素红染色、碱性磷酸酶（ALP）活性检测、油红染色、酶联免疫吸附法（ELISA）、聚合酶链反应（RT-QPCR）、蛋白免疫印迹法（WB）等检测方法进行成骨/成脂诱导及Wnt/β-Catenin分析，结果表明，老年和去卵巢诱导的骨质疏松组BMSCs成骨分化减弱，成脂分化增强，Wnt/β-Catenin通路受到抑制，伴随OPG/RANKL比例降低。药物干预方面：骨质疏松模型构建成功后，分别分离各组BMSCs细胞，RNA干扰技术沉默β-Catenin基因，并以不同浓度的骨碎补含药血清进行干预，分析细胞增殖特性，茜素红染色、ALP活性检测、油红染色、ELISA、RT-QPCR、WB等检测方法进行成骨/成脂诱导及Wnt/β-Catenin分析，研究表明骨碎补含药血清可改善BMSCs分化，促进成骨，抑制成脂分化，激活Wnt/β-Catenin通路；其次以不同浓度的骨碎补水煎液灌胃骨质疏松模型大鼠，分离培养各组BMSCs，观察其成骨/成脂能力及骨碎补对Wnt/β-Catenin通路改变的影响，进一步证实骨碎补可通过激活Wnt/β-Catenin通路，有效改善骨质疏松大鼠体内BMSCs成骨/成脂分化，升高OPG/RANKL比例，间接抑制破骨细胞分化。据本研究成果，揭示了Wnt/β-Catenin在老年和去卵巢相关骨质疏松中的作用机制，是髓枯骨痿的细胞分子生物学基础，揭示骨碎补可能通过激活Wnt/β-Catenin通路改善BMSCs成骨/成脂分化，调节OPG/RANKL比例，间接抑制破骨，进而发挥抗骨质疏松作用。为深入挖掘骨碎补抗OP作用机制、开发新的药物、改善常规抗OP药物治疗OP的缺陷和瓶颈等提供了策略，具有较好的临床及研究意义。