Lgr4对骨髓间充质干细胞成骨和成脂分化平衡的调控及其在骨质疏松发病机制中的作用

The balance between osteoblastic and adipocytic differentiations in the bone mesenchymal stem cell (BMSCs) is critical for the bone homeostasis. However, the underlying mechanism for the regulation of this balance remains unclear. Recent studies demonstrated that Lgr4 could modulate bone metabolism and the translation of white fat to brown fat through the cAMP and the Wnt/β-catenin signaling pathways. The role of Lgr4 in regulating the osteoblastic and adipocytic differentiations in BMSCs has also been identified in our previous studies and preliminary experiments. Thus, we speculate that Lgr4 regulates the balance between the osteoblastic and adipocytic differentiations in BMSCs and hence has important roles in the pathogenesis of osteoporosis. To prove our hypothesis, we observed the effect of Lgr4 on the osteoblastic and adipocytic differentiations in BMSCs by various means such as knocking out the expression of Lgr4 in the BMSCs and pre-osteoblastic and pre-adipocytic cell lines, Transwell co-culturing to mimic the microenvironment of the bone marrow, and conditional Lgr4 gene knockout in the BMSCs of mice. The possible molecular mechanisms are also explored. The loss of balance between osteoblastic and adipocytic differentiations and the role of Lgr4 in it are investigated in ob/ob, Lgr4m/m and m/m;ob mice. Finally, Lgr4 over-expressed adenovirus is injected into the aged mice, into the OVX mice and into the corticosteroid used mice. Whether the imbalance between osteoblastic and adipocytic differentiations in the BMSCs of the osteoporotic bones can be corrected or not is examined. We believe this study will deepen our understanding on the pathogenesis and provide new methods for the prevention and treatment of osteoporosis.

BMSCs成骨和成脂分化平衡对维持正常骨量至关重要，但调控机制不明确。文献报道Lgr4通过cAMP和经典Wnt通路调控骨代谢和外周脂肪转化。我们前期研究发现Lgr4调控BMSCs成骨分化，预实验证实其调控BMSCs成脂分化。故我们推测：Lgr4参与调控BMSCs成骨和成脂分化的平衡，继而在骨质疏松发病机制中起重要作用。为验证这一假说，我们采用体外干扰BMSCs、前成骨和前成脂细胞系Lgr4表达，Transwell细胞共培养模拟骨髓微环境，小鼠BMSCs Lgr4基因条件性敲除等手段，观察Lgr4对BMSCs成骨和成脂分化的影响及其分子机制；利用ob/ob、Lgr4m/m和m/m;ob鼠，观察Lgr4在成骨和成脂分化失衡中的作用；利用老龄、去卵巢和糖皮质激素诱导的骨质疏松鼠，观察过表达Lgr4的腺病毒注射能否纠正BMSCs成骨成脂分化的失衡。本研究为骨质疏松的发病机制和防治研究开辟新思路。

骨质疏松 (osteoporosis, OP) 是一种骨骼系统代谢性疾病，以骨量减少、骨微结构破坏为特征。各种原因引起的BMSCs成骨和成脂肪分化失衡是导致骨质疏松发生的一个重要原因。深入研究BMSCs成骨和成脂肪分化平衡的机制，将为有效防治骨质疏松提供新的理论基础和药物研发靶点。.我们在前期实验中发现:富含亮氨酸重复序列G蛋白偶联受体4 (Lgr4)通过激活经典Wnt/β-catenin通路活性促进骨形成｡我们进一步研究发现:R-spondin2有促间充质干细胞成骨作用,在其作用的细胞中,活性β-catenin 蛋白表达水平明显升高,且从细胞浆向细胞核内转移｡Lgr4参与R-spondin2对Wnt信号通路的激活,对间充质干细胞的成骨分化有促进作用｡我们在应力加载实验中发现周期性牵张应力(CMS)显著上调了BMSCs中Rspo1的表达和分泌｡Rspo1过表达和重组Rspo1细胞因子均激活Wnt/β-catenin通路，促进BMSCs的成骨分化,Lgr4基因敲除后,Rspo1对BMSCs成骨分化的促进作用､对Wnt/β-catenin通路的激活作用均消失｡去应力小鼠股骨局部过表达Rspo1后,成骨细胞活性､新骨生成和骨密度均明显增加,骨显微结构参数得到明显改善｡.在间充质干细胞成脂分化的过程中，Lgr4表达量下降。抑制Lgr4表达，干细胞成脂分化增强，促进其表达时，干细胞成脂分化降低。我们还发现Sh-LGR4在脂肪生成诱导期间促进了hBMSCs中p38的磷酸化,在LGR4过表达细胞中,p-P38和PPARγ蛋白表达均显著降低｡这些结果表明LGR4通过MAPKs/P38信号通路抑制BMSC脂肪生成｡.综上，LGR4在骨髓间充质干细胞分化的成骨/成脂调控中发挥重要调控作用,可作为抗骨质疏松治疗的靶点。