CX43介导的骨髓基质细胞成骨分化及调节破骨细胞功能抗骨质疏松的机制研究

The loss of bone mass during bone remodeling is the key factor of osteoporosis. BMSCs are considered as the source of osteoblasts, are involved in the formation and activity of osteoclasts, and are widely used as experimental models of bone remodeling.Our recent studies have shown that upregulation of CX43/GJIC could promote osteogenesis of BMSCs by targeting osteogenesis associated genes and downregulated RANKL/OPG ratio by suppress the expression of RANKL and augmented the increase in the levels of osteoprotegerin. In our present project, we would investigate the anti-osteoporosis mechanism of CX43 at molecular , cellular, and in vivo levels through the research of connexin 43 on promote osteogensis and inhibit osteoclast activity. At first, we would demonstrate the regulation of CX43 on bone metabolism using conditionally knockout mice with osteoporosis induced by ovariectomy,then, we would to elucidate the regulation mechanism of CX43 on bone remodeling by the differentiation phenotype analysis, differentiation genes screen, cell signaling pathway blocking，and knock-down or overexpression of connexin 43 on BMSCs. Our project would provide the treatment strategy on osteoporosis by deepen our knowledge and understanding of it.

骨重塑过程中骨量的丢失是骨质疏松发生的关键。骨髓基质细胞是成骨细胞的主要细胞来源，能调节破骨细胞的生成及活性，是骨重塑研究中最重要的细胞模型。我们最近研究显示：上调骨髓基质细胞的CX43/GJIC可促其成骨分化并抑制RANKL/OPG表达比率。为了探讨骨髓基质细胞CX43/GJIC的抗骨质疏松作用及机制。我们在BMSCs成骨分化过程中分别加用GJIC、Notch及IHH信号通路阻滞剂18α-glycyrrhetinic acid、DAPT及Cyclopamine预处理。通过分化表型分析、分化相关基因筛选、细胞信号阻滞，研究CX43/GJIC对骨髓基质细胞的成骨分化的调节及机制。从促进成骨的角度探讨CX43/GJIC抗骨质疏松的机制。本课题的开展能深化骨质疏松的发病机制，为骨质疏松的防治提供策略。

第一部分 柚皮苷对大鼠骨髓基质细胞体外成骨分化的影响 目的：研究柚皮苷对BMSCs体外增殖和成骨分化的影响。方法和结果：细胞在基本培养液，成骨诱导液和柚皮苷加入成骨诱导液培养，MTT法检测细胞增殖，茜素红染色和ALP活性评估细胞的成骨分化，RT-PCR检测成骨及成脂基因mRNA水平。结果显示基本培养液中加入柚皮苷能明显增加细胞增殖；柚皮苷能增加诱导成骨分化细胞的ALP活性、茜素红染色及ALP, Cbfa 1和 BSP的mRNA水平，抑制PPARγ2的转录水平；柚皮苷的生物学效应呈剂量依赖性。结论：柚皮苷呈剂量依赖性刺激BMSCs增殖、上调成骨基因、抑制成脂基因的表达。因此，柚皮苷能通过调节BMSCs的成骨分化治疗骨质疏松。第二部分 柚皮苷调节GJIC功能和MAPK、Wnt、Notch及IHH信号通路促进BMSCs的成骨分化促进大鼠骨髓基质细胞的成骨分化.目的：研究柚皮苷对大鼠BMSCs的GJIC、MAPK、Wnt、Notch及IHH信号通路和成骨分化相关基因表达的影响。方法和结果：茜素红染色和ALP活性评估细胞的成骨分化，RT-PCR检测成骨及成脂基因mRNA水平。ALP活性和茜素红染色显示柚皮苷刺激ALP活性和钙沉积 ；划痕标记/染料示踪技术显示，柚皮苷增加GJIC功能，同时增加CX43的mRNA、蛋白水平和MAPK、Wnt、Notch及IHH活性；RT-PCR结果显示柚皮苷能增加细胞的ALP，cbfa1和BSP的mRNA水平，抑制PPARγ2mRNA水平；GJIC、MAPK、Wnt、Notch及IHH信号通路抑制剂能逆转柚皮苷对BMSCs的生物学作用。结论：柚皮苷通过调节细胞的GJIC活性和、、MAPK、Wnt、Notch及IHH信号通路上调成骨相关基因表达，进而促进BMSCs的成骨分化。