载脂蛋白E在雌激素介导骨代谢调控中的作用及其机制

Osteoporosis and atherosclerosis are among the most frequent morbidities in postmenopausal women because of the disorder occurred in lipid metabolism and bone metabolism due to estrogen deprivation. Estrogen replacement therapy is one of the effective ways for prevention and treatment of postmenopausal osteoporosis (PMO) and atherosclerosis. Estrogen could up-regulate the apolipoprotein E (ApoE) at peripheral level, which plays an important role in lipid metabolism. Both ApoE and its receptors such as LRP5 as well as LDLR are expressed in either osteoblasts or osteoclasts. Decreased bone mass were showed in ApoE gene deficient mice feed with high-fat chow. Thus, it suggested that ApoE might be involved in estrogen mediated bone metabolism..In current study, wild-type (WT) osteoblasts derived from skull of new born mice would be treated with serial concentrations estrogen, and gene chip would be conducted to analyze the expression of apolipoprotein clusters and low density lipoprotein receptor related proteins (LRPs). Then in presence or absence of inhibitor of estrogen receptor α or β, ApoE and LRP5 in osteoblasts would be detected by real-time PCR and western blot. Afterwards, ApoE-/-, LRP5-/- or WT osteoblasts would be treated with Dkk1 then analyzed by alkaline phosphatase staining and assay, calcium nodules staining, real-time PCR and FCM to measure the biological characteristics accounted for ApoE gene deficiency and Wnt signaling pathway. .Osteoclasts would be treated with estrogen, then in presence or absence of inhibitor of estrogen receptor α or β, ApoE and LDLR would be detected by real-time PCR and western blot. Co-culture system of osteoblasts and bone marrow-derived macrophages derived from ApoE-/-, LRP5-/-, LDLR-/- or WT mice would be built to analyze the ApoE’s effect on osteoclastogenesis identified by TRAP stain, and NFATc1 would be measured by real-time PCR..Through in vitro and in vivo experiments, we try to address the modulatios of ApoE on osteoblasts’ and osteoclasts’ biological characteristics and the related signaling pathways via comparing the differences of osteoblasts and osteoclasts derived from ApoE-/-, LRP5-/-, LDLR-/- or WT mice..In vivo, the ovariectomized (ovx) ApoE-/- mice were feed by high cholesterol chow or standard chow. Micro CT, high performance liquid chromatography and immunohistochemical analyses are used to characterization and quantification of the modulation of ApoE on the bone phenotype and ApoE level in blood or liver. .Our investigation aiming to explore the underlying mechanism and molecular foundation of ApoE's modulation on estrogen mediated bone metabolism, in order to probe new target and provide a novel strategy for the therapeutics of PMO.

绝经后女性中常见骨质疏松症与动脉粥样硬化并存的现象，脂代谢与骨代谢之间存在密切联系，雌激素补充疗法是防治这两种疾病的共同有效手段。雌激素可上调脂代谢中关键分子ApoE水平。成骨及破骨细胞表达ApoE及其受体LRP5、LDLR。高脂饮食的ApoE-/-鼠骨量下降。提示ApoE 可能参与雌激素调控的骨代谢。本研究拟通过分析雌二醇是否经其受体α或β途径调控成骨和破骨细胞ApoE及其受体LRP5、LDLR表达，并探讨其分子机制。比较ApoE-/-鼠、LRP5-/-鼠、LDLR-/-鼠及WT鼠成骨和破骨细胞分化及生物学功能差异，探讨ApoE作用于成骨及破骨细胞的不同下游信号转导通路。比较高脂或普通饮食下去势对ApoE-/-小鼠骨微结构，骨代谢参数的影响。本研究旨在探讨ApoE在雌激素介导骨代谢调控中的作用机理及分子基础，为临床上雌激素补充疗法防治绝经后骨质疏疏松症提供新的科学依据。

绝经后女性中常见骨质疏松症与动脉粥样硬化并存的现象，脂代谢与骨代谢之间存在密切联系，雌激素补充疗法是防治这两种疾病的共同有效手段。一方面，雌激素缺乏，在骨代谢中，表现为破骨细胞形成增加、活性增强、寿命延长，机体骨重吸收增加，使体内骨重吸收大于骨形成，导致骨质疏松发生。另一方面，雌激素可上调脂代谢中关键分子ApoE水平，而成骨及破骨细胞表达ApoE及其受体LRP5、LDLR，高脂饮食的ApoE-/-鼠骨量下降，提示ApoE可能参与雌激素调控的骨代谢。本研究拟通过分析雌二醇是否经其受体α或β途径调控成骨和破骨细胞ApoE及其受体LRP5、LDLR表达，并探讨其分子机制。比较ApoE-/-鼠、LRP5-/-鼠、LDLR-/-鼠及WT鼠成骨和破骨细胞分化及生物学功能差异，探讨ApoE作用于成骨及破骨细胞的不同下游信号转导通路。比较高脂或普通饮食下去势对ApoE-/-小鼠骨微结构，骨代谢参数的影响。本研究结果证明了雌激素通过上调ApoE基因表达来促进成骨细胞的分化，ApoE基因缺陷促进去势雌鼠骨丢失；成骨细胞分化过程中大多LDLR家族的基因都得到表达，在雌二醇作用下，LRP6表达被上调而LRP5，LRP4和Apoer2被下调；雌激素在成骨细胞分化发育早期通过作用于LRP6发挥骨代谢调控作用；LDLR基因敲除小鼠下调成骨相关转录因子表达，使成骨细胞分化成熟受阻。LRP5基因敲除可以通过调节Wnt信号通路影响成骨细胞的分化和干扰骨髓和脾脏免疫细胞的平衡。本研究阐述了ApoE在雌激素介导骨代谢调控中的作用机理及分子基础，为临床上雌激素补充疗法防治绝经后骨质疏疏松症提供新的科学依据。