脂肪细胞RANKL/OPG系统的表达调控及其影响骨塑建/骨再建的遗传模型研究

Recent publications and our previous work showed that adipocytes might directly regulate osteoclast formation by expressing RANKL/OPG system. We also found that RANKL and OPG expression was altered in preadipocytes after treatment with 1,25 dihydroxy vitamin D3. These may suggest a novel mechanism that controls osteoclast development and modeling/remodeling of bone that is so far not demonstrated by in vivo studies. Moreover, using miRNA microarray scanning, qPCR validation and bioinformatics analysis, we found out several miRNAs were abnormally expressed during adipogenesis and some of them had the potential of targeting RANKL or OPG, suggesting that they may modulate adipogenesis and moreover, they may have the ability to regulate osteoclast formation by directly blocking the translation of RANKL or OPG in adipocytes. To demonstrate the hypotheses, we will develop adipocyte-specific RANKL transgenic and silencing mice and analyze their phenotypes. Micro-CT scanning and quantification, alcian blue/hematoxylin and orange G staining of histological slides will be carried out to measure the changes in bone mass. Tartarte resistant acid phophatase staining and calcein double labeling will be performed to analyze the function of osteoclasts and osteoblasts in the genetic animal models. Osteoclast formation assay, using the bone marrow cells isolated from the RANKL transgenic or silencing mice, will also be carried out to demonstrate if the changes in the expression of RANKL in adipocytes result in altered differentiation of osteoclasts. If the above mentioned measurements give us positive results, we will perform ovariectomy on the female adult RANKL silencing mice and study if the RANKL knockdown in adipocytes helps to block the development of osteoporosis. Furthemore, we will study if and how the expression of RANKL/OPG system is modulated in adipocytes by miRNAs. The procedures include: first, actions of miRNA candidates on adipocyte differentiation will be investigated by overexpressing or inhibiting the miRNAs in preadipocytes. Then, timing expression patterns of miRNAs and RANKL/OPG protein during adipocyte differentiation will be analyzed. Finally, targeting of RANKL/OPG by the the miRNAs will be confirmed by using reporter techniques. The present study aim to unravel the mechanisms that control the expression levels of RANKL and OPG system in adipocytes, and clarify how this system mediates the interaction of adipocytes and preosteoclasts and regulates osteoclast development thus modulating the modeling and remodeling of bone.

近年文献和我们的前期工作提示脂肪细胞通过表达RANKL/OPG而直接调节破骨细胞发育，但缺少体内实验证据。我们运用芯片/qPCR验证结合生物信息学分析筛选到若干随脂肪细胞分化而表达异常且可能靶向RANKL或OPG的miRNA，提示它们在调节脂肪细胞分化（或随分化而变化）的同时可能直接影响RANKL/OPG的蛋白翻译而调节破骨细胞发育。为证明此假设，本项目拟建立脂肪细胞特异性RANKL转基因鼠和基因沉默鼠，分析其骨量变化等表型，观察基因沉默鼠抗骨质疏松能力；用miRNA过表达和沉默技术探讨候选miRNA与脂肪细胞分化关系，分析候选miRNA与RANKL/OPG基因表达时相关系，运用报告基因技术验证miRNA与RANKL/OPG的靶向关系等。通过本研究，将揭示脂肪细胞RANKL/OPG系统的调控机制，阐明该系统是否介导脂肪细胞和破骨细胞相互作用而直接影响破骨细胞发育，进而调节骨塑建和再建过程。

前期工作提示脂肪细胞通过表达RANKL/OPG而直接调节破骨细胞发育。本项目建立脂肪细胞特异性Rankl转基因鼠和基因沉默鼠，表型分析结果表明脂肪细胞Rankl转基因鼠（aP2-Rankl）胫骨干骺端骨量显著降低，破骨细胞增多。相反， Rankl基因沉默鼠（aP2-Rankl KD）干骺端骨量增加，破骨细胞减少。分离aP2-Rankl鼠及其野生对照鼠的骨髓细胞进行破骨细胞诱导培养，结果表明aP2-Rankl组破骨细胞样细胞数量较对照组增加，qRT-PCR检测显示TRACP-5b、CTR和Cathepsin K表达水平显著高于对照组。相反，aP2-Rankl KD组破骨细胞样细胞数量较对照组减少，TRACP-5b、CTR和Cathepsin K表达水平低于对照组。骨形态计量学动力学参数显示aP2-Rankl鼠矿化沉积率（MAR）显著增加，而aP2-Rankl KD鼠MAR显著降低。ELISA结果显示aP2-Rankl鼠血清Osteocalcin高于对照鼠，而aP2-Rankl KD鼠血清Osteocalcin低于对照鼠。进一步研究提示基因沉默鼠对抗因去卵巢而导致的骨量丢失。.此外，本项目研究了脂肪细胞中对破骨细胞分化可能起调节作用的miRNA。双荧光素酶报告基因实验提示miR-30e为靶向调节OPG表达的miRNA。进一步研究显示miR-30e在前体细胞诱导成脂后表达上升，诱导成骨后表达下降。在3T3-L1细胞内增强miR-30e表达，3T3-L1细胞生长受抑制而分化加强，脂肪细胞分化关键转录因子PPARγ、C/EBPα、C/EBPβ和表征基因aP2等表达显著升高。相反，抑制3T3-L1细胞内源性miR-30e表达则可促进细胞增殖并抑制成脂分化。此外，抑制内源性miR-30e表达促进了MC3T3-E1成骨分化，Runx2、Osx、Osteocalcin、ALP和BSP等基因表达显著升高。通过TargetScan等软件预测结合荧光素酶报告基因测定证实了miR-30e能够结合Lrp6的3’UTR而阻断其表达。在3T3-L1细胞内下调Lrp6表达后，β-catenin/TCF信号减弱，成脂分化加强， PPARγ、C/EBPα、C/EBPβ和aP2等的表达显著升高。.本研究揭示了脂肪细胞可通过表达RANKL/OPG系统直接影响破骨细胞发育及其可能机制。