珍珠质是一种广泛被应用的矿物质药物，之前的研究发现，珍珠质可以促进骨重建过程，且不会引发机体的免疫反应。经验证，珍珠质中的可溶性珍珠因子可以促进新骨形成，为珍珠质中发挥作用的主要活性成分；然而，可溶性珍珠因子这一混合物中的活性成分仍然未知。我们实验室的前期工作中发现，PFMG蛋白家族中的PFMG3、PFMG5等蛋白均表现出了良好的成骨活性。这一家族中的PFMG1蛋白，首先发现于合浦珠母贝的外套膜之中，具有两个EF-hand钙结合位点，且可以在体外影响碳酸钙晶体的生长和形成，但关于其是否具有成骨活性，尚未有报道明确指出。在本研究中，我们从细胞水平和动物水平两个方面出发，对PFMG1蛋白的功能进行验证，并探究了PFMG1蛋白在骨重建过程中发挥作用的机制。 细胞实验中，我们使用成骨细胞前体细胞MC3T3-E1细胞为研究对象，通过体外培养的方法检测PFMG1蛋白对体外细胞增殖、分化、矿化等过程的影响。结果显示，PFMG1蛋白可以促进成骨细胞前体细胞的增殖、改变细胞增殖周期分布；PFMG1蛋白可以通过激活一系列成骨相关基因opn、ocn、osx、runx2的表达、激活Erk信号通路来促进成骨细胞前体细胞的分化以及胞外基质矿化；此外，在体外培养基中，PFMG1蛋白还可以促进钙晶体的聚集；在破骨细胞诱导实验中我们发现，PFMG1蛋白能够抑制破骨细胞的形成。动物实验中，我们建立了骨质疏松小鼠模型，并建立慢病毒过表达体系，通过静脉注射的方式打入小鼠体内。动物实验结果表明，PFMG1蛋白可以缓解由于卵巢切除术引起的骨质疏松骨流失症状，这一抑制作用主要体现在抑制骨小梁流失方面，而非皮质骨。在此基础上，我们进行了RNA-seq分析来寻找PFMG1蛋白调控机制，通过对差异基因进行分析，发现了一系列与组织发育、骨化作用密切相关的基因例如Fzd5、Grem1、VdR等在PFMG1处理后表达量发生了显著改变。我们的研究结果为珠母贝基质蛋白促进成骨作用提供了新的有力证据；为珠母贝基质蛋白PFMG1转化为抗骨质疏松药物提供了理论依据。
Nacre is a widely used mineral medicine and has been reported to have beneficial effects in bone remodelling without increasing inflammation. The water-soluble nacre matrix has been demonstrated to promote new bone formation. However, the core active ingredients in nacre were unknown. Our previous study showed that PFMG3 and PFMG5, which belong to the PFMG family, can enhance the osteogenic activity. PFMG1, found in the mantle of Pinctada fucata, has two EF-hand calcium-binding domains and affects the growth of calcium carbonate crystals, but whether this protein has any effect on osteogenesis was unknown. In this study, we explored the function of PFMG1 in bone remodelling by conducting both cellular experiments and animal experiment, and discussed the possible mechanism how PFMG1 enhanced osteogenesis.In the cellular experiment, MC3T3-E1 cell line was used as a model, our result showed that PFMG1 promotes osteoblast proliferation and affects cell cycle distribution. PFMG1 accelerates osteoblast differentiation and matrix mineralization by increasing the expression level of osteoblastic marker genes like opn, ocn, osx, runx2 and activating Erk signaling pathway. PFMG1 also accelerates calcium crystal aggregation in culture medium and suppresses osteoclast formation.In the animal experiment, we first built ovariectomized mice model and the lentivirus expression system. Our result showed that PFMG1 prevents bone loss caused by ovariectomy without affecting the cortical bones. Base on what we have observed, RNA-seq analysis was conducted and found that PFMG1 stimulates genes associated with tissue development and ossification, like Fzd5, Grem1 and VdR. Our findings show the therapeutic potential of PFMG1 from nacre as a novel drug for osteoporosis.
