I型胶原通过其受体OSCAR调节早期骨细胞分化及破骨细胞形成参与骨重建的机制与应用

Collagen type I is widely used in our daily life and research, but it was usually looked as a structural protein although 90% of the bone matrix are collagens type I. Recently, the receptor of collagen in osteoclast, osteoclast-associated receptor (OSCAR), was found to bind to specific motifs within fibrillar collagens，then initiated the important signal pathway in cell growth,differentiation and activity. In our previously study, we found that collagen type I promoted the differentiation of osteoblasts into osteocytes, and enhanced the activities of osteoclasts. In this study, we will mimic the different phase of the differentiation of osteocytes through the contorted three-dimensional cell culture in vitro, analyze the expression of OSCAR in osteoblast, pre-osteocytes and osteocytes. Then we will validate the receptor-specifically regulation of collagen in cell differentiation by conditional OSCAR knock-out mice in osteoblast and osteocyte respectively, and discover the involved signal pathway after collagens type I binding to its' receptor OSCAR by synapses specific PCR array and transcription factors PCR array analysis, and also by inhibiting or recovering the related signal moleculars in the signal transductional pathway. The aim of the above mentioned studies was to validate our hypothesis: collagen type I regulates the bone remodeling through controlling the differentiation of pre-osteocyte and osteoclastogenesis by binding to it's receptor OSCAR. Finally, we will evaluate the potential roles of collagen type I peptides in the therapy of osteoporosis.

因作用机制未明，既往认为I型胶原是一种结构蛋白而难以决定细胞命运；事实上它是骨吸收与骨形成的主要有机成分，能包裹成骨细胞（OB）并为骨细胞分化所必须；最近其受体OSCAR的发现为它启动细胞内信号转导并决定细胞命运提供了依据；我们进一步研究发现：I型胶原能促进OB向骨细胞转化，初步证实成骨谱系细胞表达OSCAR，二者结合的受体后信号可能参与了OB向骨细胞转化以及破骨细胞的形成。本研究拟通过三维旋转培养模拟体内OB向骨细胞分化的不同阶段，检测胶原受体的表达规律并设计活性胶原肽；再分别用OB以及骨细胞特异性OSCAR剔除小鼠来确认I型胶原调节细胞分化的受体特异性；骨细胞突触与转录因子PCR阵列、通路信号分子特异性阻滞或恢复来探讨胶原与OSCAR结合后的受体后信号通路，最终验证我们的假设：I型胶原通过受体OSCAR调节前骨细胞分化与破细胞形成参与骨重建，并考察I型胶原多肽在骨质疏松中的应用价值。

骨重建失调是骨质疏松等代谢性疾病发生的根本原因，而骨吸收与骨形成是骨重建的基础。Ⅰ型胶原对细胞的增殖、生长、分化以及功能的行使等发挥重要作用。近来胶原蛋白的受体OSCAR首先在破骨细胞上被发现，我们的前期研究也确定成骨谱系细胞上存在胶原受体，因此需进一步确定Ⅰ型胶原在骨组织细胞上的受体及其作用方式，找出并设计与受体结合的核心活性多肽，阐明受体后的信号通路。我们通过三维培养重现成骨细胞向骨细胞转变的分化过程。骨细胞在体内表现出不同的功能状态，通过该模型建立了相对静息状态下在体骨细胞的体外模型并能在体外模拟其体内生存的微环境，证实Notch1可作为判断骨细胞不同功能状态的标志分子。此外通过三维培养确认了胶原受体OSCAR在成骨细胞向前骨细胞、骨细胞分化过程中的变化规律。设计并合成可与OSCAR结合的I型胶原多肽，利用原代骨髓单核细胞（BMMs）-小鼠颅骨骨片三维培养体系中收集的条件培养基（骨吸收上清），发现骨吸收上清中存在多条可与OSCAR结合的目的I型胶原多肽。用不同浓度多肽干预原代前成骨细胞并进行成骨诱导，发现其可低浓度抑制、高浓度促进成骨细胞分化。在体外破骨细胞分化和骨吸收模型中，用不同浓度的I型胶原多肽干预BMMs，发现其可低浓度促进、高浓度抑制多核破骨细胞的形成和骨吸收，该作用是通过与OSCAR结合调控下游转录因子NFATc1实现的。采用(D-Asp)8连接Ⅰ型胶原多肽的方法构建骨靶向Ⅰ型胶原多肽，体内动物实验证明了高浓度骨靶向I型胶原多肽可使去卵巢手术（OVX）骨质疏松小鼠骨量增加。建立OSCAR基因剔除小鼠，高浓度骨靶向I型胶原多肽干预OVX骨质疏松OSCAR基因敲除同窝野生型、杂合子、纯合子小鼠，发现高浓度多肽能使野生型OVX小鼠骨吸收降低、骨量增加，而OSCAR敲除后该作用消失，说明多肽通过与OSCAR结合调控骨吸收和骨量。本研究揭示了Ⅰ型胶原在骨重建中的新作用，进一步完善骨代谢调节的新机制，更为抗骨质疏松靶向治疗药物开发提供新的靶点和思路，具有极其重要的理论意义与临床应用价值。