Exosome负载的RBP4蛋白在颅缝早闭症中的分子机制研究

Crouzon syndrome is one of the frequent congenital deformities with the premature fusion of calvarial sutures. Althought the syndrome is known as a disorder caused by mutation in FGFR2 gene, its molecular mechanism is still largely unkown. Our previous microarray analysis identified a exosome-enriched RBP4 (Retinol binding protein 4), which was differentially expressed in in unfused sutures. The differential expression in RBP4 was further verified in mice model of Crouzon symdrome. Subsequently, our group successfully isolated and identified the exosome from mouse osteoblasts. All these results indicated exosome-enclosed RBP4 might play a important role in Crouzon syndrome. Based on the hypothesis, we firstly attempt to investigate the function of exosome in the fusion of calvarial sutures by FGFR2C342Y osteoblasts lenti virus-mediated and mouse model of Crouzon symdrome. Additionally, the role of exosome-enclosed RBP4 will be evaluated by targeting interference or overexpression of RBP4 in the process of sutures fusing. The project is planed to elucidate the molecular mechanism of Crouzon syndrome from the view of protein transfer between cells and provide a scientific basis for the development of targeting therapy for the desease.

Crouzon综合征是由FGFR2突变所导致的一种常见的先天性颅缝早闭症，但其分子机制目前尚不明了。前期我们在受累患儿颅缝组织的基因芯片中筛选出一种富含于Exosome中的差异表达蛋白—RBP4(Retinol binding protein 4)，且该差异表达在Crouzon综合征模型小鼠中进一步被证实。随后，本课题组还从小鼠成骨细胞中分离并鉴定出Exosome。上述结果提示Exosome负载的RBP4可能在Crouzon综合征中发挥重要作用。基于此，本项目拟先从慢病毒介导FGFR2C342Y突变型成骨细胞系、模型小鼠两个方面，探讨Exosome在颅缝闭合过程中的作用。其次，通过靶向干扰和过表达RBP4蛋白，从体内外水平分析RBP4对Exosome介导颅缝闭合过程的影响。本项目旨在从细胞间蛋白转运的角度揭示Crouzon综合征的分子机制，并为开展该类疾病的靶向治疗提供一定的理论依据。

FGFR2C342Y突变是导致Crouzon综合征的颅缝提前闭合的关键性治病因素，但其分子机制目前尚不明了。前期通过基因芯片在受累的颅缝组织中筛选出一个富含于Exosome中的差异表达蛋白RBP4(Retinol binding protein 4)，且该差异表达在Crouzon综合征的模型小鼠中进一步被证实，提示RBP4可能参与FGFR2C342Y突变所致的Crouzon综合征发病过程。基于此，本项目①通过慢病毒过表达系统，研究FGFR2野生型和C342Y突变型成骨细胞功能差异，并采用iTRAQ技术对野生型和突变型成骨细胞的蛋白表达进行差异分析，以全面分析FGFR2C342Y突变对成骨细胞功能的影响及其可能机制。结果显示：FGFR2野生型成骨细胞生长缓慢/细胞出现明显凋亡，KEGG pathway分析结果表明野生型FGFR2通过ECM-receptor interaction信号通路分子（FN1/COL2A1）调控成骨细胞周期，而 FGFR2C342Y突变通过激活Stat3转录因子调控成骨细胞增殖。②在鉴定出小鼠成骨细胞分泌的Exosome基础上，采用Nano-LC-ESI-MS/MS技术检测成骨细胞分泌的exosome包裹的蛋白组分。结果提示：Exosome负载的蛋白主要的生物学功能是核酸和蛋白结合的分子；它们涉及的主要pathway为EIF2信号通路。IPA进一步挖掘出exosome负载的参与调控骨发育的重要蛋白：EFNB1和TGFBR。③通过慢病毒过表达载体系统研究RBP4对成骨细胞功能的影响。结果研究发现：RBP4过表达可促进成骨细胞增殖/上调碱性磷酸酶水平，促进成骨细胞分化。上述研究结果，从新的视角揭示FGFR2C342Y突变所致的Crouzon综合征的潜在分子机制，为开展该类疾病的靶向治疗奠定理论依据。