GPC3在crouzon综合征中的颅缝早闭过程中的作用及分子机制

Crouzon syndrome is one of the frequent congenital deformities with the premature fusion of calvarial sutures. Althought the syndrome is identified as a disorder caused by mutation in FGFR2 gene, its molecular mechanism is still largely unkown. Our previous microarray analysis screened a FGFR2-binding partner (glypican-3，GPC3), which was differentially expressed in cranial sutures with craniosynostosis. The differential expression in GPC3 was subsequently verified in mice model of craniosynostosis, suggesting GPC3 may be a potential important gene in Crouson syndrome.Based on the hypothesis, we firstly attempt to investigate the function of GPC3 in the premature fusion of calvarial sutures by calvarial sutures of patients with Crouzon syndrome, cells and Fgfr2c(C342Y/+) Crouzon mouse model. Secondly, GPC3-binding proteins will be screened by co-immunoprecipitation following nano-liquid chromatography tandem mass spectrometry (LC-MS/MS) and the role of the GPC3-binding protein in Crouzon syndrome and its related important signal will be further elucidated by molecular techniques including PCR array. Additionally, the regulation of FGFR2 mutation in GPC3-mediated craniosynostosis will be explored. The project is planed to reveal the molecular mechanism of Crouzon syndrome and provide a scientific basis for the development of targeting therapy for the desease.

Crouz综合征是由FGFR2突变所致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。我们前期通过基因芯片在受累的颅缝组织中筛选出一个能与FGFR2相互作用的差异表达蛋白GPC3(glypican-3)，且该蛋白的差异表达随后在颅缝早闭的小鼠模型中得到证实。上述结果提示GPC3可能成为Crouzon综合征的潜在致病基因。基于此，本项目拟先通过患者的颅缝组织、细胞以及Fgfr2c（C342Y/+）小鼠模型，研究GPC3在Crouzon颅缝早闭过程中的作用。其次，采用免疫共沉淀-蛋白组学筛选GPC3相互作用的靶蛋白，并进一步通过PCR array等技术探讨GPC3相互作用的靶蛋白在Crouzon颅缝早闭中的作用及其相关的关键信号通路。此外，本项目还将探讨FGFR2突变对GPC3调控颅缝早闭过程的影响。本项目旨在揭示Crouzon综合征的分子机制，并为开展该类疾病的靶向治疗提供理论依据。

本项目立项于前期通过基因芯片在颅缝早闭症中筛选到的差异表达蛋白GPC3，旨在探究GPC3蛋白在FGFR2突变所致先天性颅缝早闭症中的作用机制。项目主要对以下几个内容进行了研究：（1）GPC3对于成骨细胞的功能影响。通过在小鼠成骨细胞中过表达GPC3可以促进细胞分化，抑制增殖。反之则相反。（2）筛选并鉴定与GPC3相互作用的靶蛋白,并初探其调控机制。通过Co-IP技术鉴定出谷氧还蛋白3（GLRX3）可能作为GPC3相互作用蛋白并通过Wnt信号通路进行调控。（3）FGFR2突变对GPC3 介导的成骨细胞增殖/分化调控的影响。FGFR2 突变型成骨细胞系细胞增殖能力明显增强。其突变可能涉及的关键信号通路为PI3Kpathway。通过本研究，基本明确了GPC3在颅缝早闭症中颅缝异常成骨的作用机制。进一步为研究该疾病的靶向治疗提供了理论依据。