Foxc1在颅面部骨组织再生和雌激素响应中的作用及机制研究

Recent researches demonstrated a close relation between Forkhead box C1（Foxc1） and the development, differentiation and metabolic homeostasis of craniofacial bone and eyes. However, The detailed role and mechanism of Foxc1 remain to be elucidated. In our previous researches, we examined the cellular phenotype, gene expression profile and osteogenic-adipogenic differentiation of a novel Foxc1-over-expression cell line, and conducted a preliminary study on the role of Foxc1 in estrogen response of bone homeostasis using an ovariectomized mice model. We speculated that Foxc1, as downstream target of BMP-2 and estrogen, may play an important role in the process of bone tissue regeneration and estrogen response of bone homeostasis through promoting osteogenic-differentiation and inhibiting adipogenic-differentiation of precursor cells. This project aims to perform a series of cell and molecular biology experiments such as lentivirus transfection, flow cytometry, western blot, realtime-PCR,immunofluorescence,gene chip,chromatin immunoprecipitation and dual luciferase labeling, combined with animal studies using subcutaneous ectopic osteogenesis model, critical sized mice parietal bone defect model and ovariectomized mice model, to take sights into the role and mechanism of Foxc1 in craniofacial bone regeneration and estrogen response of bone homeostasis.

近期研究表明，Foxc1基因与颅面部骨骼、眼等组织的发育、分化及代谢稳态密切相关，但其具体作用及机制尚有待阐明。本课题组在前期工作中对Foxc1过表达细胞株的细胞表型、基因表达谱及成骨、成脂分化功能进行了部分研究，并利用卵巢切除去势（Ovx）小鼠模型对Foxc1在骨稳态雌激素响应中的作用进行了初步探索，我们推测Foxc1可能作为BMP-2、雌激素等生物活性因子的下游作用靶点，通过促进前体细胞成骨分化、抑制其成脂分化，参与调控骨组织再生和骨稳态雌激素响应过程。针对该假说，本项目拟利用慢病毒转染、流式细胞术、免疫印迹试验、Realtime-PCR、免疫荧光、表达谱基因芯片、染色质免疫共沉淀、双荧光素酶标记等一系列细胞分子生物学实验，并结合裸鼠皮下成骨模型、小鼠颅顶骨临界缺损模型及Ovx小鼠模型研究，从体外实验和体内实验两个层面探讨Foxc1在颅面部骨组织再生和骨稳态雌激素响应中的作用及其机制。

近期研究表明，Foxc1基因与颅面部骨骼、眼等组织的发育、分化及代谢稳态密切相关，但其具体作用及机制尚有待阐明。本课题研究通过构建Foxc1过表达及敲除慢病毒载体，研究了Foxc1对小鼠前成骨细胞系MC3T3-E1细胞细胞形态、细胞周期、细胞增殖、凋亡及骨向分化的影响，发现过表达Foxc1基因可促进细胞成骨分化；我们利用Agilent mRNA基因芯片检测、生物信息学分析和实时定量PCR技术对比MC3T3-E1 Foxc1过表达稳转系细胞和对照组细胞基因表达谱差异，初步筛选出Foxc1基因在细胞体外成骨分化过程中可能调控的数个成骨分化相关靶基因，如Runx2、IBSP、Bmp4等；通过Luciferase实验及CHIP实验，突破性的发现Foxc1可以通过结合于Runx2的启动子调控Runx2表达从而直接促进细胞成骨分化，同时抑制miRNA-103-3p表达水平从而间接调控细胞成骨分化。通过构建大鼠颅顶骨缺损模型，进一步证实Foxc1参与了PTH调控的骨组织再生和Runx2表达。该研究为Foxc1促进骨形成机制提供新的理论依据。