Foxc2基因对MC3T3-E1细胞成骨能力影响的实验研究

Experimental study on the role of Foxc2 in the osteogenic differentiation of MC3T3-E1 cells

目的探讨Foxc2基因对MC3T3-E1细胞成骨能力的影响,并初筛其下游的靶基因。方法将多个成骨诱导因子作用于MC3T3-E1细胞,明确Foxc2的表达。采用慢病毒plenti-Foxc2及plenti-EGFP转染MC3T3-E1细胞,构建MC3T3-E1^(Foxc2+)及MC3T3-E1^(EGFP)。检测细胞增殖、细胞周期和凋亡的变化。采用real-time PCR和Western bolt检测成骨生物标志物Runx2的水平。通过ALP和ARS染色观察Foxc2过表达对成骨分化的影响。利用siRNA构建MC3T3-E1^(si-Foxc2)进行反向验证。通过基因芯片和生物信息学分析,检测MC3T3-E1^(Foxc2+)和MC3T3-E1^(EGFP)基因的整体表达谱。结果Foxc2基因在成骨诱导刺激下出现表达上调。成功构建的MC3T3-E1^(Foxc2+)过表达细胞组,细胞增殖受到抑制,Runx2表达上调。利用基因芯片和生物信息学分析比较MC3T3-E1^(Foxc2+)和MC3T3-E1^(EGFP)的整体基因表达谱差异,初步筛选出可能受Foxc2调控的成骨分化差异表达基因,如mmp9、Ereg等。结论Foxc2基因可通过抑制MC3T3-E1细胞增殖,上调Runx2表达,促进MC3T3-E1细胞成骨分化。mmp9和Ereg等基因可能参与这一成骨调控过程。

Objective To investigate the effects of Foxc2 in the process of osteogenic differentiation of MC3T3-E1 cells and preliminarily screen candidate target genes.Methods Foxc2 expression pattern in MC3T3-E1 cells was observed under the stimulation of osteogenesis related factors.MC3T3-E1 cells were transfected with plenti-Foxc2 and plenti-EGFP to construct MC3T3-E1^(Foxc2+)and MC3T3-E1^(EGFP).Cell proliferation,cell cycle and apoptosis changes were detected.The level of osteogenic biomarkers Runx2 was quantified by real-time PCR and Western bolt.ALP and ARS staining was conducted to evaluate the effect of Foxc2 overexpression on osteogenic differentiation.siRNA was used to construct MC3T3-E1^(si-Foxc2) as a reverse validation.The global gene expression profiles of MC3T3-E1^(Foxc2+)and MC3T3-E1^(EGFP) were evaluated by gene chip and bioinformatics analysis.Results Foxc2 gene expression level was increased during the osteogenic differentiation of MC3T3-E1 cells in vitro.MC3T3-E1^(Foxc2+)cell line was successfully constructed and verified.Foxc2 over-expression inhabited the cell proliferation and up-regulated Runx2 expression during osteogenic differentiation.The overall gene expression profiles of MC3T3-E1^(Foxc2+)and MC3T3-E1^(EGFP) were analyzed and compared by gene chip and bioinformatics,and the differential expression genes of esteogenic differentiation,such as Mmp9,Ereg,which may be regulated by Foxc2,were preliminary screeneed out.Conclusion Foxc2 can effectively promote MC3T3-E1 cells osteogenic differentiation in vitro by inhibition of cell proliferation,activation of Runx2 expression.Mmp9 and Ereg may participate in Foxc2-regulates osteogenic differentiation.