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目的 :探讨Foxc2基因过表达对体外培养的小鼠胚胎成纤维细胞系C3H10T1/2细胞生物学行为及分化能力的影响。方法:通过慢病毒转染构建Foxc2过表达C3H10T1/2细胞系,采用实时定量PCR和Western免疫印迹法检测转染后Foxc2蛋白的表达。应用CCK-8试剂盒检测细胞增殖,流式细胞仪检测细胞周期及凋亡,实时定量PCR和Western免疫印迹法检测过表达Foxc2对成骨、成脂相关基因(Runx2、OPN、OCN、PPARγ)表达的影响,分别通过碱性磷酸酶(ALP)染色和油红染色检测细胞成骨和成脂分化能力。采用SPSS 17.0软件包对数据进行t检验。结果:成功构建Foxc2稳定过表达的C3H10T1/2细胞系,发现Foxc2过表达阻滞细胞于G1期,抑制细胞增殖。在成骨诱导过程中,过表达Foxc2可以显著上调Runx2、OPN、OCN等成骨相关基因的表达。ALP染色显示,Foxc2稳定过表达细胞较对照组细胞染色深。在成脂诱导过程中,过表达Foxc2显著下调PPARγ基因的表达;油红染色显示,成脂分化进程被部分抑制。结论:Foxc2过表达抑制细胞增殖,促进细胞分化。其机制是上调Runx2、OPN、OCN成骨相关基因的表达,下调PPARγ的表达,促进C3H10T1/2细胞的成骨分化,抑制其成脂分化。
AIM: To investigate the effect of Foxc2 gene overexpression on the biological behavior and differentiation of mouse embryonic fibroblast cell line C3H10T1 / 2 cultured in vitro. Methods: The Foxc2 overexpression C3H10T1 / 2 cell line was constructed by lentiviral transfection. The expression of Foxc2 protein was detected by real-time PCR and Western blotting. Cell proliferation and apoptosis were detected by CCK-8 kit. Cell cycle and apoptosis were detected by flow cytometry. Real-time quantitative PCR and Western blotting were used to detect the expression of Foxc2 in osteoblast and adipogenesis related genes (Runx2, OPN, OCN, PPARγ) The effect of osteoblast differentiation and adipogenic differentiation were detected by alkaline phosphatase (ALP) staining and oil red staining, respectively. Data was t-tested using SPSS 17.0 software package. Results: The C3H10T1 / 2 cell line stably overexpressed by Foxc2 was successfully constructed and found that overexpression of Foxc2 blocked cells in G1 phase and inhibited cell proliferation. Overexpression of Foxc2 significantly up-regulated the expression of osteoblast-related genes such as Runx2, OPN and OCN during osteogenic induction. ALP staining showed that Foxc2 overexpression cells stained darker than control cells. Overexpression of Foxc2 significantly down-regulated the expression of PPARγ gene during adipogenic induction; oil red staining showed that adipogenic differentiation was partially inhibited. Conclusion: Overexpression of Foxc2 inhibits cell proliferation and promotes cell differentiation. The mechanism is to up-regulate the expression of osteoblast-related genes of Runx2, OPN and OCN, down-regulate the expression of PPARγ, promote the osteogenic differentiation of C3H10T1 / 2 cells and inhibit their adipogenic differentiation.