Foxc2 稳定转染对C3H10T1/2细胞成骨性能的影响  

The effect of Foxc2 overexpression on the osteogenic properties of C3H10T1/2 cells

作　　者：王敏娇[1] 司家文[1] 李洪亮[1] 欧阳宁鹃 沈国芳[1] 

机构地区：[1]上海交通大学医学院附属第九人民医院·口腔医学院口腔颅颌面外科,上海市口腔医学重点实验室,上海200011

出　　处：《上海口腔医学》2016年第4期385-390,共6页Shanghai Journal of Stomatology

基　　金：国家自然科学基金(81371122,81570947)

摘　　要：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。PURPOSE： To investigate the effect of Foxc2 overexpression on osteogenic and adipogenic differentiation of C3H10T1/2 cells. METHODS： C3H10T1/2 cells were transfected with plenti-Foxc2 and selected with puromycin for stable clones. The expression of Foxc2 was determined by real-time PCR and Western blot. Cell proliferation was detected by CCK-8 kit. Cell cycle and apoptosis were detected by flow cytometry. The level of osteogenic biomarkers Runx2, OPN, OCN and adipogenic biomarker PPARγ were quantified by real-time PCR and Western blot. Alkaline phosphatase （ALP） staining and oil red staining were conducted to evaluate the effect of Foxc2 overexpression on osteogenic and adipogenic differentiation. Statistical analysis was performed using SPSS 17.0 software package. RESULTS： C3H10T1/2-Foxc2 cell line was successfully constructed and verified by direct sequencing and Foxc2 overexpression in vitro. Cell proliferation was reduced and cell cycle was blocked in G1/G0 phase. Enhanced ALP staining and reduced oil red staining were observed in C3H10T1/2-Foxc2 cells as compared with the control. Foxc2 overexpression up-regulated Runx2, OPN, OCN during osteogenic differentiation and down-regulated PPARγduring adipogenic differentiation. CONCLUSIONS： C3H10T1/2 cell line stably expressing Foxc2 gene was successfully established, cell proliferation was reduced, osteogenesis biomarkers were up-regulated during the osteogenesis by overexpression Foxc2, PPARγwas down-regulated during adipogenesis.

关 键 词：Foxc2基因 成骨分化 成脂分化 C3H10T1/2细胞 

分 类 号：R78[医药卫生—口腔医学]