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Title: [ROLE OF FORKHEAD/FOX TRANSCRIPTION FACTOR 2 OVER-EXPRESSION IN REGULATING OSTEOGENIC DIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS BY Wnt SIGNALING PATHWAYS]. Author: You W, Wang J, Huang G, Zhang Y. Journal: Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi; 2016 Oct 08; 30(10):1276-1281. PubMed ID: 29786210. Abstract: OBJECTIVE: To investigate the role of the forkhead/Fox transcription factor 2 (Foxc2) over-expression in regulating osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by Wnt-β-catenin signaling pathways in vitro so as to provide the experimental basis for repairing osteonecrosis of the femoral head. METHODS: The recombinant lentivirus carrying green fluorescent protein (group A) or Foxc2 (group B) were used to transfect the fifth generation rabbit BMSCs, and untransfected BMSCs served as a control (group C). The cell viability was measured with water soluble tetrazolium-1 (WST-1) regent at 72 hours after transfection. After 2 weeks of transfection, the expression of β-catenin in BMSCs was detected by real time fluorescence quantitative PCR, Western blot, and immunofluorescence staining. Meanwhile, the β-catenin inhibitors XAV-939 (0, 0.1, and 1.0 μmol/L) was added in group B; at 2 weeks after osteogenic and adipogenic induction, the gene and protein expressions of collagen type I (COL I), osteocalcin (OCN), and peroxisome proliferator activated receptor gamma 2 (PPARγ-2) were detected by real time PCR and Western blot. RESULTS: WST-1 results showed that the cell viability of group B (130.85%±0.15%) was significantly higher than that of group A (100.45%±0.35%) (t=7.500, P=0.004) at 72 hours after transfection. At 2 weeks after transfection, the gene and protein expressions of β-catenin in group B were significantly higher than those in group A (P<0.01). After XAV-939 was added in group B, the mRNA and protein expressions of OCN and COL I gradually decreased; the mRNA and protein expressions of PPARγ-2 significantly increased (P<0.05), showing a dose-dependent manner. CONCLUSIONS: The over-expression of Foxc2 gene in BMSCs may promote osteogenic differentiation by Wnt-β-catenin signaling pathway. 目的: 观察过表达插头框转录因子C2(forkhead/Fox transcription factor 2,Foxc2)经Wnt-β链蛋白(β-catenin)信号通路调节兔BMSCs成骨分化,为基因转染BMSCs修复股骨头坏死提供理论依据。. 方法: 利用慢病毒携带Foxc2或绿色荧光蛋白(green fluorescent protein,GFP)基因的重组慢病毒载体Lv-GFP(A组)及Lv-Foxc2(B组)转染第5代兔BMSCs,以未转染BMSCs为对照(C组)。慢病毒转染后72 h采用水溶性四氮唑-1(water soluble tetrazolium-1,WST-1)法检测细胞活性;慢病毒转染2周,通过免疫荧光染色、Western blot及实时荧光定量PCR检测过表达Foxc2对β-catenin表达水平的影响。然后,在B组中添加不同剂量(0、0.1、1.0 μmol/L)β-catenin抑制剂XAV-939,成骨、成脂诱导2周后,采用Western blot和实时荧光定量PCR检测各组成骨因子Ⅰ型胶原(collagen typeⅠ,COLⅠ)、骨钙素(osteocalcin,OCN)及成脂因子过氧化物酶体增殖体激活受体γ2(peroxisome proliferator activated receptor gamma 2,PPARγ-2)蛋白和基因的表达。. 结果: WST-1检测示,转染72 h B组细胞活性为130.85%±0.15%,显著高于A组的100.45%±0.35%,差异有统计学意义(t=7.500,P=0.004)。转染2周后,B组β-catenin蛋白和基因表达均明显高于A组(P<0.01)。添加β-catenin抑制剂XAV-939后,细胞中成骨因子OCN、COLⅠ蛋白和mRNA相对表达量均逐渐减少,而成脂因子PPARγ-2蛋白和mRNA相对表达量明显增高,且表达具有剂量依赖性,差异均有统计学意义(P<0.05)。. 结论: 过表达Foxc2通过调节Wnt-β-catenin信号通路促进BMSCs成骨分化。.[Abstract] [Full Text] [Related] [New Search]