过表达插头框转录因子C2经Wnt信号通路调节BMSCs成骨分化的实验研究

ROLE OF FORKHEAD/FOX TRANSCRIPTION FACTOR 2 OVER-EXPRESSION IN REGUL ATING OSTEO GENIC DIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS BY Wnt SIGNALING PATHWAYS

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LvFoxc2(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、1.0μmol/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m RNA相对表达量均逐渐减少,而成脂因子PPARγ-2蛋白和m RNA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。

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 ~tmol/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 （PPARy-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 PPARy-2 significantly increased （P〈0.05）, showing a dose-dependent manner. Conclusion The over- expression of Foxc2 gene in BMSCs may promote osteogenic differentiation by Wnt-β-catenin signaling pathway.