甲基转移酶抑制剂5-Aza-dC对膝关节纤维化中肌成纤维细胞活化增殖的影响

摘要

Objective To investigate the effect of methyltransferase inhibitor 5-Aza-dC (5-aza-2’-deoxycytidine) on proliferation and activation of myofibroblasts in arthrofibrosis of the knee. Methods The model of arthrofibrosis of rat knee was firstly established.Myofibroblasts were isolated and cultured from the posterior capsule of rat knee. Cells were identified by immunofluorescence.Myofibroblasts were treated with 2 μmol/L 5-Aza-dC. The expression levels ofα-SMA、col1A1 mRNA and protein in myofibroblasts before and after the treatment with 5-Aza-dC were detected by RT-PCR and Western blotting,respectively.The proliferation rate of these myofibroblasts were detected by MTT method,and the cell cycle was detected by flow cytometry of DNA content in each phase. Results The extension of rat knee in arthrofibrosis model was significantly decreased. α-SMA protein,the representational protein of myofibroblast,was largely expressed in the isolated cells from model knees. The expression levels ofα-SMA、col1A1 mRNA and protein in myofibroblasts were decreased after the treatment with 5-Aza-dC and the growth of myofibroblasts was also slowed down. Conclusion The methyltransferase inhibitor 5-Aza-dC may become a potential therapeutic drug in the treatment of arthrofibrosis of the knee through inhibiting the proliferation of myofibroblasts.%目的：探讨甲基转移酶抑制剂5-氮杂-2’-脱氧胞苷（5-aza-2-deoxycyfidine，5-Aza-dC）对大鼠膝关节纤维化中肌成纤维细胞增殖的影响。方法：膝关节固定法建立大鼠膝关节纤维化模型，取膝关节后关节囊，组织块培养法分离培养肌成纤维细胞。采用免疫荧光技术完成肌成纤维细胞的鉴定。用终浓度为2μmol/L 5-Aza-dC 处理肌成纤维细胞，四甲基偶氮唑盐（MTT）法检测肌成纤维细胞增殖变化。应用Quantitative Real-Time PCR 和蛋白质印迹法检测经5-Aza-dC 处理前后肌成纤维细胞中α-SMA、col1A1 mRNA和蛋白的表达情况。流式细胞术检测肌成纤维细胞周期的变化。结果：大鼠膝关节纤维化模型中膝关节伸直角度明显减少。细胞免疫荧光观察到从纤维化模型中分离培养的细胞大量表达标志性的α-SMA蛋白。5-Aza-dC处理肌成纤维细胞后，α-SMA、col1A1 mRNA及蛋白表达明显降低；5-Aza-dC处理组的肌成纤维细胞活化增殖受到明显抑制。结论：甲基转移酶抑制剂5-Aza-dC可能通过抑制肌成纤维细胞活化增殖而成为潜在的膝关节纤维化治疗药物。