DNA去甲基化对大鼠骨髓间充质干细胞增殖细胞核抗原的调节

背景：DNA去甲基化是一种重要的表观遗传修饰。 目的：观察DNA去甲基化对骨髓间充质干细胞增殖及增殖细胞核抗原蛋白表达的影响。 方法：全骨髓贴壁培养法分离培养大鼠骨髓间充质干细胞,免疫组化法鉴定骨髓间充质干细胞CD44和CD45蛋白的表达,MTT法检测5-杂氮胞苷对骨髓间充质干细胞增殖的影响,免疫组化法检测5-杂氮胞苷对骨髓间充质干细胞增殖细胞核抗原蛋白表达的影响。 结果与结论：①第3代骨髓间充质干细胞不表达或低表达CD45,高表达CD44。②与未加入5-杂氮胞苷组比较,5-杂氮胞苷干预48 h,6,12,24 μmol/L组显著促进细胞增殖活性(P < 0.05),无浓度依赖性。③与未加入5-杂氮胞苷组比较,5-杂氮胞苷干预48 h,6,12,24 μmol/L 组增殖细胞核抗原蛋白积分吸光度值显著增加 (P < 0.05),组间差异无显著性意义。结果显示在一定浓度范围内,5-杂氮胞苷发挥去甲基化作用,激活增殖细胞核抗原蛋白表达,从而促进骨髓间充质干细胞增殖。

Effect of DNA demethylation on proliferating cell nuclear antigen expression in bone marrow mesenchymal stem cells

BACKGROUND:DNA demethylation is an important epigenetic modification. OBJECTIVE:To explore the effect of DNA demethylation on proliferation and proliferating cell nuclear antigen (PCNA) protein expression in bone marrow mesenchymal stem cells. METHODS:Bone marrow mesenchymal stem cells were isolated by using the method of adhesive culture of whole bone marrow. In the third passage bone marrow mesenchymal stem cells, CD44, CD45 and PCNA protein were dectcted by immunocytochemistry, and effect of DNA demethylation on proliferation was detected by MTT method. RESULTS AND CONCLUSION:①The third passage bone marrow mesenchymal stem cells expressed CD44 postively and CD45 negatively. ②Compared with control group, after 48-hour intervention of 5-azacytidine, 5-azacytidine with 6, 12, 24 μmol/L increased proliferation of cells without concentration-dependence significantly (P < 0.05). ③ Compared with control group, after 48-hour intervention of 5-azacytidine, 5-azacytidine with 6, 12, 24 μmol/L increased PCNA protein expression significantly in bone marrow mesenchymal stem cells without concentration-dependence (P < 0.05). In certain concerntration, 5-azacytidine can increase PCNA protein expression thirough DNA demethylation, which is the key to improve proliferation of bone marrow mesenchymal stem cells.