【摘要】 目的:培养人骨髓间充质干细胞(Human mesenchymal stem cell,hMSCs),探讨hMSCs体外向心肌细胞(Cardio-myocytes,CM)定向诱导分化的实验研究。方法:取人的骨髓血,用Percoll(1.073g/ml)密度梯度离心及贴壁筛选结合的方法体外培养扩增hMSCs,并进行流式细胞仪分析鉴定其免疫学表型,以未加一抗只加二抗的hMSCs作为平行对照组。选用生长良好、纯度达到95%的P5代hMSCs,用不同诱导浓度的5-氮杂胞苷(5-Azacytidine)1、5、10和20μmol/L进行诱导,对诱导后的细胞进行心肌特异性标志TroponinⅠ及Desmin的免疫组化鉴定。结果:体外分离纯化培养扩增出hMSCs,其CD44阳性率平均为93.26%±2.48%,与平行对照组(3.42%±1.09%)相比有明显差异(P<0.01)。经5和10μmol/L5-Aza诱导分化的hMSCs表达心肌特异性标记TroponinⅠ、Desmin;10μmol/L5-Aza诱导分化的hMSCs阳性率明显高于5μmol/L组;在20μmol/L组中,诱导后超过50%的细胞脱落死亡。结论:hMSCs可体外分离培养扩增,并具有向心肌细胞分化的潜能,5-Aza最佳诱导浓度为10μmol/L。更多还原

【Abstract】 Objective:To culture human mesenchymal stem cells(hMSCs)and study their myogenic differentiation in vitro.Methods:Human bone marrow mononuclear cells were selected by gradient centrifugation on percoll at a density of 1.073 g/ml.hMSCs were futher selected and expanded by adhereing peculiarity.The phenotypes of hMSCs were identified by fluorescent-activated cell sorting(FACS).Fifth passage hMSCs,having good growth characteristics and >95% purity were treated for 24 h with 1,5,10 and 20 μmol/L 5-azacytidine to induce cellular differentiation.Expression of the cardiac-specific marker-troponin Ⅰ and desmin,identified by immunohistochemistry,was used to identify cardiac muscle differentiation.Results:hMSCs expressed a high level of CD44(hMSCs 93.26%±2.48% vs 3.42%±1.09% in a control group,P<0.01).hMSCs treated with 5 or 10 μmol/L 5-azacytidine were demonstrated that troponin Ⅰ and desmin were positive.The number of troponin Ⅰ and desmin-positive cells in the culture treated with 10 μmol/L 5-azacytidine was visually greater than that in culture treated with 5 μmol/L 5-azacytidine.Over 50% of cells became necrotic after culture with 20 μmol/L 5-azacytidine.Conclusion:hMSCs can be successfully cultured and expanded in vitro.They have the potential to differentiate into cardiomyocytes.The optimal concentration of 5-azacytidine for induction of myocyte differentiation is 10 μmol/L.更多还原