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目的探讨不同浓度的5-氮胞苷(5-aza)对人骨髓间充质干细胞(hMSCs)向心肌细胞分化的影响。方法抽取人胸骨骨髓3~5 ml,采用密度梯度离心法和贴壁法进行hMSCs的分离培养。于第3代生长状态良好的hMSCs中,分别加入3、5、10、20和40μmol/L 5-aza处理24 h,更换新鲜的培养基继续培养;对照组的细胞始终用完全培养基培养。光镜下动态观察细胞形态的变化,4周后用免疫组化染色法检测心肌肌钙蛋白I(cTnI)、连接蛋白43(Cx43)的表达。结果经5-aza诱导处理后,hMSCs的形态变长、增大,排列更加紧密、整齐。免疫组化染色法检测表明,对照组与3μmol/L 5-aza组cTnI、Cx43的表达呈阴性,5、10、20和40μmol/L 5-aza组cTnI和Cx43的表达均呈阳性。在一定范围内(<10μmol/L的5-aza),随着5-aza浓度的增加,hMSCs向心肌细胞的转化率逐渐增高;但高浓度(>10μmol/L)5-aza的毒性可造成细胞大量死亡。结论hMSCs经适当浓度的5-aza处理后,可向心肌细胞分化,10μmol/L的5-aza为最适诱导浓度。
Objective To investigate the effects of different concentrations of 5-aza on the differentiation of human bone marrow mesenchymal stem cells (hMSCs) into cardiomyocytes. Methods Human sternal bone marrow (3 ~ 5 ml) was isolated and cultured by density gradient centrifugation and adherence method. The third generation of hMSCs with good growth condition were treated with 3, 5, 10, 20 and 40 μmol / L 5-aza for 24 h, respectively, and replaced with fresh medium for further culture. Cells in the control group were always cultured with complete medium. The changes of cell morphology were observed under light microscope. The expression of cardiac troponin I (cTnI) and connexin 43 (Cx43) was detected by immunohistochemistry 4 weeks later. Results After induced by 5-aza, the morphology of hMSCs became longer and larger, and arranged more closely and neatly. Immunohistochemical staining showed that the expression of cTnI and Cx43 in the control group and 3μmol / L 5-aza group was negative, while the expressions of cTnI and Cx43 in 5,10,20 and 40μmol / L 5-aza group were all positive. In a certain range (<10μmol / L 5-aza), the conversion of hMSCs into cardiomyocytes gradually increased with the increase of 5-aza concentration; however, the toxicity of high concentration (> 10μmol / L) A large number of cells died. Conclusions hMSCs can differentiate into cardiomyocytes after treatment with appropriate concentration of 5-aza, and 10μmol / L 5-aza is the optimal inducing concentration.