5-氮胞苷浓度对体外骨髓间充质干细胞诱导分化为心肌样细胞的影响

Effect of different concentrations of 5-azacytidine on cells differentiation of swine bone marrow mesenchymal stem cells into cardiomyocyte-like cells in vitro

目的 探讨不同浓度5-氮胞苷(5-aza)体外诱导小型猪骨髓间充质干细胞(MSCs)向心肌样细胞分化的可能性及其对细胞扩增速度的影响。方法 10 ml骨髓，1．077g／ml Ficoll密度梯度离心法分离骨髓单个核细胞(MNCs)，第2代传代细胞分别加入3，5，10μmol／L浓度5-aza化学诱导24 h，与对照组连续培养4周，细胞爬片免疫荧光法鉴定结蛋白desmin及心肌特异性肌钙蛋白I(cTnI)的表达，电镜观察诱导细胞的超微结构变化。结果 5-aza化学诱导后MSCs形态变长、增大，排列更加紧密、整齐，5和10μmol／L组可见较多的多核细胞，部分管状结构，诱导率分别为36％和31％，无显著性差异；3 μmol／L组上述变化的细胞极少，3种浓度及对照组4周后细胞扩增数量分别为(3．81±0．40)×10~6、(3．76±0．62)×10~6、(3．67±0．80)×10~6及(2．90±0．21)×10~6，10μmol／L组增殖速度明显减慢(P＜0．05)。各组诱导后的细胞均未见自发搏动。免疫组化提示desmin及cTnI表达阳性，5和10μmol／L组电镜下出现肌丝样结构、心房颗粒及细胞间的缝隙连接。结论 小型猪MSCs经5、10μmol／L 5-aza化学诱导可分化为心肌样细胞，其中5 μmol／L 5-aza不仅可以诱导心肌样细胞而且对细胞增殖速度影响小。

Objective To investigate the potential of differertiation of mesenchymal stem cells （MSCs） derived from swine bone marrow into cardiomyocyte-like cells after exposure to different concentrations of 5-azacytidine （5-aza） in vitro. Methods Ten ml of bone marrow was taken from swine, and mononuclear cells （MNCs） were fleshly isolated by 1.077 g/ml Ficoll lymphocyte separating medium using density-gradient centrifugafion. The 2nd-generafion MSCs were exposed to the medium containing different concentrafions of 5-aza （3,5,10μmol/L） for 24 h for induction cellular diferentafion,then cultured in complete of medium for 4 weeks together with control group. Desmin and myocardium-specific cardiac troponin I （cTnI） were examined with irr^nunohischemistry on day 28 and the uhrastructure of induced cells were observed under dec＇Iron microscope. Results The MSCs were enlarged, lengthened and closely arranged after chemical inducement. Mulfinuclear cells and myotube structures were found more in 5 and 10μmol/L 5-aza treatment groups than in 3μmol/L and control groups. （3.81±0.40）×10^6、（3.76±0.62）×10^6、（3.67±0.80）×10^6 and（2.90±0.21）×10^6 cells were harvested from 3, 5, 10 μmol/L 5-aza and control groups respectively and no spontaneous beat was observed in all groups after 28 days of culture. The proliferation slowed down obviously in 10μmol/L group （ P 〈 0.05）. Immunohistoehemieal examination revealed positive expression of desmin and cTnI in 5 and 10μmol/L groups, but no heart-specific protein was found in 3μmol/L group or in control group. Transmission electron microscopy showed that the differentiated cells had myofilaments, atrial granule structures and crevice connection between two cells. Conclusion Purified MSCs from swine bone marrow can differentiate into cardiac-like muscle cells by 5, 10μmol/L 5-aza inducement in vitro and the 5 μmol/L 5-aza has little effect on cell proliferation.