新生小鼠心肌干细胞与心肌细胞共培养诱导分化试验

Study on Differentiation of Murine Cardiac Stem Cells Co-cultrued with Cardiomyocytes

目的了解心肌条件培养液有无诱导心肌干细胞(cardiac stem cells,CSCs)分化为心肌细胞的作用,CSCs与心肌细胞接触能否影响CSCs向心肌分化。方法用免疫磁珠分选原代CSCs,得到纯化的c-Kit^+ CSC。分3组培养:条件液组用心肌条件培养液诱导CSCs 21 d,混合组分别用PKH 26或DAPI标记CSCs后与心肌细胞混合培养8 d,空白组无干预培养21 d,观察CSCs形态变化。各组完成培养后,用免疫组化法鉴定肌钙蛋白Troponin-T和缝隙连接蛋白connexin 43表达。结果心肌条件培养液不能有效诱导CSCs表达心肌细胞特异蛋白Troponin-T和connexin 43,未见自发搏动细胞。CSCs与心肌细胞接触后,两者同步搏动。CSCs可表达Troponin-T和connexin43。结论心肌条件培养液无诱导CSCs向心肌细胞分化的作用,心肌细胞与CSCs接触能有效诱导CSCs向心肌细胞分化。

Objective To investigate whether CSCs can be induced into cardiomyocytes （CM）by myocardial conditioned medium,whether contact with CM is necessary to CSCslnyocardial differentiation. Methods Primary passage of CSCs were sorted with immunobeads to obtain c-Kit ＋ CSCs. There groups were estab- lished. The first group, With myocardial conditioned medium, CSCs were induced for 21 days. The second group, CSCs labeled with PKH 26 or DAPI were co-cultured with CM for 8 days. and blank group with no in- terference for 21 days. Observing morphologic changes everyday. Performing immunohistochemistry to detect Troponin-T and connexin 43 expression at the final day. Results Myocardial conditioned medium had no effect on inducing CSCs＂ expression of Troponin- T and connexin 43, no spontaneous beating events were ob- served. While CSCs in contact with CM expressed Troponin-T and connexin 43, and beat with CM synchro- nously. Conclusions Myocardial conditioned medium have no effect on conducting CSCs into myocardial differentiation. Contact with CM is a potent factor in CSCs＇ myocardial differentiation.