体外心肌细胞共培养诱导骨髓间充质干细胞向心肌样细胞的分化

目的 探讨体外心肌细胞间接接触共培养诱导骨髓间充质干细胞(BMSCs)向心肌样细胞分化的效果，以寻找最佳的诱导条件。 方法 2~3周龄SD大鼠24只和新生1~3d SD大鼠96只。分别通过全骨髓贴壁筛选法和差速贴壁法获取BMSCs和心肌细胞（CMs）。根据诱导条件不同分成3组：A组：5-氮杂胞苷（5-Aza-CR）诱导组，取采用10μmol/L 5-Aza-CR避光诱导24h；B组：共培养CMs诱导组，实验开始时，CMs和BMSCs分开培养，待各自贴壁后进行共培养；C组：5-Aza-CR+共培养CMs诱导组，CMs接种于Transwell小室上层，下层接种5-Aza-CR诱导的BMSCs。在相差显微镜下连续观察各组BMSCs的形态变化，诱导至第2、4周时收集细胞。应用免疫组织化学法和免疫荧光方法检测诱导后的BMSCs α-横纹肌肌动蛋白（α-actin）和心肌肌钙蛋白T（cTnT）的表达情况。 结果 1.细胞形态的变化： B组细胞有聚集生长趋势，C组脱落或降解的细胞明显少于A组，但部分细胞内有脂肪空泡形成。2.免疫组织化学和免疫荧光检测结果均显示，诱导2周时，A、B、C组cTnT均呈阴性或低表达，α-actin均呈弱表达；诱导4周时各组cTnT和α-actin均呈阳性表达。A组cTnT阳性表达率和α-actin阳性表达率分别为（20.22±2.30）%和（28.05±2.45）%、B组为（21.18±1.30）%和（29.06±1.86）%、C组为（26.28±2.89）%和（33.91±2.18）%，且C组与A、B组比较差异均有统计学意义（P<0.05），但A组与B组组间差异无统计学意义（P＞0.05）。 结论 体外心肌细胞间接接触共培养可以诱导BMSCs向心肌样细胞分化，和5-Aza-CR共同诱导可提高诱导率。

Differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells via co-culture with cardiac myocytes induction in vitro

Objective To investigate the effect of indirect contact co-culture with cardiac myocytes induction in the differentiation of bone mesenchymal stem cells (BMSCs) into cardiomyocyte-like cells in order to find the optimal induction conditions. Methods 2-3 weeks old 24 SD rats and newborn 1-3 day old 96 SD rats were used. BMSCs were derived by adhesive screening, and cardiac myocytes（CMs）were isolated by differential adhesion. According to the induction conditions, the cells were divided into groups A, B, and C. In group A［the 5-azacytidine（5-Aza-CR）induction group］, 10 μmol/L 5-Aza-CR was used to induce for 24 hours under a light-free environment. In group B (the co-cultured CMs induction group), at the beginning of the experiment, the CMs and BMSCs were cultured separately; when each had attached, and they were co-cultured. In group C (the 5-Aza-CR and co-cultured CMs group), CMs were seeded in the upper side of the transwell chamber, and 5-Aza-CR-induced BMSCs were seeded in the lower side. The phase contrast microscope was used to continuously observe morphological changes of BMSCs in each group; the cells were collected in 2-4 weeks. Expression of α-sarcomeric actin and cTnT in induced BMSCs was inspected by immunohistochemistry and immunofluorescence. Results 1.Cells of group B had a trend of aggregate growth. Shedding or degrading cells of group C were significantly less than in group A, but fatty vacuolization appeared in a part of the cells. 2.Resultsof immunohistochemistry and immunofluorescence: The expression of cTnT in group A, B, and C was negative or at low levels at 2 weeks of induction, while the expression of α-actin was weak. The expression of cTnT and α-actin in each group was positive, respectively, at 4 weeks of induction. The positive expression rates of cTnT and α-actin were (20.22±2.30)% and (28.05±2.45)% in group A, (21.18±1.30)% and (29.06±1.86)% in group A, and (26.28±2.89)% and (33.91±2.18)% in group C. The difference of group A and C was statistically significant (P<0.05), but the difference of group A and B was not (P＞0.05). Conclusion CMs indirect contact co-culture can induce BMSCs to differentiate into cardiomyocyte-like cells, and induction effects could be improved by being induced with 5-Aza-CR together.