目的探讨骨形态发生蛋白2(BMP-2)诱导P19细胞形成细胞聚集体并向心肌细胞分化的作用。方法将P19细胞接种于铺有0.5%软琼脂的培养皿中,BMP-2诱导悬浮培养4d形成细胞聚集体,将细胞聚集体用生长培养基黏附培养至19d。相差显微镜观察细胞形态学变化,应用免疫细胞化学、激光扫描共焦显微镜技术检测α-横纹肌肌动蛋白(α-sarcomeric actin)和心肌特异性肌钙蛋白T(C-TnT)的表达,透射电镜观察分化细胞的超微结构。结果经BMP-2诱导悬浮培养24h后即可见多个悬浮的细胞团形成,至第4天形成细胞聚集体/类胚体(EBs)。将EBs再用生长培养基黏附培养后,可见位于EBs中央的细胞密集、颜色较深,为EBs的内核。EBs贴壁生长后8h,细胞由EBs边缘逐渐爬出,在周边形成单层的生长晕,中央细胞多层排列,增殖速度较快,细胞体积较小,具有高核质比,为未分化细胞。培养至11d后,细胞生长晕中一些细胞变形,胞体伸长,体积增大,呈放射状排列。培养至第19天,EBs边缘生长晕中变形的细胞表达α-横放肌肌动蛋白和c-TnT蛋白。免疫荧光双标染色显示α-横纹肌肌动蛋白和C-TnT蛋白均定位于细胞质并且呈共表达。透射电镜下观察到,分化细胞呈杆状,细胞核呈卵圆形,位于细胞中央,细胞器丰富,并可见到平行排列的肌丝。结论 BMP-2暴露结合悬浮培养可以诱导P19细胞向心肌样细胞分化。 Objective To investigate the role of bone morphogenetic protein 2 (BMP-2) in inducing the formation of cell aggregates in P19 cells and their differentiation into cardiomyocytes. Methods P19 cells were inoculated on petri dishes plated with 0.5% soft agar. BMP-2 was induced to grow in suspension for 4 days to form aggregates of cells. Cell aggregates were cultured in growth medium for 19 days. The morphological changes of cells were observed by phase contrast microscope. The expression of α-sarcomeric actin and cardiac Troponin T (C-TnT) were detected by immunocytochemistry and confocal laser scanning microscopy. Transmission electron microscopy Observe the ultrastructure of differentiated cells. Results After suspended in PBS-2 medium for 24 h, many suspended cell clusters were observed, and cell aggregates / embryoid bodies (EBs) formed on day 4. EBs and then growth medium adhesion culture, we can see in the center of EBs dense cells, darker color, as the core of EBs. At 8h after adherent growth of EBs, the cells gradually climbed out from the edge of EBs and formed a monolayer of corona around the periphery. The central cells were arranged in multiple layers with a faster proliferation rate, smaller cell volume, higher ratio of nuclear to cytoplasm, . After culturing for 11 days, some of the cells in the corpuscle grew out of shape, cell bodies were elongated, enlarged in size and arranged radially. On day 19, cells deformed in the halo of EBs grew a-trans-actin and c-TnT proteins. Immunofluorescence double-stained staining showed that both α-striated actin and C-TnT proteins were localized in the cytoplasm and were co-expressed. Under the transmission electron microscope, the differentiated cells were rod-shaped, the nucleus was oval, located in the central cell, organelles are abundant, and can be seen in parallel arranged myofilament. Conclusion BMP-2 exposure combined with suspension culture can induce P19 cells to differentiate into cardiomyocyte-like cells.