骨形态发生蛋白2诱导P19细胞体外向心肌样细胞分化

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.

Bone morphogenetic protein 2 inducing P19 cells differentiating into cardiomyocyte-like cells in vitro

Objective To investigate the process of cell aggregation and myocardium differentiation after the P19 cells treated with the bone morphogenetic protein 2（BMP-2）. Methods P19 cells were cultivated with BMP-2 in suspension for 4 days in dishes containing a thin layer of soft agar to form cell aggregation.The aggregates were plated on gelatin-coated culture dishes, and cultured without BMP-2 for 19 days. The cultured cells were observed using a phase-contrast microscope. Immunohistochemical technique and laser scanning confocal microscope (LSCM) were used to examine the expression of α-sarcomeric actin and cardiac-specific troponin T(C-TnT) protein. The induced cells were evaluated using a transmission electron microscope. Results Cell masses appeared after P19 cells were suspension cultured with BMP-2 for 24 hours and then globular cell masses grew into a embryonic body(EBs) after 4 days. After the aggregates were plated on gelatin-coated culture dishes, the EBs were attached regular and the core was observed to be darker and had a higher cell density. After the EBs were attached for 8 hours, cells grew from the border of the EB and formed monolayered peripheral outgrowth. Central cells were undifferentiated cells, which were multilayered,smaller and had a higher nuclear-cytoplasmic ratio and grew faster. Some cells in the peripheral outgrowth became elongated, larger and radial at day 11 and expressed α-sarcomeric actin and C-TnT protein by day 19.Immunofluorescence double staining revealed that α-sarcomeric actin and C-TnT protein co-expressed in the cytoplasm. Under the transmission electron microscope, the differentiated cells derived from Pl9 cells had a single orbicular ovate cell nucleus and plenty of cell organs, including chondriosomes, endoplasmic reticulum and ribosome. Paralleled myofilaments were seen in the cytoplasma or beside the edge of membrane. Conclusion The combination of B