间接共培养法兔BMMSCs诱导分化为兔VSMCs

摘要

Objective Indirect co-culture of the different rabbit source of maturation vascular smooth muscle cells(VSMCs) and bone marrow mesenchymal stem cells(BMMSCs) to induce BMMSCs to differentiate into vascular smooth muscle-like cells and provide seed cells for tissue engineered blood vessels.Methods The BMMSCs(Hoechst33342 nuclear marked) and the different sources of rabbits’ VSMCs were indirectly co-cultured by the same percentage in the specially made culture flask.Immunofluorescence analysis was performed by using monoclonal antibodies against smooth muscleα-actin and calponin.The ultrastructure of BMMSCs were observed by transmission electron microscopy,to detect the orientation differentiation of BMMSCs.Results After the BMMSCs and VSMCs of different rabbit sources were indirectly co-cultured for 7 days,immunofluorescence staining results were visible,the nucleus blue dyed BMMSCs with anti the SM-α-actin(fluorescein Rhodamine marked appear orange-red) and anti-FITC-labeled calponin(fluorescein FITC marked appear green).Contrary cultured BMMSCs alone anti-SM-α-actin and anti-calponin was negative.After BMMSCs were indirectly co-cultured for 7 days the muscle iflamentous structures were visible under TEM.Conclusion Rabbit vascular smooth muscle cells from different sources can induce BMMSCs differentiation into vascular smooth muscle-like cells by indirect contact with the culture medium.%目的将分化成熟的不同兔来源的兔血管平滑肌细胞（vascular smooth muscle cells，VSMCs）与兔骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMMSCs）间接共培养，诱导BMMSCs分化为血管平滑肌样细胞，为组织工程血管提供种子细胞。方法将BMMSCs（Hoechst33342核标记）与不同兔来源的血管平滑肌细胞按相同的比例于特制共培养瓶中间接共培养7d后，用平滑肌细胞抗体SM-α-actin、calponin免疫荧光染色，并用透射电镜观察BMMSCs的超微结构改变，检测BMMSCs的定向分化情况。结果BMMSCs与其他兔来源的血管平滑肌细胞间接共培养7d后免疫荧光染色，可见胞核蓝染的BMMSCs与抗SM-α-actin（荧光素Rhodamine标记呈橘红色）、抗calponin（荧光素FITC标记呈绿色）的双标细胞出现；而单独培养的BMMSCs抗SM-α-actin和抗calponin为阴性。间接共培养后的BMMSCs透射电镜下可见肌丝状结构。结论不同来源的兔血管平滑肌细胞通过培养液间接接触可以诱导BMMSCs向血管平滑肌样细胞分化。