骨髓间充质干细胞向血管平滑肌样细胞的诱导

Induction of bone marrow mesenchymal stem cells to vascular smooth muscle-like cells

背景：已经证实应用维甲酸和双丁酰环磷酸腺苷作为诱导剂,可以在体外建立胚胎干细胞分化为平滑肌细胞的模型。目的：尝试应用维甲酸和双丁酰环磷酸腺苷诱导犬骨髓间充质干细胞向血管平滑肌样细胞分化,评价其作为血管壁种子细胞的可行性。设计、时间及地点：随机对照,体外诱导细胞学实验,于2006-05/2007-12在北京协和医院胸心外科实验室和桂林医学院附属医院中心实验室完成。材料：成年杂种犬10只,雌雄不拘,用于取骨髓。方法：骨髓穿刺法获取犬骨髓,密度梯度离心和贴壁生长的方式提取单个核细胞;取第2-4代细胞,用含维甲酸和双丁酰环磷酸腺苷的培养液诱导、分化为血管平滑肌样细胞。主要观察指标：相差倒置显微镜观察细胞形态学;免疫荧光显微镜观察β肌动蛋白的表达;电镜观察细胞超微结构的改变。结果：骨髓间充质干细胞经苷诱导2周后细胞融合成片形成峰谷样外观,β肌动蛋白呈阳性表达。透射电镜显示胞浆内可见肌丝形成,表现出比较原始的血管平滑肌细胞的特点。结论：维甲酸和双丁酰环磷酸腺苷可诱导骨髓间充质干细胞在体外定向分化为具有平滑肌细胞特性的细胞,将其作为构建组织工程化血管的种子细胞是可行的。

BACKGROUND： Embryonic stem cells could be induced to differentiate into smooth muscle cells by AT-RA and dibutyryl cyclic amp （db-cAMP）. OBJECTIVE： To explore the induction and differentiation of bone marrow derived mesenchymal stem cells （BMSCs） into vascular smooth muscle-like cells （VSMLCs） in vitro by AT-RA and db-cAMP. DESIGN, TIME AND SETTING： A randomized control experiment was performed at the laboratory of Department of Cardiothoracic Surgery of PUMCH and Central Laboratory of Hospital of Guilin Medical College from May 2006 to December 2007. MATERIALS： Ten adult hybrid dogs, irrespective of gender, were used for marrow collection. METHODS： The canine bone marrow was aspirated from sternum, BMSCs were isolated and purified by density gradient centrifugation and attachment method. The second to fourth passages of BMSCs were cultured in DMEM-LG with AT-RA and db-cAMP to induce differentiation of VSMLCs. MAIN OUTCOME MEASURES： Phase-contrast inverted microscope and electron microscope were used to observe morphological and ultrastructural changes. Fluorescence microscope was used to observe express of β -aetin. RESULTS： BMSCs showed peak-valley-like morphology after 2 weeks of induction. Immunofluorescence results showed β - action positive of VSMLCs. Primary muscle filament was observed by transmission electron microscope, which was initial characteristic of VSMLCs. CONCLUSION： BMSCs can be induced into VSMLCs with the characteristic of vascular smooth muscular cells by cultured with AT-RA and db-cAMP.