成年鼠骨髓间质细胞在体外培养中分化为平滑肌细胞

目的　探讨小鼠骨髓间质细胞在体外经条件培养基诱导定向分化成平滑肌细胞的可行性 ,为研究血管损伤后新生内膜形成机制奠定基础。方法　采用骨髓干细胞培养基和贴壁法从骨髓中分离骨髓间质细胞 ,通过补加人血管平滑肌条件培养基诱导骨髓间质细胞分化成平滑肌细胞 ,采用形态学和免疫荧光染色分析分化后的细胞特异性标志物 ,并测定 1μmol/L血管紧张素Ⅱ对细胞胞液钙离子浓度及细胞收缩功能的影响 ,通过加入氯沙坦抑制血管紧张素Ⅱ受体验证血管紧张素Ⅱ特异性激活钙通道引发的细胞收缩。结果　骨髓间质细胞经诱导在体外可传代生长 ,稳定传代 6代后 ,分化的细胞形态呈梭形平滑肌样 ,融合后形成峰谷状排列。表达平滑肌特异性蛋白标志物α 肌动蛋白、肌钙结合蛋白和平滑肌肌球蛋白。分化后的细胞经血管紧张素Ⅱ刺激产生瞬间胞液钙离子转移并偶联细胞收缩反应 ,细胞 Ca2 + ]i呈现瞬间增高变化 ,Ca2 + ]i为 2 131nmol/L± 14 0nmol/L ,经用氯沙坦后 Ca2 + ]i被阻断 ,为 4 0 2nmol/L± 31nmol/L。结论　骨髓间质细胞经体外诱导可分化为有收缩功能的平滑肌细胞 ,可用于平滑肌细胞分化和血管损伤后新生内膜形成起源等研究。

Bone marrow stromal cells of adult mice differentiate into smooth muscle cells in vitro

Objective To explore the potential of murine bone marrow stromal cells (MSCs) to differentiate into vascular smooth muscle cells so as to contribute to neointimal formation after vascular injury. Methods Murine MSCs were isolated from bone marrow and cultured in M199 with 10% FBS supplemented with medium conditioned from human smooth muscle cell culture to induce differentiation. Immunofluorescent technique was used to observe the morphology of cells to examine the smooth muscle characters. Vasoactive substance was added onto the cultured smooth muscle like cells. Inverted microscopy was used to observe the contractile changes of the cells. Results After 6 passages of subculture in differentiation medium, MSCs demonstrated smooth muscle cell like spindle shaped morphology and assumed a hill and valley growth pattern. Immunofluorescent technique revealed positive signals for alpha smooth muscle actin, calponin and smooth muscle myosin heavy chains. The smooth muscle cell like cells displayed robust calcium transients. In response to stimulation of vasoactive substance the Ca 2+ ] of the cells instantaneously increased which was coupled by prominent cell contraction. Conclusion MSCs can differentiate into vascular smooth muscle cells with contractile ability. This model may be useful in study of smooth muscle cell differentiation and the origin of neointimal cells after vascular injury.