小鼠骨髓基质细胞的生物学特性及血管新生能力

目的 探讨体外培养的骨髓基质细胞的一些生物学特性及体内移植后在缺血区新血管生成中的作用。方法分离5—6周龄的小鼠胫骨、股骨，用预冷的DMEM／F12培养基冲洗出骨髓，经密度梯度离心分离出骨髓单个核细胞，接种后12～16d形成单层贴壁的成纤维样细胞。体外诱导分化鉴定分离的细胞，用传代的细胞进行生长曲线测定，观察其接种贴壁率、分裂指数，检测细胞周期和超微结构，并建立下肢缺血模型。荧光标记的体外扩增的骨髓单个核细胞被移植入缺血组织。移植后2周，荧光显微镜及内皮细胞碱性磷酸酶染色，检查荧光阳性细胞与染色阳性细胞的时空关系。结果体外传代培养的单个核细胞倍增时间约为42h。传代10h贴壁率达90％以上。分裂指数曲线与生长曲线相似。细胞周期显示约83％的细胞处于G1期。结论 体外培养的骨髓基质细胞生长稳定，传代后的细胞适应性强，增殖较快，表现出较早期细胞特点，在体外及移植入体内缺血区能分化为血管内皮细胞，有望用于改善组织缺血。

Biological properties and vasculogenesis of mouse bone marrow-derived stromal cells

Objective To establish a method of isolating, culturing and identifying mouse bone marrow stromal cells(MSCs) in vitro and vasculogenesis in vivo and explore their biological properties in order to provide an experimental foundation for applying MSCs to improve ischemia Methods After the tibias and femurs were dissected from 5 to 6 week old mice, the marrow was flushed out with ice cold DMEM/F12 medium The mononuclear cells of the marrow were obtained with density gradient centrifugation and the plated and cultured in DMEM/F12 medium The cultured cells in vitro were identified with their potential differentiation, growth curve, adhesive rate, cleavage index, cell cycle, ultrastructure and vasculogenesis were observed Results The adherent fibroblast shaped cells approached confluence in single layer 12 to 16 d after plating The cultured MSCs in vitro differentiated into endothelium The double time of subcultured MSCs was about 42 h More than 90% of subcultured MSCs were adhesive in 10 h Cleavage index curve was similar with the growth curve The cell cycle analysis showed that about 83% of MSCs was in G 1 phase MSCs transplanted into ischemic tissues took part in vasculogenesis Conclusion The subcultured MSCs possess potential differentiation into vascular endothelium cells MSCs show stable growth in vitro, easy survival in the subculture and rapid proliferation in present culture condition MSCs may be able to be used in therapy of myocardium ischemia in the future