中文摘要：

背景：近几年关于鼠、兔等小型动物骨髓间充质干细胞的研究较多,如何获得大型动物羊的骨髓间充质干细胞的报道还较少。目的：观察全骨髓培养法和密度梯度离心法对羊的骨髓间充质干细胞生物活性的影响。方法：取健康2月龄的阿勒泰大尾羊,麻醉后于髂后上棘穿刺抽取骨髓,分别采用全骨髓培养法和密度梯度离心法提取羊骨髓间充质干细胞,倒置荧光显微镜观察原代骨髓间充质干细胞的细胞形态及贴壁情况,记录两组细胞首次传代时间,流式细胞仪检测CD44抗原表达,MTT法测定细胞的生长曲线,VonKossa’s染色检测成骨诱导的分化潜能。结果与结论：两种分离方法均能得到较均一的梭形,三角形和多边形的BMSCs,胞核大胞浆丰富,CD44抗原表达阳性。全骨髓培养法较密度梯度离心法的细胞数量较多,传代时间略早。骨髓间充质干细胞诱导培养至第14～21天,两组的VoKossa染色均阳性。其中首次传代全骨髓贴壁法的传代细胞较密度梯度离心法的细胞贴壁较快,活力较好。提示全骨髓培养法是一种简单有效的提取方法。

英文摘要：

BACKGROUND：In recent years,there are numerous studies on bone marrow mesenchymal stem cells（BMSCs） from small animals such as rats and rabbits,but reports addressing BMSCs from large animals,such as sheep,are rare.OBJECTIVE：To observe the effect of two isolated methods on the biological activity of sheep BMSCs.METHODS：Fresh bone marrow of 10 mL was extracted from the posterior superior iliac spine of a healthy Altay sheep aged two months by puncture following anesthesia,and BMSCs were obtained by the whole bone marrow culture method and density gradient centrifugation method.The shape and adherent growth of primary BMSCs was observed under inverted fluorescence microscope.Primary culture time in two groups was recorded,and the CD44 antigen expression was identified by flow cytometry.Cell growth curve was measured by MTT assay.The potential of osteogenic differentiation was examined by Von Kossa＇s staining.RESULTS AND CONCLUSION：Using both of these two separation methods,more uniform spindle,triangle and polygon BMSCs could be harvested,the nuclei were big and cytoplasm was rich,which were positive for CD44 antigen.The whole marrow culture was better than the density gradient centrifugation in cells quantity,slightly early in culture time.Von Kossa staining was positive in the two groups after BMSCs were cultured for 14-21 days.The adherent growth of passaged BMSCs obtained by using the whole bone marrow culture method was faster than that using the density gradient centrifugation culture method,and the cell vitality was also better in the former.It is indicated that the whole marrow culture is an effective method of extraction.