中文摘要：

目的：分离、纯化培养大鼠BMSCs，对其生物学特性进行检测和鉴定。方法：取大鼠长骨中的骨髓组织，以淋巴分离液梯度离心获得有核细胞层，分别以本实验室BMSCs专用培养基接种培养，经多次换液得到较纯的BMSCs，倒置相差显微镜下观察细胞形态。选择CD44、CD31、CD45、CD29、CD34及flk1，应用流式细胞仪对细胞的表面抗原标志进行检测。结果：倒置相差显微镜观察发现，接种后的24h内，有核细胞（包含骨髓基质细胞）开始贴壁，4-7d，贴壁细胞出现明显分裂增殖，10-14d细胞增殖旺盛，可见明显的细胞克隆团，贴壁牢靠。20d左右，在本实验室专用BMSCs培养基培养下，绝大多数细胞逐渐分化呈梭形、三角形或多角形，核为圆形或类圆形、居中，这些细胞发出的突起逐渐生长延伸并彼此相连，形态学上具有神经系细胞的明显特征。细胞表面抗原检测显示所培养的BMSCs为：CD34^-、CD45^-、CD31^-、flk1^-、CD29^＋、CD44^＋，与文献报道一致。结论：所采用的细胞分离培养方法简便可行，所获得的细胞其表型特征与文献报道一致，确为BMSCs。

英文摘要：

Objective： To culture rat BMSCs in vitro and explore its biologic characteristics. Methods： The nucleated cells in rat long bone marrow tissue were separated by gradient centrifugation iin lymophocyte separating liquid （density 1.077） and incubated randomly in the special BMSCs culture medium and DMEM/F12 respectively. The BMSCs were purified by discarding suspended cells through exchanging medium. The morphology of cultured BMSCs was observed under inverted phase contrast microscope. For identifing BMSCs, cell surface antigens, including CD44, CD31, CD45, CD29, CD34 and flkl were detected by flow cytometry. Results：Under microscope, the dissociated cells were round in shape with integral and clean outline. The attachment of bone marrow nucleated cells including BMSCs started ahead of 24h and finished within 72h. After eliminating suspended cells, the attached cells were purified and displayed round shape and reflected light well. During day 4 to 7, the cell division and proliferation were observed. During day 10 to 14, proliferation and distinct clones of cells were observed. Till 20d, the BMSCs cultured in special medium differentiated gradually and cell shape changed to shuttle, triangle or polygon. Cell processes spreaded and connected with each other, displaying morphologic features of neurocyte. Immuophenotye studies presented that the cultured BMSCs were CD34, CD45, CD31, flkl, CD29^＋ and CD44 positive.Conclusions： The results suggest that it is easy and feasible to dissociate and culture rat BMSCs, which can be identified by immuophenotye study.