大鼠骨髓基质干细胞体外培养及诱导分化.docVIP

大鼠骨髓基质干细胞体外培养及诱导分化 　　[摘要] 目的 观察全骨髓贴壁培养法培养骨髓基质干细胞的效果。 方法 将4周龄健康SD大鼠脱颈处死后，无菌条件下取出股骨和胫骨，收集骨髓腔细胞悬液，L-DMEM培养基培养，通过全骨髓贴壁培养法将细胞纯化传代，观察其生长特性，绘制生长曲线。流式细胞仪检测细胞表面标记物CD29、CD45、CD90并作同型对照，细胞分别向成骨、成脂方向诱导分化，观察诱导结果。 结果 经全骨髓贴壁培养法培养的SD大鼠BMSCs呈集落状贴壁生长，镜下可见细胞成簇生长，类似鱼群状、菊花瓣状或漩涡状。BMSCs增殖速度快，细胞形态稳定，生长状态较好。生长曲线显示BMSCs符合正常细胞生长特征且生长活跃。流式细胞仪鉴定CD29和CD90呈阳性表达，CD45呈阴性表达。BMSCs能向成骨、成脂方向诱导分化。 结论 全骨髓贴壁培养法简单易行，可获得纯度高，活性好，性状均一的骨髓基质干细胞。 　　[关键词] 骨髓基质干细胞；分离纯化；培养；分化；鉴定 　　[中图分类号] R414.4 [文献标识码] A [文章编号] 1673-9701（2015）32-0030-05 　　[Abstract] Objective To observe the effect of BMSCs through the method of whole bone marrow adherent culture. Methods After 4-weeks-old healthy SD rats were killed by cervical dislocation， the femur bone and tibia bone were taken out under sterile conditions. Marrow cell suspensions were collected then cultured in L-DMEM medium. The method of the whole bone marrow adherent culture was used to passage purified. The growth characteristic was observed and the growth curve was drawn. CD29， CD45， CD90 of the cell surface markers were detected by flow cytometry and made an isotype control. The cells could be induced to differentiate into osteogenic and adipogenic direction respectively and the results were observed. Results Cultured by the method of the bone marrow adherent， the BMSCs of SD rat showed colony adherent growth and other sharp like cell clusters， similar fish，daisy-like petals or whirlpool under microscope. BMSCs had high rate of proliferation， stabilization of morphology and good growth condition. BMSCs showed the growth characteristics of normal cells and actively growing in the growth curve. The expression of CD29 and CD90 were identified positive by flow cytometry. The expression of CD45 showed negative. BMSCs could be induced to differentiate into osteogenic and adipogenic direction， respectively. Conclusion The method of whole bone marrow adherent culture is simple and can get bone marrow stromal cells of high purity， activity， and homogeneous traits. 　　[Key wo