人脐血间充质干细胞分离培养及向成骨细胞分化的研究.DOCVIP

临床医学论文-人脐血间充质干细胞分离培养及向成骨细胞分化的研究 ?????????????? 作者：农丕地，黄海玲，解继胜?? 【摘要】? 　　目的 拟建立一套较为简便、有效和实用的人脐血间充质干细胞（MSCs）体外分离培养体系，探讨其向成骨细胞分化的可行性。方法 由人脐静脉血获得MSCs，纯化培养后用含地塞米松、β-甘油磷酸钠和维生素C的培养液诱导，通过倒置显微镜观察、碱性磷酸酶染色检测诱导后细胞。结果 来源于脐血的单个核细胞经体外培养贴壁后出现形态学上可见间充质样的细胞，间充质样细胞为类似成纤维细胞样的细胞形态。诱导培养的细胞碱性磷酸酶染色呈阳性。结论 人脐血MSCs经合理的体外诱导培养后，可以分化为成骨细胞。 【关键词】? 胎血 间质干细胞 成骨细胞 细胞分化 地塞米松 β-甘油磷酸钠 　　?Abstract:? Objective? To establish a more simple, effective and practical isolation and culture system for human umbilical cord blood (HUCB)-derived mesenchymal stem cells (MSCs), and investigate the feasibility of turning them into osteoblasts.?Methods?MSCs isolated from HUCB with Percoll density gradient solution were culture-expanded because the cells attached and grew on dishes, then MSCs were subcultured in a condition medium including dexamethasone, vitamin C and ?-glycerophosphate. An inverted microscope, immunocytochemical staining for alkaline phosphatase (ALP) were applied to observe the MSCs proliferation capability and osteogenic property.?Results? The HUCB-derived mononuclear cells, when placed in culture, gave rise to adherent cells, which exhibited either a fibroblast-like or mesenchymal-like phenotype. The MSCs in induced culture showed? immunocytochemical staining for ALP.? Conclusion? The HUCB-derived MSCs can be cultured and differentiated into osteoblasts in vitro.?? 　　Key words:? fetal blood; mesenchymal stem cells; osteoblasts; cell differentiation; dexamethasone;? ?-glycerophosphate 　　脐血(human umbilical cord blood,HUCB)是胎儿出生时脐带内及胎盘近胎儿一侧血管内的血液，含有丰富的干细胞和祖细胞。目前研究证实脐血中存在的间充质干细胞(mesenchymal stem cells, MSCs)在一定条件下进行体外培养和诱导分化后能成为神经细胞(神经元和神经胶质细胞)、心肌细胞、软骨细胞［1～4］，可作为细胞治疗新的种子细胞和基因治疗新的载体细胞。随着组织工程学研究的深入，对于相关功能细胞研究逐渐扩展到干细胞领域，MSCs对骨组织修复有重要意义，本实验主要探索人脐血MSCs分离培养及分析其向成骨细胞分化的可行性。 　　1? 材料与方法 　　1.1? 材料? 　　主要试剂和仪器：Dulbecco改良培养基(DMEM)，胎牛血清（HYCLONE公司）；β-甘油磷酸钠、地塞米松（美国SIGMA公司）；Percoll分离液(PHARMACIA公司)；碱性磷酸酶（ALP）试剂盒（博士德公司）；其他试剂均为国产分析纯；CO2培养箱（SHELDONA公司）；AE31型倒置显微镜（MOTIC公司）。 　　1.2? 方法 　　1.2.1? 脐血MSCs的分离培养? 　　从胎盘脐静脉穿刺抽