脐血间充质干细胞分离,培养与鉴定.docVIP

脐血间充质干细胞分离、培养与鉴定 李启明1 程天民1 李宁2? 通信作者 粟永萍1 ? 通信作者 (1.第三军医大学全军复合伤研究所，创伤、烧伤与复合伤国家重点实验室 2.第三军医大学新桥医院, 高压氧中心 重庆400038) 摘 要 ：目的 研究脐带血(HUCB)中含有的间允质干细胞(MSCs)体外分离、培养条件，探讨其作为种子细胞用于下一步实验研究的稳定性与实用性。方法 无菌条件下取正常足生产的脐带血，经CPDA-1复合抗凝剂抗凝，然后经过去红细胞处理，再用淋巴细胞分离液分离脐血的单个核细胞，DMEM/F12培养基进行培养和纯化获得贴壁细胞层，采用定向诱导分化方法检测鉴定获得的MSCs 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，问充质样细胞为类似成纤维样的细胞形态，但偏向半月形弯曲状，经过成肌、成脂肪、成神经细胞诱导分化成功后确定为间充质干细胞。结论 脐血来源的间充质干细胞能够在体外分离、培养出有用的进一步实验所需要的种子细胞。 关键词：脐带血；间充质干细胞 ；细胞培养；诱导分化鉴定 中图法分类号： 文献标识码： THE ISOLATION CULTURE AND IDENTIFICATION OF HUCA MSCs LI Qi-ming, CHEN Tian-min LI ning, SU Yong-ping, RUAN xin-ze (1.State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, 2. Hyperbaric Third Military Medical Abstract: Objective To investigate the isolation，cultivation, differentiation and identification of human umbilical cord blood(HUCB) mesenchymal stem cells(MSCs) in vitro，and to observe the feasibility of using MSCs as seed cells in experimental furthermore． Methods HUCB were collected from full term childbirth scheduled for Cesarean section and vagina deliveries, a11 specimens was obtained sterilely with Preservative-free CPDA-1 compound natrium citricum. After lyse red blood cell, the cord blood mononuclear cell was isolated by human lymphocyte separating medium. culture with dulbeccos modified eagles medium(DMEM/F12) medium to produce adherent layer and identification with orientation differentiation results. Results The UCB-derived mononuclear cells，when set in culture，gave rise to adherent cells, which exhibited either a fibroblast-1ike but crescent-shaped or bending-shaped morphology or mesenchymal-like phenotype. It can be affirmed as the MSCs which characterized with multi-differentiating potentiality after the myogenic、lipogenic and neurogenic differentiation. Conclusion MSCs in HUCB can be isolated and cultivated in vitro，which could be regarded as an alternative source of MSCs for further experi