人脐带血间充质干细胞的体外分离、培养及鉴定

发布时间：2018-11-27 09:18

【摘要】：【目的】研究脐带血间充质干细胞体外分离、培养条件，探讨脐带血间充质干细胞作为细胞治疗种子细胞的可行性。【方法】无菌条件下取正常足月产脐带血36份，CAPD-1复合抗凝剂抗凝。其中18份脐血采用直接分离法，即将脐血稀释后，直接用Ficoll分离单个核细胞，分别采用含5％、10％、20％血清浓度的L-DMEM常规间充质干细胞贴壁培养；其余18份脐血采用间接分离法，即将脐血用5g／L的甲基纤维素先沉降出红细胞，然后将富集到的白细胞用Ficoll进行密度梯度离心，获得单个核细胞，同样分别采用含5％、10％、20％血清浓度的L-DMEM进行培养；另外，无菌条件下取11份骨髓，按常规间充质干细胞分离培养法，分离培养骨髓作为阳性对照。对不同方法富集到的脐血单个核细胞数量进行对比，对脐血原代和传代细胞的贴壁时间、贴壁细胞类型、生长速度进行观察记录，对各脐血组成功培养出间充质干细胞的例数进行统计分析，，对分离出的脐血间充质干细胞进行鉴定并与骨髓间充质干细胞对比，采用流式细胞仪考察脐血及骨髓中单个核细胞的细胞亚群以及间充质干细胞存在情况。
[Abstract]:[objective] to study the isolation and culture conditions of umbilical cord blood mesenchymal stem cells in vitro, and to explore the feasibility of using umbilical cord blood mesenchymal stem cells as cells to treat seed cells. [methods] 36 cases of normal term cord blood were collected under aseptic condition. CAPD-1 combined with anticoagulant was used. 18 cord blood samples were separated by direct separation method. Mononuclear cells were separated directly by Ficoll after dilution of cord blood. The normal mesenchymal stem cells (MSCs) were cultured with L-DMEM containing 5% 10% serum concentration and 20% serum concentration respectively. The other 18 cord blood samples were separated indirectly, that is, the cord blood was precipitated out of red blood cells with methyl cellulose from 5g/L, and then the enriched white blood cells were centrifuged by density gradient centrifugation with Ficoll to obtain mononuclear cells. L-DMEM with 20% serum concentration was cultured. In addition, 11 samples of bone marrow were isolated and cultured as positive control according to the routine method of mesenchymal stem cell isolation and culture under aseptic condition. The number of cord blood mononuclear cells enriched by different methods was compared. The adherent time, adherent cell type and growth rate of umbilical cord blood primary and passage cells were observed and recorded. The cases of mesenchymal stem cells cultured successfully in each cord blood group were statistically analyzed. The isolated mesenchymal stem cells were identified and compared with bone marrow mesenchymal stem cells. The subsets of mononuclear cells and mesenchymal stem cells in cord blood and bone marrow were investigated by flow cytometry.
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329

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