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为了体外同时获得符合鉴定标准的造血干/祖细胞(hematopoietic stem/progenitor cell,HSC/HPC)和间充质干细胞(mesenchymal stem cell,MSC),本研究采用Ficoll淋巴细胞分离液方法从脐带血中分离出脐带血单个核细胞,MACS免疫磁珠法分离出CD34~+的造血干细胞。分离出的细胞与MSC同时接种在培养瓶中,利用15%AB型脐血浆的IMDM培养基添加白细胞介素3(interleukin-3,IL-3)、IL-6、血小板生成素(thrombopoietin,TPO)、干细胞因子(stem cell factor,SCF)和Flt-3配体(FMSlike tyrosine kinase 3 ligand,Flt-3L)因子培养体系来同时培养扩增HSC/HPC和MSC。为了评估扩增出的HSC/HPC和MSC是否符合细胞鉴定标准,本研究通过倒置显微镜观察HSC/HPC和MSC生长状态,并计数细胞。流式细胞仪检测HSC/HPC表面抗原CD34阳性百分率,检测第4代(P4)MSC表面抗原CD105、CD90、CD73、CD45、CD34和HLA-DR阳性表达率。半固体集落培养检测HSC/HPC的粒细胞-巨噬细胞集落形成能力。将共培养至P4的MSC行成骨、成软骨、成脂肪诱导鉴定。通过秋水仙素法进行MSC核型分析。结果显示,体外共培养的HSC/HPC依然有高增殖能力、集落形成能力和CD34阳性百分率。MSC具有向成骨细胞、脂肪细胞和软骨细胞诱导分化的能力。MSC染色体核型维持稳定。以上结果提示本研究成功建立了一种合适的MSC和HSC/HPC混合培养体系,通过该体系可同时获得两种干细胞,共培养后的MSC依然有典型的MSC的生物学功能;共培养后的HSC/HPC的粒细胞-巨噬细胞集落培养、细胞数量和流式表型符合鉴定标准,HSC/HPC生物学功能没有受到影响。
In order to obtain both hematopoietic stem / progenitor cells (HSC / HPC) and mesenchymal stem cells (MSCs) in vitro and in vitro, Ficoll lymphocytes were isolated from cord blood Isolated cord blood mononuclear cells, MACS immunomagnetic beads method to isolate CD34 ~ + hematopoietic stem cells. The isolated cells were seeded into culture flasks simultaneously with MSCs. Interleukin-3 (IL-3), IL-6, and thrombopoietin (TPO) were added into IMDM medium supplemented with 15% AB- ), Stem cell factor (SCF) and Flt-3 ligand (Flt-3L) factor culture system were used to simultaneously culture and amplify HSC / HPC and MSC. In order to assess whether the expanded HSCs / HPCs and MSCs meet the criteria of cell identification, we observed the growth status of HSC / HPC and MSCs by inverted microscope and counted the cells. The positive percentage of HSC / HPC surface antigen CD34 was detected by flow cytometry, and the positive expression rates of CD105, CD90, CD73, CD45, CD34 and HLA-DR on the 4th generation (P4) MSC were detected. Semi-solid colony culture assay of granulocyte-macrophage colony-forming ability of HSC / HPC. MSC co-cultured to P4 line osteogenic, cartilage, adipogenic induction identification. MSC karyotyping was performed by the colchicine method. The results showed that in vitro co-cultured HSC / HPC still have high proliferative capacity, colony formation ability and percentage of CD34 positive. MSCs have the ability to induce differentiation into osteoblasts, adipocytes and chondrocytes. MSC chromosome karyotype remained stable. The above results suggest that this study successfully established a suitable MSC and HSC / HPC mixed culture system, through which two kinds of stem cells can be obtained at the same time, and the MSC still have the typical biological function of MSC after co-culture HSC / HPC granulocyte-macrophage colony culture, cell number and flow phenotype met the identification criteria, HSC / HPC biological function was not affected.