猕猴间充质干细胞的培养及生物学特性研究

Study on Biological Characteristics of Cultured Rhesus Mesenchymal Stem Cells

本研究的目的是分离、培养猕猴的骨髓间充质干细胞(MSC),分析其表型及生物学特性。从猕猴股骨抽取骨髓,分离单个核细胞,24小时后去除非贴壁细胞,当贴壁细胞生长到80%融合时进行传代,传至5代后用流式细胞术检测细胞表型,在不同条件下诱导其多向分化,用特异性染色鉴定,用RTPCR法检测MSC表达的细胞因子mRNA。结果表明:贴壁生长的猕猴MSC主要为典型的成纤维细胞样形态,在对数生长期细胞的倍增时间约31-40小时,可以连续传代、成功扩增。第5代猕猴MSC细胞高表达Flk1、CD29、CD105和CD166,低表达造血细胞标记CD34、CD45和CD33。在不同诱导分化条件下,猕猴的MSC可以分化为成骨细胞及脂肪细胞。用RTPCR法检测,猕猴的MSC高表达IL-6、TGF-β,弱表达TNF-α,而I-L2、FasL和IFN-γ未被检出。结论:猕猴的MSC可以成功分离、培养,其生物学特性与人的MSC相似。

This study was aimed to isolate and culture rhesus mesenchymal stem cells (MSC), and to analyze its phenotype and biological characteristics. Bone marrow was aspirated from femur of rhesus, mononuclear cells were extracted, the nonadherent cells were removed after 24 hours, adherent cells were subcultured when they grew 80% confluence. After the fifth passage, the cells were used for examination. The phenotypes of MSC were analyzed by flow cytometry, differentiated cells were identified by relevant specific staining. Cytokines' mRNA expressed by MSC were detected by RT-PCR. The results showed that rhesus MSC gave rise to a population of adherent cells characterized by the presence of a predominant cell type with a typical fibroblast-like morphology. During the log phase of growth, MSC proliferated to a two-fold population at 31-40 hours. MSC could be ex vivo expanded by successive cycles of trypsinization, seeding, and culture. The phenotypes expressed on rhesus MSC were Flk-1,CD29, CD105, CD166 positive and CD34, CD45,CD33 nearly negative. In various induction differentiation conditions, rhesus MSC could differentiate into the osteoblast and lipocyte. IL-6, TGF-β were highly expressed,TNF-α was lowly expressed and IL-2, Fas-L, IFN-γ were not detected on rhesus MSC using RT-PCR method. It is concluded that rhesus MSC can be successfully isolated and culture-expanded and the biological characteristics of rhesus MSC are similar to those of human MSC.