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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。
The purpose of this study is to isolate and culture the cynomolgus monkey bone marrow mesenchymal stem cells (MSCs) and analyze its phenotype and biological characteristics. Bone marrow was extracted from the macaque femurs, mononuclear cells were isolated, non-adherent cells were removed 24 hours later, passaged when adherent cells grew to 80% confluence, and passaged to passage 5 to detect cell phenotype by flow cytometry. Induced by multi-directional differentiation conditions, identified by specific staining, using RTPCR method to detect the expression of cytokines mRNA in MSC. The results showed that the adherent growth of Macaca mulatta MSCs was a typical fibroblast-like morphology. The doubling time of cells in logarithmic growth phase was about 31-40 hours, which could be continuously passaged and amplified successfully. The fifth generation of rhesus monkey MSC cells highly expressed Flk1, CD29, CD105 and CD166, low expression of hematopoietic cells labeled CD34, CD45 and CD33. Under different inducing and differentiation conditions, cynomolgus monkey MSC can differentiate into osteoblasts and adipocytes. By RTPCR method, the expression of IL-6, TGF-β in MSCs and the weak expression of TNF-α in MSC were not detected in I-L2, FasL and IFN-γ. Conclusion: Macaque MSCs can be successfully isolated and cultured, and their biological characteristics are similar to human MSC.