骨髓间充质干细胞传代扩增培养与干细胞干性的维持关系的研究

The relationship between the cell culture periods and the stem character of bone marrow mesenchymal stem cells

目的建立一种体外分离、培养骨髓间充质干细胞（BMMSCs）的方法，研究BMMSCs传代扩增培养与干细胞干性的维持关系。方法采用全骨髓细胞贴壁培养法对大鼠BMMSCs进行分离、培养、传代。采用倒置显微镜观察各代细胞的形态，通过流式细胞术（FACS）观察各代BMMSCs干细胞干性，通过免疫荧光检测各代BMMSCS在肝硬化模型的表达。结果大鼠BMMSCs呈均一的成纤维细胞样。连续传代干细胞始终保持着旺盛的生长力及扩增力。FASC结果表明，CD29、CD44、CD90的表达率随着传代的进行逐渐增高，P5-P6时表达最高，之后其表达率逐渐下降，免疫荧光检测发现P5-P6代干细胞在大鼠肝硬化模型中表达最强。结论随着传代BMMSCs的干性逐渐增强，传代至P5-P6时干细胞干性最强，随着传代的继续其干性逐渐下降。

Objective To explore a method of the isolation and the amplification of bone marrow mes- enchymal stem cell （BMMSCs）in vitro, and study on the relationship between cells culture and the stem charac- ter of BMMSCs; Methods The BMMSCs were isolated, cultured and extended by adherent culture method. The cell morphology of each generation was observated by inverted microscope. The character of BMMSCs by fluorescence-activated cell sorting （FACS） and its expression in an animal model of liver cirrhosis of different generations BMMSCs was observed by immunofluorescence analysis. Result The morphology and histology of BMMSCs were identical to fibra-like shape. The stem cells still kept a higher ability of growth and amplification following subculture. The results of FACS showed that the expression of CD29, CD44, CD90 was gradually in- creased and the highest expression was in P5-P6, then the expression gradually decreased. The expression of BMMSCs of P5-P6 was also the highest in liver cirrhosis model. Conclusions The character of BMMSCs was gradually strengthen followed by stem cell extension and the P5-P6 were the most strongest, and its stem charac- ter was gradually declined following subculture.