Bmi-1基因表达与人骨髓间充质干细胞增殖关系的探讨

Relationship of Bmi-1 gene expression and human mesenchymal stem cell replication

目的:建立人骨髓间充质干细胞(hMSCs)的体外培养体系,研究hMSCs表面抗原的表达情况,以及体外传代培养对Bmi-1基因表达变化的影响,为进一步探讨Bmi-1基因在hMSCs增殖中的作用及调控机制提供依据。方法:骨穿抽取骨髓,以1.077g/ml的Ficoll分离液梯度密度离心,收集单个核细胞进行培养和传代,观察细胞形态和生长周期。取P4代的hMSCs,流式细胞仪检测细胞表面抗原的表达情况。通过SA-β-gal的染色检测细胞的老化程度,通过抽提各代次细胞的RNA,逆转录PCR定量Bmi-1基因量的变化,分析与细胞倍增代数的关系。结果:体外培养的hMSCs贴壁生长,呈长梭形,可增殖形成克隆。经分离纯化培养后,流式细胞仪显示细胞表型为CD13,CD105,少量表达CD33,不表达造血细胞的标志CD34及CD117。SA-β-gal的染色显示随细胞培养时间延长,SA-β-gal染色更明显和清晰。逆转录PCR显示随传代次数的增加,hMSCs的增殖能力逐渐下降,Bmi-1基因表达随培养时间而减少。结论:本培养体系可获得纯度较高的hMSCs,具有强大的增殖能力。Bmi-1基因极可能是影响MSC增殖能力的一个重要基因。

Objective：To establish the cultural system of hMSCs in vitro,investigate the changes of Bmi-1 gene expression in different passages,and explore the effect of Bmi-1 in hMSCs replicative senescence. Methods：hMSCs were isolated from the femur bone marrow using adherence method and cultured in L-DMEM with 10% fetal bovine serum（FBS）. The 4 th passage hMSCs were picked up for analyzing expressions of cell surface phenotype,and the 1 st,3 rd,6 th,9 th,12 th,15 th and 20 th passages were examined of Bmi-1 gene expression by RT-PCR. Results：hMSCs were successfully isolated from the femur bone marrow which were positive for CD13,CD105,CD33 and negative for CD34,CD38,CD117,CD45. The SA-β-gal staining showed that the senescent cells were more suffused than the vigorous cells. The Bmi-1 mRNA was detected in hMSCs, and the gene expression decreased related to the cell passage. Conclusion：hMSCs that can be successfully isolated from femur bone marrow are easy to proliferate. The replicative senescence of hMSCs has correlation with the expression level of Bmi-1 gene.