摘要目的探讨骨髓来源的人间叶干细胞(mesenchymal stem cells, MSCs)生物学特性.方法利用Percoll梯度离心分离骨髓单个核细胞,将之接种于含10%已筛选胎牛血清的低糖DMEM培养基中.台盼蓝拒染法测定细胞增殖状态,MTT实验检测不同细胞因子对细胞增殖的影响.应用流式细胞学技术测定细胞周期及细胞表面抗原特征.同时观察了MSCs细胞组织化学特点.结果作者建立了一种简单的从骨髓中分离纯化及培养扩增MSCs的方法.MSCs具有独特的表征,即CD29,CD44及CD166阳性,CD34,CD45,HLA-DR和荆豆素阴性.细胞化学结果显示,几乎所有细胞酸性(萘酚醋酸酯酶(ANAE)及糖原(PAS反应)阳性,酸性磷酸酶(ACP)及苏丹黑反应(SB)阴性,而少数细胞碱性磷酸酶(ALP)阳性.细胞倍增时间约为30小时,细胞周期分析显示约10%细胞处于S期.MTT结果表明,MSCs对多种细胞因子的增殖反应性不同,TNF-α, IFN-γ, 干细胞因子 (SCF)及胰岛素样生长因子-1 (IGF-1)明显促进细胞的增殖,所试其它因子对细胞生长影响不大.结论 MSCs是一群均一的细胞,具有独特的增殖、表征及组织化学特征.MSCs对细胞因子的不同反应性,为筛选合适的细胞体外扩增及维持体系提供了有意义的线索,同时也将加深人们对MSCs生物学特性的了解.

abstractsObjective　To study the biological characteristics of mesenchymal stem cells (MSCs) from human bone marrow. Methods　A culture of mesenchymal stem cells was initiated from bone marrow low-density mononuclear cells separated by Percoll Centrifugation and maintained in low-glucose Dulbecco's modified Eagle's medium (DMEM) with 10% selected fetal calf serum. Cell growth pattern and its responses to cytokines were evaluated by trypan blue exclusion and MTT test, respectively. Cell cycle and surface antigenic features were analyzed by flow cytometry technique. Cytochemistry characteristics of MSCs were determined. Results　Easy-handling methods to isolate and culture expand MSCs were developed in this study. MSCs were unique in their phenotypes. They were positive for CD29, CD44, CD166, and negative for CD34, CD45, HLA-DR and Ulex europaeus. Cytochemistry evaluation showed that MSCs were homogeneously positive for acid α-naphthl acetate esterase (ANAE), glycogen (periodic acid Schiff reaction, PAS), and negative for acid phosphatase (ACP) and the Sudan black reaction (SB). Around 5% of them were positive for alkaline phosphatase (ALP). The cells had a population doubling time of 30 hours and cell cycle analysis showed that approximately 10% of them were in S phase. MSCs grew at significantly different rates when incubated in the presence of various recombinant human cytokines, of which interferon γ, tumor necrosis factor α, stem cell factor and insulin-like growth factor promoted the proliferation of MSCs dramatically, while others tested had no effects on cell growth. Conclusions　MSCs are a homogenous population of cells that have unique growth, phenotypical and cytochemical characteristics. Furthermore, the diverse responses of MSCs to different cytokines provide a clue for the selection of optimal expansion and maintenance of MSCs.