摘要
从人骨髓中分离人间充质干细胞 (MSCs) ,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并观察细胞组织化学特点。结果 :MSCs具有独特的表征 ,即CD2 9,CD44 ,CD16 6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 ,约5 %细胞碱性磷酸酶 (ALP)阳性。细胞倍增时间约为 30h ,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。提示 :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。
Culture of mesenchyma stem cells was initiated from bone marrow low-density mononuclear cells separated by Percoll centrifugation and maintained in low-glucose DMEM with 10% selected fetal calf serum. Cell growth pattern and its response to cytokines were evaluated by trypan blue exclusion and MTT test respectively. Cell cycle and surface antigenic features were analyzed by flow cytometry technique. Likewise, cytohistochemical characteristics of MSCs were determined. Results: Easy-handling methods to isolate and culture MSCs were developed in this study. The cells 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), Sudan black reaction (SB) and that approximately 5% of them was positive for alkaline phosphatase (ALP). The cells had a population doubling time of about 30 hours and cell cycle analysis showed that 10% of them was in S phase. MSCs grew significantly at different rates when incubated in the presence of various cytokines, of which TNF-α, IFN-γ, SCF, IGF-1 dramatically promoted cell growth. Conclusions: MSCs are a homogenous population of cells that have unique growth, phenotype and Cytohistochemical characteristics. Furthermore, the diverse responses of MSCs to different cytokines provide a clue for selection of optimal expansion and maintenance of MSCs.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2000年第5期346-348,共3页
Medical Journal of Chinese People's Liberation Army
基金
国家"973"基金!资助课题 (编号G19990 5 430 2 )
