人树突状细胞的大量培养及鉴定

摘要 目的：探讨人树突状细胞体外大量培养及鉴定方法。方法：采用免疫磁珠法分离纯化CD34⁺干细胞；采用含有TPO、SCF、Flt3L和IL-3的扩增培养基培养1周，以及含有SCF、Flt3L、GM-CSF和IL-4的分化培养基培养2-3周，获得CD34⁺细胞来源树突状细胞。采用普通光学显微镜观察细胞形态，牛鲍氏血细胞计数板进行细胞计数，荧光抗体标记、流式细胞仪检测细胞纯度和细胞表面共刺激分子的表达情况。结果：以含有TPO、SCF、Flt3L和IL-3的培养基扩展培养一周，及含有SCF、Flt3L、GM-CSF和IL-4的培养基诱导分化3周，可获得大量悬浮细胞；细胞数目扩增倍数约达50倍；普通光学显微镜下可见悬浮细胞有明显的树突状凸起；流式细胞术检测结果显示悬浮细胞中CD141和CD11c双阳性细胞（等同于单核细胞来源树突状细胞）比例达30%，此群细胞高表达HLA-DR和CD209，低表达共刺激分子CD80和CD86；细胞寿命较短，40天时培养体系中悬浮细胞和CD34⁺细胞来源树突状细胞数目急剧减少。结论：采用多细胞因子联合刺激可获得大量的树突状细胞，为树突状细胞的特性及功能学研究奠定了基础。

Culture and Identification of Human CD34+ Stem Cell Derived Dendritic Cells

ABSTRACT Objective: To explore a method to obtain mass human dendritic cells in vitro, and suitable method to identify them. Methods: CD34⁺ stem cells were isolated and purified from stem cell collection using immunomagnetic bead method, which was then cultured in the expansion medium containing TPO, SCF, Flt3L and IL-3, for one week, and differentiation medium containing SCF, Flt3L, GM-CSF and IL-4 for two to three weeks, CD34⁺ stem cells were induced to CD34⁺ derived dendritic cells in vitro. The morphology of these cells was observed using optical microscopy, and the cell numbers were counted using NiuBao''s blood count plate. After multi-color immunofluorescence labeling, purity of dendritic cell and expression of costimulatory molecules on cell membrane were detected by flow cytometry. Results: When cultured in the expansion medium containing TPO, SCF, Flt3L and IL-3 for one week, and differentiation medium containing SCF, Flt3L, GM-CSF and IL-4 for three weeks, a large number of suspension cells were obtained. The numbers of cell increased about by 50 times compared with the initial number. The dendritic bumps were observed obviously on cells under optical microscope. Among them, the proportion of CD141 and CD11c double-positive cells, which are similar to monocyte derived dendritic cells, is more than 30% of total cells by flow cytometry. These cells highly express HLA-DR and CD209, and express low levels of co-stimulatory molecules CD80 and CD86. The cell life span of cultured dendritic cells is short. The cell number of suspension cells and dendritic cells (CD141⁺ CD11c⁺ cells) decreases sharply by 40 days. Conclusion: A large number of dendritic cells could be obtained by stimulating CD34⁺ stem cells separated from stem cell collection by expansion and differentiation medium with multiple cytokines, which may provide foundation for the characteristics and functional study of dendritic cells.