小鼠树突状细胞的体外培养与鉴定

In vitro culture and characterization of mouse spleen dendritic cells

目的探索、优化体外诱导和扩增小鼠树突状细胞(DC)的方法,并进行形态学观察和生物学鉴定.方法应用环磷酰胺200mg/kg经尾静脉注射Balb/c小鼠,8d后处死小鼠,常规培养脾细胞,第3日加入含有IL-4,GM-CSF细胞因子的条件培养液,第5日补加TNF-α,第8日制备标本进行相差显微镜、扫描电镜、透射电镜观察,并分别在培养第3,5,7日经流式细胞仪检测成熟细胞表面标志.结果刺激培养细胞经相差显微镜、扫描电镜、透射电镜观察,具有典型的DC形态,流式细胞仪检测发现细胞表面表达CD11c,CD86,ⅠAb,H2D6标志物.结论在小鼠脾脏细胞培养中加入IL-4,GM-CSF,TNF-α等刺激,可获得足量、较高纯度的DC,为DC的基础研究与临床应用奠定了基础.

AIM： To explore and optimize the methods for in vitro culture of mouse dendritic cells （DC） that were then observed morphologically and identified biologically, METHODS： Mice were injected with 200 mg/kg cyclophosphamide through the tail vein, and were sacrificed 8 d later. Spleen cells were cultured with ordinary methods firstly, and 3 d later conditional medium containing IL-4, GM-CSF was added, and TNF-α was added at day 5. At day 8, cells were subjected to phase-contrast microscope, scanning electron microscope, and transmission electron microscope analysis. At day 3, 5, 7, cells were subjected to FACS for detection of cell surface markers. RESULTS： Cultured ceils displayed a typical DC phenotype in morphological analysis, and FACS showed these cells expressing such DC markers as CDllc, CD86, Ⅰ Ab, H2D6. CONCLUSION： Using of IL-4, GM-CSF, TNF-α as stimulators in the mouse spleen cell culture can obtain DC with high quality and high purity, which makes a foundation for future research on the basic and clinical usage of DC.