小鼠骨髓源性树突状细胞体外诱导培养方法的比较研究

目的探讨最佳体外诱导培养小鼠成熟树突状细胞（dendritic cells，DC）的方法。方法分离、纯化6周龄C57BL／6小鼠骨髓单核细胞，以含10％胎牛血清、20ng／ml重组小鼠粒细胞-巨噬细胞集落刺激因子（GM—CSF）和10ng／ml重组小鼠白细胞介素-4（IL-4）的RPMI-1640培养基培养7d，然后将细胞分成对照未刺激组、肿瘤坏死因子-α（TNF-α）刺激组和TNF-α＋脂多糖（lipopolysaccharides，LPS）刺激组。继续培养48h后，观察各组细胞形态，检测IL-12、IL-6浓度及细胞表面标志CD11c、CD80、CD86和MHC II。结果培养9d后，两刺激组培养的细胞经相差显微镜观察有DC生长。TNF—α刺激组细胞培养上清液中IL-6、IL-12含量显著高于对照组（P〈0．01），但显著低于TNF—α＋LPS刺激组（P〈0．05）。3组均高表达CD11c，各组间无显著差异；而CD80、CD86和MHC II表达阳性率TNF-α刺激组显著高于对照组（P〈0．01），TNF-α＋LPS刺激组显著高于单纯TNF—α刺激组（P〈0．05）。结论联合使用TNF-α与LPS刺激可使DC成熟度提高，分泌IL-6、IL-12增加。

Comparison of in vitro Culture Methods of Mouse Bone Marrow Derived Dendritic Cells

Objective To explore optimal culture methods of dendritic cells （DCs） derived from mouse bone marrow in vitro. Methods Bone marrow-derived monocytes from 6-week-old C57BL/6 mice were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 20ng/ml recombinant murine granulocyte macrophage-colony stimulating factor （GM-CSF）, and 10 ng/ml recombinant murine interleukin （IL） -4. After 7 days, the cells were randomly divided into three groups： non-treated control group, TNF-α group, and TNF-α plus Lipopolysaccharides （LPS） group. The cells then were continuously cultured for 48h. Cellular morphological features of each group were observed. Cellular surface markers such as CD11c, CD80, CD86 and MHC-II were detected. IL-12 and IL-6 in supematant were tested by ELISA. Results After 8 days, the cells of two treated groups displayed typical ecphymata and of CD1 lc showed the same level in both control morphological characteristics of DCs. The expression and treated groups. In TNF-α group the expressions of CD80, CD86 and MHC-II were significantly higher than that of control group （P 〈0. 01 ） , but significantly lower than that of TNF-α plus LPS group （P 〈0.05） . In addition, IL-12 and IL-6 in TNF-α group was expressed significantly higher than that in control group （P 〈 0.01 ） , whereas lower than that in TNF-α plus LPS group （ P 〈 0.05） . Conclusion Under the condition of TNF-α plus LPS treatment, degree of maturity of DCs was most improved, with increase in secretion of IL- 6 and IL-12.