小鼠骨髓来源树突状细胞体外诱导培养及其不同生长状态差异性探索

In vitro induction and amplification of marrow-derived dendritic cells in mice and exploring difference under different growth conditions

摘要:

目的：体外诱导扩增获取大量高纯度小鼠骨髓来源树突状细胞（dendritic cells，DC），并研究DC不同生长状态、不同培养时间的生物学特性差异。方法：重组小鼠粒细胞巨噬细胞集落刺激因子（recombinant-murine granulocyte-macrophage colony-stimulating factor，rmGM-CSF）体外诱导小鼠骨髓细胞分化为DC，在培养接种后0 h及1 d、4 d、7 d、9 d、11 d用倒置光学显微镜动态观察DC的数量、存活率及形态学变化。分别收集9 d不加入重组小鼠白细胞介素-4（recombinant mouse interleukin-4，rmIL-4）及加入rmIL-4刺激的悬浮细胞和贴壁细胞，流式细胞术（flow cytometry，FACS）检测悬浮细胞和贴壁细胞表面分子CD11c、CD80、CD86、MHC-Ⅱ的表达水平。收集培养9 d、11 d的DC，利用FACS分析BMDC细胞CD11c表达水平。采用SPSS 17.0软件进行统计学分析，两独立样本间的比较采用t检验。结果：培养0 h及1 d、4 d、7 d、9 d、11 d的BMDC成活率均为90%～95%。培养4 d，大量集落细胞团形成并逐渐增多。培养9 d，细胞明显变大，表面可见明显不规则树突或伪足。培养9 d，流式细胞术显示悬浮细胞表面标志CD11C+ CD80+、CD11C+CD86、CD11C+MHC-Ⅱ的表达比例分别为（88.10±2.41）%、（84.60±3.26）%、（92.90±3.93）%，高于贴壁细胞的（79.17±2.32）%、（75.40±4.41）%、（82.77±4.80）%，差异有统计学意义（P<0.05）。加入rmIL-4刺激与不加入rmIL-4 BMDC中CD11c+表达差异无统计学意义（P>0.05）。培养11 d，悬浮细胞及贴壁细胞的CD11c+表达明显高于培养9 d的表达（P<0.05）。结论：体外简易诱导培养可以获得高纯度小鼠骨髓来源的各具特性DC；rmGM-CSF单独或者联合rmIL-4诱导刺激对DC特异表面标志物CD11c+无明显影响；悬浮细胞的成熟度高于贴壁细胞；延长培养时间可提高CD11c+ DC的产量及纯度。

Abstract:

Objective：To obtain a large number of high-purity mouse marrow-derived dendritic cells（DC） through in vitro induction and amplification，and to explore its biological characteristics with different cultivating time and under different growth conditions. Methods：Marrow-derived cells of mouse were induced to differentiate into DCs in vitro by using recombinant-murine granulocyte-macrophage colony-stimulating factor（rmGM-CSF）. The cell count，survival rate and morphology changes of DCs were observed under the light inverted Microscope at 0 h，1 d，4 d，7 d，9 d and 11 d. On day 9，suspended cells and adherent cells without stimulation of mouse interleukin-4（rmIL-4） or with stimulation of rmIL-4 were collected，respectively；expression levels of CD11c，CD80，CD86 and MHC-Ⅱ on the surface of suspended cells and adherent cells by Flow Cytometry. On day 9 and 11，the DC was collected and the expression level of CD11c of BMDC was analyzed by FACS. SPSS 17.0 software was used for statistical analysis，and t-test was used for comparison between two independent groups. Results：On days 0 h，1 d，4 d，7 d，9 d and 11 d，the survival rate of BMDCs ranged from 90% to 95%. On day 4，a large number of colony cells were formed and gradually increased. On day 9，BMDCs became significantly larger and atypical protrusions and pseudopodia were able to be obviously found on the surface. On day 9，flow cytometry showed that expression ratio of cell-surface markers including CD11c+ CD80+，CD11c+CD86 and CD11c+MHC-Ⅱin suspended cell population were significantly higher than those in adherent cell population [（88.10±2.41）% vs. （79.17±2.32）%，（84.60±3.26）% vs. （75.40±4.41）%，（92.90±3.93）% vs. （82.77±4.80）%]，with significant differences（all P<0.05）. The expression of CD11c+ in BMDCs had no significant dif－ference in rmIL-4 groups and non-rmIL-4 groups（P>0.05）. After cultivating for 11 days，both the suspended CD11c+ cells population and adherent CD11c+ cells population were significantly higher than those after cultivating for 9 days（P<0.05）. Conclusion：A simple induced cultivation in vitro can obtain highly purified marrow-derived DC with different characteristics. There is no difference of the CD11c+ cells between the differentiation of DCs with rmGM-CSF alone and combination of rmGM-CSF and rmIL-4. The maturity of suspended cells population is higher than that of adherent cells population and prolonged cultivating time can increase the production and purity of CD11c+ DC.