Flt3L刺激体外培养小鼠骨髓源CD8α^+树突状细胞及其成熟状态分析

Ex vivo Flt3L stimulation of CD8α^+ dendritic cells derived from mouse bone marrow and analysis of their maturity

目的建立体外快速制备大量具有免疫活性的小鼠骨髓来源不成熟CD8α+DCs方法。方法实验分2组。Flt3L组:将小鼠骨髓前体细胞用含终浓度为100ng/ml Flt3L的BMDC培养基混悬后接种于细胞培养皿中,每平皿10ml,37℃培养至第7d收集细胞;GM-CSF+IL-4组:将骨髓前体细胞用含终浓度为20ng/ml GM-CSF、5ng/ml IL-4的BMDC培养基混悬后接种于细胞培养皿中,每平皿10ml,分别于37℃培养第3d和第5d半量换液,培养第7d收集细胞,采用磁选法分离CD11c+BMDCs,用流式细胞仪检测。结果 Flt3L刺激组CD11c+BMDCs得率为(95.07±0.09)%,GM-CSF+IL-4刺激组为(83.97±0.43)%,差异有统计学意义(P<0.01)。Flt3L刺激组CD11c+CD8α+BMDCs得率为(9.83±0.33)%,GM-CSF+IL-4刺激组刺激组为(4.06±0.17)%,差异有统计学意义(P<0.01)。Flt3L刺激组CD11c+CD8α+BMDCs表面几乎不表达CD40、CD80、CD86,仅低表达MHCI、MHCII,处于未成熟阶段。结论采用Flt3L刺激小鼠骨髓前体细胞的方法可获得大量未成熟的CD11c+CD8α+BMDCs,方法简单,得率较高,为开展CD8α+DCs相关基础和临床研究奠定了基础。

Objective To establish a method using ex vivo stimulation to rapidly prepare a large number of CD8α＋ im- mature dendritic cells that are immunocompetent from mouse bone marrow. Methods For cells stimulated with Flt3L, precursor cells from bone marrow were cultured in BMDC medium containing Flt3L at a final concentration of 100 ng/ml. Half of the 10 ml of medium was replaced with fresh medium on Days 3 and 5. Cells were collected on Day 7. For cells stimulated with GM-CSF ＋ IL-4, precursor cells were cultured in BMDC medium containing GM-CSF at a final concen- tration of 20 ng/ml and IL-4 at a final concentration of 5 ng/ml. Results Fh3L stimulation generated CD11c＋ BMI）Cs of more than 95% purity （95.07 ± 0. 09 %）, a rate significantly higher than cells stimulated with GM CSF＋ IL-4 （83.97 ~±0.43%）. Fh3L stimulation generated CD11c＋ CDSα＋ BMDCs at a rate close to 10% （9.83 ± 0.33%）, which is significantly higher than that （4. 06 ± 0. 17%） generated by GM-CSF ＋ IL-4 stimulation. Flt3L-induced CD11c＋ CD8α＋ BMDCs expressed low levels of CD40, CD80, CD86, MHCI, and MHCII, indicating the cells were immature. Conclusion Flt3L can stimulate precursor cells in mouse bone marrow to generate a large number of immature CD11c＋ CD8α＋ BMDCs. This is a simple method of preparing CD11c＋ CD8α＋ BMDCs with a high yield and can aid in basic and clinical research on CD8α＋ DCs.