培养条件对小鼠髓源性树突状细胞生物特性影响的观察  

Effect of cultural methods on cytobiological characteristics of mouse bone marrow-drived dendritic cell in vitro

作　　者：何秋山[1] 张燕[1] 何敬波[1] 龚志敏[1] 易铁男[1] 

机构地区：[1]湖北文理学院医学院附属医院肿瘤中心,湖北襄阳441000

出　　处：《中华肿瘤防治杂志》2013年第18期1391-1394,共4页Chinese Journal of Cancer Prevention and Treatment

基　　金：湖北省卫生厅青年科技人才项目(QJX2012-40)

摘　　要：目的:明确GM-CSF浓度、IL-4及细胞起始培养密度对培养的小鼠髓源性DC生物特性的影响。方法:分离小鼠骨髓细胞,用100、20和2ng/mL GM-CSF联合或不联合IL-4以及起始细胞培养密度为2.5×105和7.5×105 mL-1进行体外诱导DC细胞培养,培养7d后收集细胞进行计数,同时用流式细胞仪检测CD11c+及CD11c+CD80+细胞的比例,并对GM-CSF联合或不联合IL-4培养的DC细胞吞噬肿瘤细胞的能力进行检测。此外以混合淋巴细胞反应(MLR)检测培养的DC细胞对淋巴细胞的剌激能力。结果:2ng/mL GM-CSF扩增的DC细胞为起始培养细胞浓度的0.21±0.05倍,与100ng/mL的0.52±0.10倍(t=10.311,P<0.001)及20ng/mL的0.46±0.04倍(t=6.934,P<0.001)差异均有统计学意义,但100ng/mL与20ng/mL组之间差异无统计学意义,t=1.333,P=0.152。此外,100ng/mL GM-CSF组CD11c细胞的比例低于低2ng/mL组,t=2.571,P=0.034;但100ng/mL与20ng/mL组差异无统计学意义,t=0.913,P=0.39。在20ng/mL GM-CSF时,IL-4的加入与否对培养的DC细胞扩增、纯度、成熟、吞噬及活化淋巴细胞的能力均无影响,起始培养细胞密度可影响细胞的扩增。结论:小鼠髓源DC细胞的培养中,GM-CSF浓度对细胞的扩增及成熟均很重要,高浓度GM-CSF有利于DC的扩增及成熟,而在GM-CSF为20ng/mL时,IL-4对DC细胞的培养并不是必需的。OBJECTIVE：To study the impact of cultural methods on biological characteristics of mouse bone marrow derived dendritic cells in vitro. METHODS：Bone marrow cells （BM） from C57BL mice were resuspended in RPMI1640 com- plete medium supplemented with different concentration GM CSF,and/or IL-4. At day 0,BM cells were seeded at 0.75 × 10^6 or 0.25 × 10^6 per 100 mm dish in 10 mL medium. At day 3 and day 5 another 10 mL and 5 mL complete medium containing the same concentration cytokines were added to the plates respectively. The expression of CD11c and CD11c＋ CD80＋ ,endocytosis and allo- geneic mixed lymphocyte reaction（MLR） capacity of DC were analysised at day 7. RESULTS： The amplification of cell was 0.52±0.10 and 0.46±0.04 times of initiation cultured cell in 100 ng/mL and 20 ng/mL respectively, which were higher than that of 2 ng/mL group（0.21 ± 0.05 ; t= 10.311, P〈0. 001; t= 6. 934, P〈0.001）. There was no significant difference between 100 and 20 ng/mL（t= 1. 333,P=0. 152）. For the percentage of CDllc＋ ,GM-CSF 100 ng/mL group was less than 2 ng/mL group （t= 2.571, P=0. 034）, but there were no difference between 100 ng/mL and 20 ng/mL （t= 0.913, P=0.39）. Ib4 didn＇t effect the amplifcation, purity, mature, endocytosis and MLR of DC induced by 20 ng/mL GM CSF. CONCLUSION：In the culture of mouse BM-DC,the concentration of GMCSF is very important for amplification and maturation of DC,but IL-4 is not necessa- ry in the culture of 20 ng/mL GM-CSF.

关 键 词：树突状细胞 骨髓 细胞因子 动物 实验 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]