摘要
目的:探讨在不同成熟状态下树突状细胞(dendritic cell,DC)分泌的外泌体(exosomes,Dex)的制备、提取及体外功能鉴定。方法:F344大鼠骨髓来源DC培养6后分为2组,一组为未成熟树突状细胞组(immature DC,imDC):直接收集上清;一组为成熟树突状细胞组(mature DC,mDC):每孔加入终浓度为1μg/ml的LPS培养48h后,收集上清。然后用多步离心法分离得到Dex,流式细胞术检测两组Dex的表型,酶联免疫吸附测定(ELISA)检测两组Dex与Wistar大鼠骨髓来源的imDC共培养上清的白细胞介素10(IL-10)、白细胞介素12(IL-12p70)的含量,混合淋巴细胞实验(MLR)检测共培养DCs刺激T细胞增殖能力。结果:与mDC分泌的exosomes相比,imDC分泌的exosomes表面中度表达MHC-Ⅱ类分子、低表达CD80、CD86、CD40共刺激分子,而且与Wistar大鼠骨髓来源的imDC共培养后刺激IL-12p70分泌能力较低(P<0.05),刺激T淋巴细胞增殖的能力也较mDC分泌的exosomes组弱(P<0.05)。结论:不同成熟状态下的DC分泌的exosomes功能各异,可能在宿主的免疫反应中发挥不同的作用。
Objective：To investigate the preparation,extraction and identification of exosomes （Dex） derived from the different maturity dendritic cells（DCs） in vitro. Methods：F344 rat bone marrow-derived DCs were cultured 6 days then divided into 2 groups, the immature dendritic cells group （immature DC,imDC）：the supernatant was collect;the mature dendritic cells group （mature DC, mDC）：the concentration of 1μg / ml of LPS was added,and after cultured for 48 hours,the supernatant was collected. The Dex was isolated by multi-step centrifugation. Flow cytometry was used to detecte Dex phenotype. Enzyme-linked immunoassay （ELISA） was used to detected the IL-10 and interleukin-12 （IL-12p70） levels of in the supernatant. Both groups of Dex were co-cultured with imDC derived from Wistar bone marrow. The co-cultured DCs ability to stimulate T cell proliferation was detected by mixed lymphocyte reaction （MLR）. Results：Compared with exosomes secreted from mDc,the imDC secreted exosomes expressing moderate levels of MHC-Ⅱ,low levels of CD80,CD86,CD40. And it has a weak stimulation to IL-12p70 secretion after co-cultured with Wistar rat bone marrow-derived imDC（P〈0.05）,as to as stimulation to T lymphocyte proliferation（P〈0.05）. Conclusion：The exosomes secreted by different maturity of DCs have different functions,may play different roles in the host immune response.
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
北大核心
2010年第3期324-327,333,共5页
Journal of Nanjing Medical University(Natural Sciences)
基金
国家自然科学基金面上项目(30872532)
