文章摘要
孟〓松,杨晓俊,徐〓皓,朱春富,姜〓红,吴文溪.大鼠骨髓来源树突状细胞外泌体的提取及功能研究[J].南京医科大学学报,2010,(3):324~327333
大鼠骨髓来源树突状细胞外泌体的提取及功能研究
Preparation,extraction and identification of exosomes derived from the different maturity dendritic cells
  
DOI:10.7655
中文关键词: 树突状细胞  外泌体  混合淋巴细胞反应  免疫反应
英文关键词: dendritic cells  exosomes  mixed lymphocyte reaction  immune response
基金项目:国家自然科学基金面上项目(30872532)
作者单位
孟〓松 南京医科大学第一附属医院普外科,江苏 南京〓210029 
杨晓俊  
徐〓皓  
朱春富  
姜〓红  
吴文溪  
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中文摘要:
      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1 μg/ml的LPS培养48 h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?
英文摘要:
      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.
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