摘要
本研究旨在探讨胞壁酰二肽(muramyldipeptide,MDP)激活NOD2信号通路对白血病抗原致敏的树突状细胞(dendritic cells,DC)的免疫调控影响。采用梯度离心法获取健康人外周血单个核细胞(peripheral blood mononuclear cells,PBMNC),体外给予3种细胞因子诱导培养7 d,第5 d给予白血病细胞株HL-60冻融抗原致敏DC,DC诱导成熟后,给予MDP(2000 ng/ml,24 h)刺激各组细胞。应用RT-PCR和Western blot检测NOD2 mRNA和蛋白表达,流式细胞仪分析各组DC表面分子,ELISA法检测各组DC培养上清中IL-12和p40表达。结果显示:MDP作用于经不同方式处理的DC后,可以刺激NOD2 mRNA和蛋白的表达,并以负载白血病细胞株HL-60冻融抗原并给予MDP刺激DC组(致敏DC+MDP组)最高,其显著高于仅给予MDP刺激无负载抗原DC组(DC+MDP组)和无MDP刺激致敏DC组,差异有统计学意义(P<0.05);DC表面分子(HLA-DR、CD80、CD83、CD86、CD40)在致敏DC+MDP组表达明显高于DC+MDP组和致敏DC组,未处理DC表达最低,差异有统计学意义(P<0.05);同样地发现,致敏DC+MDP组分泌细胞因子IL-12 p40最高为(898.30±61.08)pg/ml,显著高于DC+MDP组(573.86±32.09)pg/ml和致敏DC组(365.03±28.86)pg/ml,差异有统计学意义(P<0.05)。结论:MDP可明显上调致敏DC中NOD2 mRNA和蛋白表达,同时促进致敏DC表面HLA-DR、协同共刺激分子、黏附分子表达及炎性因子IL-12和p40分泌。本研究有望为DC在白血病免疫治疗中的应用提供新思路。
The purpose of this study was to explore the effect of NOD2 signalling pathway activated by muramyl dipeptide （MDP） on the immunomodulation effect of human monocyte-derived dendritic cells （DC） loaded with leukemia cell lysates. Peripheral blood mononuclear cells （PBMNC） were isolated by density gradient centrifugation, These cells were cultured with three cytokines for 7 days to induce their maturation. On the 5th day, cells were loaded with leukemia cell HL-60 lysates. NOD2 expression was detected by RT-PCR and Western blot. The phenotype of DC were analyzed by flow cytometry, and ELISA was used to assay levels of IL-12 （p40） . The results showed that MDP could trigger NOD2 mRNA and protein expression in different groups of DC, especially in sensitized DC ＋ MDP group, which was significantly higher than that in the DC ＋ MDP group and sensitized DC without MDP stimulation, the difference was statistically significant （ P 〈 0.05 ）. Besides, the expression of surface molecules （ HLA-DR, CD80, CD83, CD86, CD40） in the group of DC loaded with leukemia cell lysate and stimulated by MDP（ sensitized DC ＋ MDP） reached the highest level, followed by the group of DC loaded with leukemia cell lysate without MDP and DC only stimulated by MDP, non-treated DC were the lowest（P 〈 0.05）. Similarly, compared with untreated unstimulated DC, after loading with HL-60 lysates or only stimulating with MDP, the secretion of IL-12p40 increased, but IL-12p40 level （ 573.86 ± 32.09 pg/ml） in DC ＋ MDP group was higher than that in group of sensitized DC （ 365.03 ±28.86 pg/ml ） （P 〈 0.05 ）, and it in sensitized DC ＋ MDP group reached the highest （898.30 ± 61.08 ） pg/ml, compared to other groups （ P 〈 0.05 ）. It is concluded that MDP can significantly enhance the NOD2 mRNA and protein expression in sensitized DC, promote the expression of HLA-DR, synergistic costimulatory molecules and adhesion molecules of DC,at the same time, MDP can increase secretion of inflammatory factors IL-12p40. This study will provide a new ideas for DC application in leukemia immunotherapy.
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2013年第6期1591-1596,共6页
Journal of Experimental Hematology
基金
湖北省自然科学基金(编号2011CD012)
宜昌市科技项目(编号A11301-02)
作者简介
通讯作者：郭静明，教授，主任医师．E-mail：gjm425@163．com
