大鼠骨髓树突状细胞的分离培养及HMGB1/TLR4信号通路的调控作用  

Study on isolation and culture of rat bone marrow-derived dendritic cells: regulatory effect of HMGB1/TLR4 signaling pathway

作　　者：王涵磊 薛继阳 葛捍伟 夏杰[1] 林炜 赵琦峰[1] WANG Hanlei;XUE Jiyang;GE Hanwei;XIA Jie;LIN Wei;ZHAO Qifeng(Department of Cardiovascular and Thoracic Surgery,Pediatric Heart Center,the Second Affiliated Hospital ＆ Yuying Children＇s Hospital of Wenzhou Medical University,Wenzhou,32502)

机构地区：[1]温州医科大学附属第二医院育英儿童医院心胸外科儿童心脏中心,浙江温州325027

出　　处：《温州医科大学学报》2018年第7期508-516,共9页Journal of Wenzhou Medical University

基　　金：浙江省科技厅实验动物科技计划项目(2015C37133)

摘　　要：目的:研究HMGB1/TLR4信号通路对大鼠骨髓树突状细胞(mDCs)MyD88、NF-κBP65、细胞因子、共刺激分子表达的影响,探讨此信号通路是否对m DCs具有调控作用。方法:mDCs分离、培养、纯化、鉴定后,随机分为5组,每组各设24、48、72h3个时间点。C组:m DCs加于RPMIl640完全培养基中;H组:加入HMGB1;H-Ab组:加入HMGB1后30min再加入HMGB1特异性中和抗体;TLR4-A组:加入HMGB1后30min再加入TLR4拮抗剂;IgG组:加入HMGB1后30min再加入对照IgG抗体。检测各组各时间点m DCsMy D88、NF-κBP65蛋白和基因、共刺激分子(CD80、CD86)的表达及上清液中细胞因子(IL-6、IL-8、IL-12、TNF-α)的浓度变化。结果:24h、48h、72h各组HMGB1刺激后MyD88、NF-κBP65蛋白和基因、细胞因子、共刺激分子表达明显升高,使用HMGB1特异性中和抗体、TLR4拮抗剂后明显下降,差异均有统计学意义(P<0.01)。结论:HMGB1通过m DCs的受体TLR4影响下游MyD88、NF-κBP65蛋白和基因的表达,并刺激细胞因子的分泌,同时促进共刺激分子CD80、CD86的表达,对mDCs起一定的调控作用。Objective： To investigate the regulatory effect of HMGB1/TLR4 signaling pathway on the expression of My D88, NF-κB P65, cytokine and costimulatory molecules in rat bone marrow-derived dendritic cells（m DCs）. Methods： Dendritic cells were isolated, cultured, purified and identified in vitro from bone marrow stem cells of rats, then randomly divided into 5 groups： m DCs cultured with HMGB1 only（H group）, HMGB1 specific neutralizing antibody（H-Ab group）, TLR4 antagonist（TLR4-A group） and control Ig G（Ig G group） 30 min after HMGB1, separately, or with RPMI l640 serum-containing medium as control group（C gruop）. Each group was observed at different time points of 24 h, 48 h and 72 h by detecting the expression of My D88, NF-κB P65 protein and m RNA, costimulatory molecules（CD80, CD86） and the levels of cytokine（IL-6, IL-8, IL-12, TNF-α） in the supernatant. Results： My D88 and NF-κB P65 protein and m RNA, costimulatory molecules and cytokine levels in m DCs stimulated with HMGB1 were significantly higher than control group at each time point（P〈0.01）. The levels were significantly decreased after being treated with HMGB1 specific neutralizing antibody or TLR4 antagonist（P〈0.01）. Conclusion： Data analysis suggests that HMGB1 plays a regulatory role in m DCs by affecting the downstream My D88, NF-κB P65 protein and m RNA expression through TLR4, stimulating the secretion of cytokine and promoting the expression of costimulatory molecules（CD80, CD86）.

关 键 词：高迁移率族蛋白B1 TOLL样受体4 信号通路 树突状细胞 大鼠 

分 类 号：R392.12[医药卫生—免疫学]