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

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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-κB P65、细胞因子、共刺激分子表达的影响，探讨此信号通路是否对mDCs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、72 h 3个时间点。C组：mDCs加于RPMI l640完全培养基中；H组：加入HMGB1；H-Ab组：加入HMGB1后30 min再加入HMGB1特异性中和抗体；TLR4-A组：加入HMGB1后30 min再加入TLR4拮抗剂；IgG组：加入HMGB1后30 min再加入对照IgG抗体。检测各组各时间点mDCs MyD88、NF-κB P65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：24 h、48 h、72 h各组HMGB1刺激后MyD88、NF-κB P65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mDCs的受体TLR4影响下游MyD88、NF-κB P65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。

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	王涵磊
	薛继阳
	葛捍伟
	夏杰
	林炜
	赵琦峰

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

Abstract：Objective: To investigate the regulatory effect of HMGB1/TLR4 signaling pathway on the expression of MyD88, NF-κB P65, cytokine and costimulatory molecules in rat bone marrow-derived dendritic cells (mDCs). Methods: Dendritic cells were isolated, cultured, purified and identified in vitro from bone marrow stem cells of rats, then randomly divided into 5 groups: mDCs cultured with HMGB1 only (H group), HMGB1 specific neutralizing antibody (H-Ab group), TLR4 antagonist (TLR4-A group) and control IgG (IgG 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 MyD88, NF-κB P65 protein and mRNA, costimulatory molecules (CD80, CD86) and the levels of cytokine (IL-6, IL-8, IL-12, TNF-α) in the supernatant. Results: MyD88 and NF-κB P65 protein and mRNA, costimulatory molecules and cytokine levels in mDCs 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 mDCs by affecting the downstream MyD88, NF-κB P65 protein and mRNA expression through TLR4, stimulating the secretion of cytokine and promoting the expression of costimulatory molecules (CD80, CD86).

Key words： high mobility group box 1 protein Toll-like receptor-4 signaling pathway dendritic cells rats

收稿日期: 2017-11-26

基金资助:浙江省科技厅实验动物科技计划项目（2015C37133）

通讯作者: 赵琦峰，主任医师，Email：zhaoqfl862@163.com

作者简介: 王涵磊（1993-），男，浙江温州人，硕士生。

链接本文:

https://xb.wmu.edu.cn/CN/ 或 https://xb.wmu.edu.cn/CN/Y2018/V48/I7/508

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