高迁移率族蛋白通过Toll样受体4降低天然调节性T细胞的抑制功能及其机制  

High Mobility Group Box 1 Bond Toll Like Receptor 4 to Decrease the Suppressive Function and Its Mechanism of the Natural Regulatory T Cells

作　　者：王嘉军 骆春艳[2] 张伟[3] Wang Jiajun;Luo Chunyan;Zhang Wei(Medical College in HuBei University For Nationalities, Enshi, 445000;The First Clinical Hospital of China Three Gorges University, Yichang, 443000;Medical College of China Three Gorges University, Yichang, 443002)

机构地区：[1]湖北民族学院医学院,恩施445000 [2]三峡大学第一临床医院,宜昌443000 [3]三峡大学医学院,宜昌443002

出　　处：《基因组学与应用生物学》2017年第11期4456-4463,共8页Genomics and Applied Biology

基　　金：湖北省卫生计生委西医类科研基金一般项目(No.WJ2015MB172);湖北民族学院博士启动金(No.MY2015b027)共同资助

摘　　要：探讨Toll样受体4的内源性配体高迁移率族蛋白(high mobility group box protein 1,HMGB1)对天然调节性T细胞(natural regulatory T cells,n Tregs)抑制功能的影响及其机制。磁珠法分选健康人外周血中n Tregs,流式细胞术检测分选纯度后进行原代细胞培养。将不同浓度的HMGB1(0.01μg/m L,0.1μg/m L,1μg/m L)分别加入抗TLR4单抗封闭的n Tregs(即anti-TLR4+HMGB1组)和正常n Tregs(即Non anti-TLR4组)两组细胞、同时设定不加HMGB1作为对照组。采用实时定量PCR和酶联免疫吸附(ELISA)检测各组n Tregs中IL-10、TGF-β和IFN-γ的m RNA表达水平和细胞培养上清液中蛋白含量。采用CFSE法流式细胞术检测不同浓度HMGB1处理的n Tregs与CD4+T效应细胞混合培养后CD4+T细胞的增殖水平。Westernblotting检测HMGB1刺激后n Tregs的胞浆及胞核中NF-κBP65蛋白表达水平。结果分选得到的n Tregs纯度>82%(84.52±2.10%)。Non anti-TLR4组中CD4+CD25+Tregs的IL-10、TGF-β的RNA水平及蛋白含量均较无HMGB1刺激的对照组明显降低(p<0.05),而anti-TLR4组中较相应对照组显著增高(p<0.05);IFN-γ的RNA水平及蛋白含量在Non anti-TLR4组中较对照组增加(p<0.05),而在anti-TLR4组中明显低于相应对照组(p<0.05)。CD4+CD25+Tregs显著抑制CD4+T细胞的增殖,与CD3/CD28抗体活化的阳性对照组相比有差异(p<0.05)。经HMGB1刺激的Nonanti-TLR4组中,CD4+T细胞增殖指数较无HMGB1刺激的对照组增高(p<0.05);anti-TLR4组中,CD4+T细胞增殖指数较无HMGB1刺激的相应对照组明显降低(p<0.05)。1μg/m L HMGB1刺激两组CD4+CD25+Tregs后,Non anti-TLR4组胞浆蛋白中NF-κBp65的含量较无刺激对照组降低,而胞核中则相应增加;anti-TLR4组则无明显改变。当TLR4与内源性配体HMGB1结合后,CD4+CD25+Tregs表达及分泌抑制性因子IL-10、TGF-β降低而促炎性细胞因子IFN-γ增加,抑制CD4+T增殖能力减弱,其抑制功能减弱,功能变化机制与NF-κB信号分子的活化相关。To investigate the effects and mechanism of suppressive function and mechanism of natural regulatory T cells （nTregs） after Toll liked receptor 4 （TLR4） bond the HMGB1 （high mobility group box protein 1, HMGB1）, which is endogenous ligand of TLR4. Used magnetic bead separated nTregs from the peripheral blood in healthy people, and flow cytometry detected purity of nTregs, then for primary cell culture. Different concentrations HMGB 1 （10 μg/mL, 1μg/mL, 0.1 μg/mL） were added to the anti-TLR4 groups （anti-TLR4＋LPS group） and normal nTregs （Non anti-TLR4 group）, respectively. At the same time established as control group without HMGB 1. Real time quantitative PCR was used to detect mRNA expression level of IL-10, TGF-β and IFN-γ in nTregs. The concentration of IL-10, TGF-β and IFN-γ in cell culture supernatant were detected by enzyme-linked immunosorbent （ELISA）. Used CFSE flow cytometry to detect the level of CD4＋T cell proliferation in nTregs and CD4＋T effector cells mixed culture that added to different concentrations HMGB1. The protein expression level of NF-KBP65 in the cytoplasm and nuclear of nTregs were detected by Western blotting. The results showed that the purity of separated CD4＋CD25＋Tregs more than 82% （（84.52±2.10）%）. In Non anti-TLR4 group, IL- 10 and TGF-β mRNA expression and protein content of nTregs were significantly lower than those of the control group （p 〈0.05）, but anti-TLR4 group significantly increased （p 〈0.05）. Compared with Non anti-TLR4 group, I FN-γ mRNA expression andprotein content ofnTregs significantlyincreased in control group （p〈0.05）, while those ofthe anti-TLR4 group were significantly lower than those of control group （p〈0.05）. The suppressive experiment results showed that nTregs could effectively inhibit the proliferation of CD4＋T cells, and compared with positive control group which was activated by CD3/CD28 antibody had significantly differences （p 〈0.05）. In Non ant-TLR4 group, CD4 ＋

关 键 词：TOLL样受体4 高迁移率族蛋白 调节性T细胞 NF-ΚB 细胞因子 

分 类 号：R392[医药卫生—免疫学]