NF-κB寡核苷酸对CIA大鼠Th17/Treg平衡及细胞因子表达影响的研究
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摘要
为探讨NF-κB寡核苷酸(oligodeoxynucleotide,ODN)对CIA大鼠Th17/Treg平衡及细胞因子表达的影响,建立Ⅱ型胶原诱导大鼠关节炎模型,将NF-κB ODN注射到大鼠右后踝关节腔内,并设对照组、模型组和实验组。42d后用流式细胞仪检测各组大鼠脾脏Th17、Treg百分率、凋亡率;qRT-PCR检测IL-17、TGF-β和IL-6mRNA表达;用ELISA检测血清和关节液IL-17、TGF-β、IL-6含量。结果显示实验组大鼠脾脏Th17占CD4~+T细胞比例及IL-17和IL-6mRNA表达、血清和关节液中IL-17和IL-6的水平均低于模型组,差别有统计学意义(P<0.05);实验组大鼠脾脏Th17凋亡百分率低于模型组,差别有统计学意义(P<0.05);实验组大鼠脾脏Treg占CD4~+T细胞比例及TGF-βmRNA表达、血清和关节液中TGF-β的水平均高于模型组,差别有统计学意义(P<0.05);实验组大鼠脾脏Treg凋亡百分率与模型组相比明显降低,差别有统计学意义(P<0.05)。由此可知,NF-κB ODN调节CD4~+T细胞Th17、Treg之间细胞平衡及相关细胞因子的分泌,对CIA有较好的治疗作用。
To investigate the effects of NF-κB ODN on Th17/Treg balance and cytokine expression in rat with collagen-induced arthritis,type II collagen induced arthritic rats was established.Animals were randomly divided into control group,CIA model group and experimental group.NF-κB ODN decoy was injected into the posterior ankle joint cavity in the experimental group.42 days after the initial administration,the percentage and apoptosis rate of Th17/Treg cells were detected by flow cytometry,real-time fluorescence quantitative PCR was used to determine mRNA expression of IL-17,TGF-βand IL-6.IL-17,TGF-βand IL-6 concentrations in the serum and joint fluid were measured by ELISA.The results showed that,in the experimental group,proportion of Th17 in the CD4~+T cells,IL-17 and IL-6 mRNA levels in the spleen and the levels of IL-6,IL-17 in the serum and joint fluid were lower than those of the model group(P <0.05).The percentage of apoptotic Th17 cells in the spleen was significantly increased compared with the model group(P < 0.05).The proportion of Treg cells in CD4~+T cells,TGF-βmRNA expression in the spleen and the levels of TGF-βin the serum and joint fluid were higher than those of the model group(P< 0.05).The percentage of apoptotic Treg cells in the spleen was significantly lower compared with the model group(P <0.05).These results indicate that NF-κB decoy ODN may play a role in the treatment of RA through adjusting the balance between Th17 and Treg cells and the secretion of cytokines.
To investigate the effects of NF-κB ODN on Th17/Treg balance and cytokine expression in rat with collagen-induced arthritis,type II collagen induced arthritic rats was established.Animals were randomly divided into control group,CIA model group and experimental group.NF-κB ODN decoy was injected into the posterior ankle joint cavity in the experimental group.42 days after the initial administration,the percentage and apoptosis rate of Th17/Treg cells were detected by flow cytometry,real-time fluorescence quantitative PCR was used to determine mRNA expression of IL-17,TGF-βand IL-6.IL-17,TGF-βand IL-6 concentrations in the serum and joint fluid were measured by ELISA.The results showed that,in the experimental group,proportion of Th17 in the CD4~+T cells,IL-17 and IL-6 mRNA levels in the spleen and the levels of IL-6,IL-17 in the serum and joint fluid were lower than those of the model group(P <0.05).The percentage of apoptotic Th17 cells in the spleen was significantly increased compared with the model group(P < 0.05).The proportion of Treg cells in CD4~+T cells,TGF-βmRNA expression in the spleen and the levels of TGF-βin the serum and joint fluid were higher than those of the model group(P< 0.05).The percentage of apoptotic Treg cells in the spleen was significantly lower compared with the model group(P <0.05).These results indicate that NF-κB decoy ODN may play a role in the treatment of RA through adjusting the balance between Th17 and Treg cells and the secretion of cytokines.
引文
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[13]Figdor CG,de Vries IJ,Lesterhuis WJ,et al.Dendritic cell immunotherapy:mapping the way[J].Nature Med,2004,10(5):475-480.
[14]胡恒贵,尹向飞,蒋红梅.核因子-κB诱骗剂处理的DC对胶原诱导性关节炎大鼠外周血IFN-γ、IL-10、抗Ⅱ型胶原抗体水平的影响[J].中华微生物学和免疫学杂志,2011,31(10):870-874.
[15]Xiao S,Zhu B,Jin H,et al.Tim-1stimulation of dendritic cells regulates the balance between effector and regulatory T cells[J].Eur J Immunol,2011,41(6):1539-1549.
[16]Duarte J,Aglla-Doce A,Oliveira VG,et al.Modulation of IL-17and Foxp3expression in the prevention of autoimmune arthritis in mice[J].PLoS One,2010,5(5):e10558.
[2]王淑华,黄一虹.调节性树突状细胞与免疫耐受[J].医学综述,2010,16(10):1459-1461.
[3]王琦,曾学军.树突状细胞在类风湿关节炎中的研究进展[J].基础医学与临床,2011,31(8):951-954.
[4]蹇孝丽,尹向飞,胡恒贵,等.NF-κB寡核苷酸诱骗剂构建大鼠特异性耐受性树突状细胞[J].贵州医科大学学报,2015,40(5):442-446.
[5]蹇孝丽,尹向飞,岳萍,等.NF-κB寡核苷酸诱骗剂诱导的大鼠耐受型树突状细胞及其免疫学特性观察[J].山东医药,2016,56(2):17-19.
[6]胡恒贵,秦淑国,黄峰,等.核因子-κB诱骗剂局部应用对胶原诱导性关节炎大鼠治疗作用的研究[J].现代免疫学,2015,35(6):487-491.
[7]Bonham CA,Peng L,Liang X,et al.Marked prolongation of cardiac allograft survival by dendritic cells genetically engineered with NF-κB oligodeoxyribonucleotide decoys and adenoviral vectors encoding CTLA4-Ig[J].J Immunol,2002,169(6):3382-3391.
[8]Harrington LE,Hatton RD,Mangan PR,et al.Interleukin17-producing CD4+effector T cells develop via a lineage distinct from the T helper type 1and 2lineages[J].Nat Immunol,2005,6(11):1123-1132.
[9]Lubberts E,Koenders MI,Oppers-Walgreen B,et al.Treatment with a neutralizing anti-murine interleukin-17antibody after the onset of collagen-induced arthritis reduces joint inflammation,cartilage destruction,and bone erosion[J].Arthritis Rheum,2004,50(2):650-659.
[10]Park MK,Jung YO,Lee SY,et al.Amelioration of autoimmune arthritis by adoptive transfer of Foxp3-expressing regulatory B cells is associated with the Treg/Th17cell balance[J].J Transl Med,2016,14(1):1-11.
[11]杨明珍,吴德沛,岑建农,等.雷帕霉素诱导Balb/c小鼠CD4+CD25+foxp3+调节性T细胞增殖[J].中国药理学通报,2007,23(6):795-799.
[12]Guzman CA,Domann E,Mohde M,et al.Apoptosis of mouse dendritic cells is triggered by listeriolysin,the major virulence determinant of listeria monocytogenes[J].Mol Microbiol,1996,20(1):119-126.
[13]Figdor CG,de Vries IJ,Lesterhuis WJ,et al.Dendritic cell immunotherapy:mapping the way[J].Nature Med,2004,10(5):475-480.
[14]胡恒贵,尹向飞,蒋红梅.核因子-κB诱骗剂处理的DC对胶原诱导性关节炎大鼠外周血IFN-γ、IL-10、抗Ⅱ型胶原抗体水平的影响[J].中华微生物学和免疫学杂志,2011,31(10):870-874.
[15]Xiao S,Zhu B,Jin H,et al.Tim-1stimulation of dendritic cells regulates the balance between effector and regulatory T cells[J].Eur J Immunol,2011,41(6):1539-1549.
[16]Duarte J,Aglla-Doce A,Oliveira VG,et al.Modulation of IL-17and Foxp3expression in the prevention of autoimmune arthritis in mice[J].PLoS One,2010,5(5):e10558.