CpG-ODN对鼠巨噬细胞TLR-9及Th1/Th2类细胞因子分泌的影响
|
摘要
背景
支气管哮喘是由淋巴细胞、嗜酸性粒细胞和肥大细胞等多种炎性细胞、炎症介质和细胞因子共同参与的一种气道慢性炎症。免疫反应在哮喘的发病机制中占有重要地位。目前有人研究发现哮喘的发病的重要基础是辅助性T细胞两种亚型Th1和Th2细胞组成的比例和功能失衡。肺泡巨噬细胞可通过吞噬凋亡的嗜酸性粒细胞、中性粒细胞等而有利于哮喘气道炎症的消退,同时也可通过释放多种细胞因子和蛋白酶,参与哮喘免疫炎症反应及气道的修复和重构,为支气管哮喘的重要效应细胞之一。因此,研究巨噬细胞在支气管哮喘发病机制中的作用,调控巨噬细胞功能可能为支气管哮喘治疗提供一个新思路。
CpG-ODN是含未甲基化的CpG二核苷酸的寡核苷酸序列,和细菌DNA一样具有强烈的免疫刺激作用,能激活哺乳动物巨噬细胞、树突状细胞等多种免疫细胞。CpG-ODN还能诱导Th1细胞因子产生,抑制Th2细胞因子的释放,诱导Th1反应,下调Th2反应,并使Th2反应向Th1反应转变,使Th1/Th2免疫反应达到平衡的状态。而调节Th1/Th2平衡对哮喘的治疗具有重要意义。其激活效应细胞途径是通过Toll样受体9(Toll like-receptor,TLR-9)介导的。TLR-9在识别CpG-ODN后,通过结构域募集髓样分化因子(Myeloid differentiation factor 88,MyD88),核因子κB等多条信号途径,启动细胞活化进程,上调主要组织相容性复合物和共刺激分子表达等,进而诱导各种炎性细胞因子的表达。因此研究CpG-ODN与TLR-9相互识别后对巨噬细胞细胞因子的分泌,TLR-9自身表达的影响,对于揭开CpG-ODN调控巨噬细胞功能的分子机制具有重要意义。
目的
(1)从mRNA和蛋白水平分析CpG-ODN刺激鼠巨噬细胞后对鼠巨噬细胞TLR-9的表达。探讨CpG-ODN调控巨噬细胞TLR-9表达的特征。
(2)检测CpG-ODN刺激前后巨噬细胞上清液中Th1类细胞因子TNF-α、IL-12及Th2类细胞因子IL-6的表达水平,了解CpG-ODN刺激鼠巨噬细胞后对Th1/Th2类细胞因子分泌的影响。探讨CpG-ODN调控巨噬细胞功能及TLR-9表达的分子机制。
方法
(1)本研究采用体外培养小鼠单核-巨噬细胞系RAW264.7细胞,分别用CpG-ODN,Non-CpG-ODN,CpG-ODN+氯喹和培养基刺激巨噬细胞24小时,采用逆转录聚合酶链反应(RT-PCR)检测并比较各组细胞TLR-9mRNA的表达情况,同时用免疫印迹法(Western blotting)进一步验证TLR-9蛋白的表达。
(2)放免法检测各组巨噬细胞上清液中TNF-α、IL-6的表达水平。酶联免疫吸附法(ELISA)检测各组巨噬细胞上清液中IL-12的表达水平。
结果
(1)刺激24小时后,CpG-ODN与CpG-ODN+氯喹组巨噬细胞上清中TLR-9mRNA的表达均显著高于Non-CpG-ODN及对照组(P＜0.05),用Western blotting法进一步证实了CpG-ODN与CpG-ODN+氯喹组巨噬细胞上清中TLR-9蛋白的表达较Non-CpG-ODN及对照组明显增加(P＜0.05)。
(2)巨噬细胞上清液中TNF-α水平CpG-ODN组明显高于其他组,差异有显著性(P＜0.05)。而各组巨噬细胞上清液中IL-6,IL-12水平无显著性差异(p＞0.05)。
结论
CpG-ODN刺激鼠巨噬细胞24小时后可引起鼠巨噬细胞TLR-9无论在蛋白水平还是在核酸水平表达均增高,并且能增加巨噬细胞上清液中Th1类细胞因子TNF-α水平,但不增加Th2类细胞因子IL-6及Th1类细胞因子IL-12水平,从而调节Th1/Th2类细胞因子的分泌。CpG-ODN能很好的调控巨噬细胞功能,将为支气管哮喘治疗提供一个新思路。
Bacground
Bronchial asthma(asthma) is a inflammatory lung disease including lymphocyte、eosinophil、mast cell、inflammation matter and many cytokines.Immune response has an important position in asthma pathogenesy. Rccently,someone preferred nosogenic foundation of asthma is the constitutive ratio and function in balance of Th1,Th2 which are the subgroups of T helper cells.The removal of these apoptotic neutrophils and eosinophils through macrophages is one prerequisite of resolution of acute inflammation.Macrophages can also release many cytokines and protein enzyme,participate in asthma inflammation and airway repair and restructure.So it is necessary to study the function of macrophages in asthma and it is a good idea for asthma therapy through adjusting macrophages.
CpG-ODN are oligodeoxynucleotides including CpG.It has intensity immunity stimulation like bacteria DNA.It can arose many immune cells,like macrophage and dendritic cell of mammal.DNA.CpG-ODN can induce Th1 cytokines and reduce Th2 cytokines,down-regulate Th2 but induce Th1 response,which can also shift the Th2 responses substantially toward Th1 and modulate the Th1 / Th2 immune response balance.The balance has significant meaning in cure of asthma.The approach of stimulate functionary cell through Toll like receptor-9(TLR-9).After TLR-9 recognises CpG-ODN,it initiate cellactivation through myeloid differentiation factor 88 and nuclear factor,then upregulate major histocompatibility complex(MHC) and costimulatoty molecules expression,secrete cytokine and so on.So it is important to study cytokines secretion in macrophages after CpG-ODN meets TLR-9,and TLR-9 expression,It has signification to know how CpG-ODN adjusts macrophage.
Objective
(1) The study was designed to investigate the protein and mRNA expression of TLR-9 after CpG-ODN stimulated macrophages.To explore the character in CpG-ODN adjusting macrophages.
(2) Investigate secretion on Th1 cytokines like TNF-α、IL-12 and Th2 cytokines like IL-6 before and after CpG-ODN stimulated the murine-macrophages-like Raw264.7 Cells.To know how CpG-ODN adjusts macrophage.
Method
(1) In this study,we cultivated RAW264.7 cells.They were seeded in 6-well culture plates and grew overnight in DMEM supplemented with 10%fetal calf serum in a Rumidified 5%CO_2 in cubator at 37℃.Then they were treated with DMEM,CpG-ODN,Non-CpG-ODN or CpG-ODN+chloroquine for 24 hours.The expression of TLR-9 mRNA and protein of each group were analyzed by RT-PCR and Western blot respectively.
(2) The concentration of TNF-αand IL-6 in the supernatants were e xamined by Radioimmunoasssy(RIA) and the level of IL-12 was analyzed by ELISA.
Result
(1) After treated for 24 hours,the expression of TLR-9 mRNA in CpG-ODN group and CpG-ODN+chloroquine group were significant higher than that in other groups.(P＜0.05) Using western blotting also prived that the protein expression of TLR-9 in CpG-ODN group and CpG-ODN+chloroquine group were significant higher than that in other groups.(P＜0.05)
(2) The level of TNF-αin the supernatant in the CpG-ODN group was higher than that in other groups.(P＜0.05).The levels of IL-6 and IL-12 in the supernatants had no significant difference in these groups (p＞0.05).
Conclusions
The protein and mRNA expression of TLR-9 both incarase after CpG-ODN stimulated macrophages.CpG-ODN can induce Th1 cytokines like TNF-α,but Th2 cytokines like IL-6 and Th1 cytokines like IL-12 not.These results demonstrate that CpG-ODN can up-regulate the expression of TLR-9 and adjust the expression of Th1 /Th2 cytokines secretion from macrophages. It is a good idea for asthma therapy through adjusting macrophages.
支气管哮喘是由淋巴细胞、嗜酸性粒细胞和肥大细胞等多种炎性细胞、炎症介质和细胞因子共同参与的一种气道慢性炎症。免疫反应在哮喘的发病机制中占有重要地位。目前有人研究发现哮喘的发病的重要基础是辅助性T细胞两种亚型Th1和Th2细胞组成的比例和功能失衡。肺泡巨噬细胞可通过吞噬凋亡的嗜酸性粒细胞、中性粒细胞等而有利于哮喘气道炎症的消退,同时也可通过释放多种细胞因子和蛋白酶,参与哮喘免疫炎症反应及气道的修复和重构,为支气管哮喘的重要效应细胞之一。因此,研究巨噬细胞在支气管哮喘发病机制中的作用,调控巨噬细胞功能可能为支气管哮喘治疗提供一个新思路。
CpG-ODN是含未甲基化的CpG二核苷酸的寡核苷酸序列,和细菌DNA一样具有强烈的免疫刺激作用,能激活哺乳动物巨噬细胞、树突状细胞等多种免疫细胞。CpG-ODN还能诱导Th1细胞因子产生,抑制Th2细胞因子的释放,诱导Th1反应,下调Th2反应,并使Th2反应向Th1反应转变,使Th1/Th2免疫反应达到平衡的状态。而调节Th1/Th2平衡对哮喘的治疗具有重要意义。其激活效应细胞途径是通过Toll样受体9(Toll like-receptor,TLR-9)介导的。TLR-9在识别CpG-ODN后,通过结构域募集髓样分化因子(Myeloid differentiation factor 88,MyD88),核因子κB等多条信号途径,启动细胞活化进程,上调主要组织相容性复合物和共刺激分子表达等,进而诱导各种炎性细胞因子的表达。因此研究CpG-ODN与TLR-9相互识别后对巨噬细胞细胞因子的分泌,TLR-9自身表达的影响,对于揭开CpG-ODN调控巨噬细胞功能的分子机制具有重要意义。
目的
(1)从mRNA和蛋白水平分析CpG-ODN刺激鼠巨噬细胞后对鼠巨噬细胞TLR-9的表达。探讨CpG-ODN调控巨噬细胞TLR-9表达的特征。
(2)检测CpG-ODN刺激前后巨噬细胞上清液中Th1类细胞因子TNF-α、IL-12及Th2类细胞因子IL-6的表达水平,了解CpG-ODN刺激鼠巨噬细胞后对Th1/Th2类细胞因子分泌的影响。探讨CpG-ODN调控巨噬细胞功能及TLR-9表达的分子机制。
方法
(1)本研究采用体外培养小鼠单核-巨噬细胞系RAW264.7细胞,分别用CpG-ODN,Non-CpG-ODN,CpG-ODN+氯喹和培养基刺激巨噬细胞24小时,采用逆转录聚合酶链反应(RT-PCR)检测并比较各组细胞TLR-9mRNA的表达情况,同时用免疫印迹法(Western blotting)进一步验证TLR-9蛋白的表达。
(2)放免法检测各组巨噬细胞上清液中TNF-α、IL-6的表达水平。酶联免疫吸附法(ELISA)检测各组巨噬细胞上清液中IL-12的表达水平。
结果
(1)刺激24小时后,CpG-ODN与CpG-ODN+氯喹组巨噬细胞上清中TLR-9mRNA的表达均显著高于Non-CpG-ODN及对照组(P＜0.05),用Western blotting法进一步证实了CpG-ODN与CpG-ODN+氯喹组巨噬细胞上清中TLR-9蛋白的表达较Non-CpG-ODN及对照组明显增加(P＜0.05)。
(2)巨噬细胞上清液中TNF-α水平CpG-ODN组明显高于其他组,差异有显著性(P＜0.05)。而各组巨噬细胞上清液中IL-6,IL-12水平无显著性差异(p＞0.05)。
结论
CpG-ODN刺激鼠巨噬细胞24小时后可引起鼠巨噬细胞TLR-9无论在蛋白水平还是在核酸水平表达均增高,并且能增加巨噬细胞上清液中Th1类细胞因子TNF-α水平,但不增加Th2类细胞因子IL-6及Th1类细胞因子IL-12水平,从而调节Th1/Th2类细胞因子的分泌。CpG-ODN能很好的调控巨噬细胞功能,将为支气管哮喘治疗提供一个新思路。
Bacground
Bronchial asthma(asthma) is a inflammatory lung disease including lymphocyte、eosinophil、mast cell、inflammation matter and many cytokines.Immune response has an important position in asthma pathogenesy. Rccently,someone preferred nosogenic foundation of asthma is the constitutive ratio and function in balance of Th1,Th2 which are the subgroups of T helper cells.The removal of these apoptotic neutrophils and eosinophils through macrophages is one prerequisite of resolution of acute inflammation.Macrophages can also release many cytokines and protein enzyme,participate in asthma inflammation and airway repair and restructure.So it is necessary to study the function of macrophages in asthma and it is a good idea for asthma therapy through adjusting macrophages.
CpG-ODN are oligodeoxynucleotides including CpG.It has intensity immunity stimulation like bacteria DNA.It can arose many immune cells,like macrophage and dendritic cell of mammal.DNA.CpG-ODN can induce Th1 cytokines and reduce Th2 cytokines,down-regulate Th2 but induce Th1 response,which can also shift the Th2 responses substantially toward Th1 and modulate the Th1 / Th2 immune response balance.The balance has significant meaning in cure of asthma.The approach of stimulate functionary cell through Toll like receptor-9(TLR-9).After TLR-9 recognises CpG-ODN,it initiate cellactivation through myeloid differentiation factor 88 and nuclear factor,then upregulate major histocompatibility complex(MHC) and costimulatoty molecules expression,secrete cytokine and so on.So it is important to study cytokines secretion in macrophages after CpG-ODN meets TLR-9,and TLR-9 expression,It has signification to know how CpG-ODN adjusts macrophage.
Objective
(1) The study was designed to investigate the protein and mRNA expression of TLR-9 after CpG-ODN stimulated macrophages.To explore the character in CpG-ODN adjusting macrophages.
(2) Investigate secretion on Th1 cytokines like TNF-α、IL-12 and Th2 cytokines like IL-6 before and after CpG-ODN stimulated the murine-macrophages-like Raw264.7 Cells.To know how CpG-ODN adjusts macrophage.
Method
(1) In this study,we cultivated RAW264.7 cells.They were seeded in 6-well culture plates and grew overnight in DMEM supplemented with 10%fetal calf serum in a Rumidified 5%CO_2 in cubator at 37℃.Then they were treated with DMEM,CpG-ODN,Non-CpG-ODN or CpG-ODN+chloroquine for 24 hours.The expression of TLR-9 mRNA and protein of each group were analyzed by RT-PCR and Western blot respectively.
(2) The concentration of TNF-αand IL-6 in the supernatants were e xamined by Radioimmunoasssy(RIA) and the level of IL-12 was analyzed by ELISA.
Result
(1) After treated for 24 hours,the expression of TLR-9 mRNA in CpG-ODN group and CpG-ODN+chloroquine group were significant higher than that in other groups.(P＜0.05) Using western blotting also prived that the protein expression of TLR-9 in CpG-ODN group and CpG-ODN+chloroquine group were significant higher than that in other groups.(P＜0.05)
(2) The level of TNF-αin the supernatant in the CpG-ODN group was higher than that in other groups.(P＜0.05).The levels of IL-6 and IL-12 in the supernatants had no significant difference in these groups (p＞0.05).
Conclusions
The protein and mRNA expression of TLR-9 both incarase after CpG-ODN stimulated macrophages.CpG-ODN can induce Th1 cytokines like TNF-α,but Th2 cytokines like IL-6 and Th1 cytokines like IL-12 not.These results demonstrate that CpG-ODN can up-regulate the expression of TLR-9 and adjust the expression of Th1 /Th2 cytokines secretion from macrophages. It is a good idea for asthma therapy through adjusting macrophages.
引文
1.BramanSS.The global burden of asthma.Chest,2006,130:4S-12S
2.Herrick CA,Bottomly K.To respond or not to respond:T cells in allergic asthma.Nat Rev Immunol,2003,3:405-412
3 蒋玉红,王亚秋,孙宽秋。支气管哮喘患者TH细胞的极化状态及临床意义。中国当代儿科杂志,2006,8:379-381.
4.Marc Peters-Golden.The alveolar macrophage:the forgotten cell in asthma.American Journal of Respiratory Cell and Molecular Biology.2004;31(1):3-7
5.Krieg AM,Yi AK,Matson S,et al.CpG motifs in bacterial DNA trigger direct B.cell activation[J].Nature,1995,374(6522):546-549.
6 Hashimoto C,Hudson K L,Anderson K V.The Toll gene of Drosophila required for dorsoal-ventral embryonic polarity,appears to encode a transmembrane protein[J].Cell,1988,(52):269-279.
7 Doyle S L,O' Neil L A J.Toll-like receptors:From the discobery of NFKB to new insights into transcriptional regulations in innate immunology[J].Biochemical Pharmacology,2006,(72):1102-1113.
8 Akira S,Uematsu S,Takeuchi O.Pathogen recognition and innate immunity[J].Cell,2006,124:783-801.
9 Terry K M,Douglas T G,Matthew J F.Structure and Function of Toll-like recepto.r proteins[J].Life Science,2000,(68):241-258.
10 Michael R.Of mice and men:Species variations of Toll-like receptor expression[J].Trends in Immunology,2002,23(6):375-379.
11 Akira S,Uematsu S,Takeuchi O.Pathogen recognition and innate immunity[J].Cell,2006,124:783-801.
12 Hiroaki H,Osamu T,Taro K,et al.A Toll-like receptor recognizes bacterial DNA.Nature,2000:408(6813):740
13 Zarember KA,Godowskil PJ.Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes,their products,and cytokines.J Immunol,.2002,168(2):554-561
14 Kawai T,Akira S.TLR signaling[J].Seminar in Immunology.2007,(19)24-32.
15 Aderem A,et al.How do you see CG?,Cell,2000,103:993-996
16 KawaiT,Adachi O,OgawaT,etal.Unresponsiveness of MyD88-deficient mice to endotoxin[J].Immunity,1999(11):115
17 Takeuchi O,Takeda K,Hoshlno K,et al.Cellularresponses to bacterial cell wall components are mediated through MyD88-dependent signaling cascades[J].Immunol,2000,12(1):113
18 Burns K,Clatworthy J,Martin L.et al.Tollip:a new component of the IL-1 RI pathway,L inks IRAK to the IL-1 receptor[J]Nat Cell Biol,2000,2(6):346-35
19 Kerkmann M,Rothelafusser S,Homung V.et al.Activation with CpG-A and CpG-B oligonucleotides reveals two distinct regulatory pathways Of type Ⅰ IFN synthesis in human plasmacytoid dendritic dells.J Immunol,2003,170(9):4465
20 Takauji R.Iho S.Takatsuka H.et al.CpG-DNA-induced IFN-alpha production involves p38 MAPK-dependent STAT1 phosphorylation in human plasmacytoid dendritic cell precursors.J Leukoc Biol.2002,72(5):1011
21 Underhill DM.Toll-like receptors:networking for success.Eur J Immunol,2003,33(7):1767
22 蒋为薇,王良喜,丁国富等,CpG-N ODN对CpG-S ODN诱导hPBMC释放TNF-a的 抑制作用。中国抗生素杂志,2007,Mar,32(3):187-190
23 肖文珺,周育森,Toll样受体与新型佐剂-佐剂研究的新趋势。微生物学免疫学进展,2007,35(2):60-3
24 Kundu SD,Lee C,Billips BK,et al.The toll-like receptor pathway:A novel mechanism of infection-induced carcinogenesis of prostate epithelial cells.Prostate,2OO8,Feb 1;68(2):223-9
25 Lim EJ,Lee SH,Lee JG,et al.Toll-like receptor 9 dependent activation of MAPK and NF-kB is required for the CpG ODN-induced matrix metalloproteinase-9 expression.Exp Mol Med,2007,Apr 30;39(2):239-45
26.吴羽中,丁国富,周红,抗人TLR-9抗体对CpG寡核苷酸介导的人外周血单核细胞肿瘤坏死因子-α释放的影响。中国临床药理学与治疗学。2006,Mar;11(3):324-327
27.石永云,凌斌,周颖等,妊娠高血压综合症患者蜕膜中Th1Th2型细胞因子的变化。安徽医科大学学报,2006;41(4):456-8
28.Tsuda H,Sakai M,Michimata T,et al.Characterization of NKT cell in human peripheral blood and decidual lymphocytes.Am J Reprod Immunol,2001;45(5):295-302.
29 Dittmer U,Olbrich AR.Treatment of infectious diseases with immunostimulatory oligodeoxynucleotides containing CpG motifs.Curr Opin Microbiol,2003;6(5):472-7.
30.Fk Gibbons,E Israel,A Deykin,B Schaub,et al.The combined effects of zafirlukast,prednisone,and inhaled budesonide on IL-13 and IFN-gamma secretion. J Clin Immunol, 2005;25(5):437-44
31. KitagakiK, Jain VV, Businga TR, et al. Immunomodulatory effects of CPG oligodeoxynucleotides on established Th2 responses. [J] Clin Diagn Lab Immunol, 2002, 9:1260
1 Krieg AM,Yi AK,Matson S,et al.CpG motifs in bacterial DNA trigger direct B.cell activation[J].Nature,1995,374(6522):546-549.
2 Mosmann TR,Cherwinski H,Bond MW,etal.Two types of murine helper Tcell clone.I.Definition according to profiles of lymphokine activities and secreted proteins[J],J Immunol,1986,136(7):2348-2357.
3 吴健,徐军,钟南山。BC6与变应性哮喘TH1TH2平衡[J]。国外医学。呼吸系统分册。2001,21(3):151-153,156
4 拉杰。哮喘发病机理及TH1TH2比例的关系[J]。天津医科大学学报,2002,8(3):403-406
5 Krieg AM.lmmune effects and mechanisms of action of CpG motifs[J].Vaccine,2000,19(6):618-622.
6 Hartmann G,et al.Delinenation of a CpG phosphorothioate oligodexynucleotide for activating primate immune responses in vitro and in vivo,J Immunol.2000,164:1617-1624
7 Krieg AM,et al.Mechanism of action of CpG DNA,Curr Top Microbiol Immunol,2000,247:1-21
8 Kaisho T,et al.Dendritic-cell function in Toll-like receptor and MyDD-knockout mice,Trends Immunol,2001,22;78-83
9 O'Neill L,et al.Specificity in the innate response:pathogen recognition by Toll-like receptor combinations,Trends Immunol, 2001, 22:70
10 Ahmad-Nejad P, Hacker H, Rutz M, et al. Bacterial CpG DNA and lipopolysaecharides activate Toll—like receptors at distincct cellular compartments. Eur J Immunol. 2002. 32(7): 1958
11 Takeshita F, Gursel I, Ishii KJ, et al. Signal transduction pathways mediated by the interaction Of CpC DNA with Toll—like receptor 9. Semin Immunol, 2004, 16(1): 17
12 Hemmi H, et al. A Toll-like receptor recognizes bacterial DNA, Nature, 2000, 408 (7) :740-745
13 Gursel M, Gursel I, Mostowski HS, et al. Cxcl 16 influences the nature and Specificity of CpG-Induced Immune Activation. J Immunol. 2006 Augl;177 (3):1575-80
14 Aderem A, et al. How do you see CG?, Cell, 2000, 103:993-996
15 Kerkmann M, Rothelafusser S, Homung V. et al. Activation with CpG-A and CpG-B oligonucleotides reveals two distinct regulatory pathways Of type I IFN synthesis in human plasmacytoid dendritic cells. J Immunol, 2003, 170(9): 4465
16 Takauji R. Iho S. Takatsuka H. et al. CpG—DNA—induced IFN—alpha production involves p38 MAPK—dependent STAT1 phosphorylation in human plasmacytoid dendritic cell precursors. J Leukoc Biol. 2002. 72(5): 1011
17 Underhill DM. Toll—like receptors: networking for success. Eur J Immunol,2003,33(7):1767
18 Verthelyi C,Klinman DM.Immunoregulatory activity of CpG oligonucleotidesin humans and nonhuman primates[J].Clin lmmunol,2003.109(1):64-71.
19 李宁,范学工,汤参娥 等TLR9在CpG寡脱氧核苷酸所致种属特异性免疫效应中的意义[J]。中南大学学报(医学版),2005,30(5):533-535
20 Choudhury BK.Wild JS.Alam R.et al.In vivo role of p38 mitogen -activated protein kinase in mediating the anti-inflammatory effects of CpG oligodeoxynucleotide in murine asthma.J Immunol.2002.169(10):5955-5961
21 hiang DJ.Ye YI.Chen WL.et al.Ribavirin or CpG DNA sequence-modulated dendritic cells decrease the IgE level and airway inflammation.Am J Respir Crit Care Med.2003.168(5):575-580.
22 Jozefowski S,Sulahian T,Arredouani M,Koblzik L Role of scavenger receptor MARCO in macrophage responses to CpG oligodeoxynucleotides Journal of Leukocyte Biology.2006 Aug 1,volume 80
23 Greco E,De Spirito M,Papi M.et al.CpG oligodeoxynucleotides induce Ca(2+)-dependent phospholipase D activity leading to phagolysosome maturation and intracellular mycobacterial growth inhibition in monocytes.Biochem Biophys Res Commun.2006 Jul 18 2006 Sep 8;347(4):963-9
24 Redford TW,et al.Cyclosporine A enhances IL-12 production by CpG motifs in dacterial DNA and synthetic oligodeoxynucleotides, J Immunol, 1998, 161:3930-3935
25 Yi AK, Peckham DW , Ashman RF, et al. CpG DNA rescues B cells from apoptosis by activating NFkappaB and preventing mitochondrial membranepotential disruption via a chloroquine-sensitive pathway [J]. Int Immunolml, 1999. 11(12): 2015—2024.
26 Santeliz JV, Van Nest G, Traquina P, et al. Amb a l-linked CpG oligedeoxynucleotides reverse established airway hyperresponsiveness in a murine model of asthma[J]. J Allergy Clin Immunol, 2002, 109(3): 455-462.
27 Krieg AM , Wagner H. Causing a commotion in the blood: immunotherapy progresses from bacteria to bacterial DNA [J]. Immunol Today, 2000, 21(10): 521-526.
28 Bendigs S, et al. CpG-oligodeoxynucleotides co-stimulate primary T cells in the absence of antigen-presenting cells, Eur J Immunol, 1999, 29:1209-1218
29 Wild JS, Sur S. CpG oligonucleotide modulation of allergic inflammation[J]. Allergy, 2001, 56(5): 365-376
30 Yi AK, Yoon JG, Hong SC, etal. Lipopolysaccharide and CpG DNA synergize for tumor necrosis factor-alpha production through activation of NF-kappa B[J]. Int Immunol, 2001, 13(11): 1391-1404.
31 Hessel EM, Chu M, lizcano JO, etal. Immunostimulatory oligonucleotides block allergic airway inflammation by inhibiting Th2 cell activation and IgE-mediated cytokine induction. J Exp Med. 2005 Dec 5;202(11):1563-73
32 Amaral CC, Garcia IP, Fernandes GF, etal. Adjuvant effect of synthetic oligodeoxyribonucleotides(CpG-ODN) from the Paracoccidioides brasiliensis gp43 gene on the Th2-Th1 immunomodulation of experimental. Scand J Immunol. 2005 Oct:62(4):325-33
33 Sugai T, Mori M, Nakazawa M, etal. A CpG-containing oligodeoxynucleotide as an efficient adjuvant counterbalancing the Th1/Th2 immune response in diphtheria-tetanus-pertussis vaccine. Vaccine. 2005 Nov 16;23 (46-47):5450-6. Epub 2005 Jun 21
34 Risini D. W, M J. McCluskie. Yu Xu, et al. CpG DNA induces stronger immune responses with less toxicity than other adjuvant[J]. Vaccine. 2000, 18: 1755-1762.
35 Antoine F.Carpentier Cancer immunotherapy with CpG-ODN Medicine/Science 2005 Jan 7 , 17-dimethyltestoscerone
36 Wang H, Rayburn ER, etal. Immunomodulatory oligonucleotides as novel therapy for breast cancer: pharmacokinetics, in vitro and in vivo anticancer activity, and potentiation of antibody therapy. Mol Cancer Ther. 2006 Aug;5(8):2106-14
37 den Brok MH, Sutmuller RP, etal. Synergy between In situ Cryoablation and TLR-9 Stimulation Results in a highly effective in vivo Dendritic Cell Vaccine. Cancer Res. 2006 Jul 15;66(14):7285-92
38 Mo JH , Park SW, Rhee CS, Takabayashik, etal. Suppression of allergic response by CpG motif oligodeoxynucleotide-house-dust mite conjugate in animal model of allergic rhinitis. Am J Rhinol. 2006 Mar-Apr:20(2):212-8
39 Lazarczyk M, Grzela K, Grzela T. Immunostimulatory oligonucleotides in therapy of allergic diseases. Expert Opin Biol Ther. 2005 Apr:5(4):525-36
40 Ashino S, Nishimura T. The critical role of IFN-alpha/beta and IFN-gamma in the regulation of airway inflammation. Nippon Rinsho. 2006 Jul:64(7):1260-8
2.Herrick CA,Bottomly K.To respond or not to respond:T cells in allergic asthma.Nat Rev Immunol,2003,3:405-412
3 蒋玉红,王亚秋,孙宽秋。支气管哮喘患者TH细胞的极化状态及临床意义。中国当代儿科杂志,2006,8:379-381.
4.Marc Peters-Golden.The alveolar macrophage:the forgotten cell in asthma.American Journal of Respiratory Cell and Molecular Biology.2004;31(1):3-7
5.Krieg AM,Yi AK,Matson S,et al.CpG motifs in bacterial DNA trigger direct B.cell activation[J].Nature,1995,374(6522):546-549.
6 Hashimoto C,Hudson K L,Anderson K V.The Toll gene of Drosophila required for dorsoal-ventral embryonic polarity,appears to encode a transmembrane protein[J].Cell,1988,(52):269-279.
7 Doyle S L,O' Neil L A J.Toll-like receptors:From the discobery of NFKB to new insights into transcriptional regulations in innate immunology[J].Biochemical Pharmacology,2006,(72):1102-1113.
8 Akira S,Uematsu S,Takeuchi O.Pathogen recognition and innate immunity[J].Cell,2006,124:783-801.
9 Terry K M,Douglas T G,Matthew J F.Structure and Function of Toll-like recepto.r proteins[J].Life Science,2000,(68):241-258.
10 Michael R.Of mice and men:Species variations of Toll-like receptor expression[J].Trends in Immunology,2002,23(6):375-379.
11 Akira S,Uematsu S,Takeuchi O.Pathogen recognition and innate immunity[J].Cell,2006,124:783-801.
12 Hiroaki H,Osamu T,Taro K,et al.A Toll-like receptor recognizes bacterial DNA.Nature,2000:408(6813):740
13 Zarember KA,Godowskil PJ.Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes,their products,and cytokines.J Immunol,.2002,168(2):554-561
14 Kawai T,Akira S.TLR signaling[J].Seminar in Immunology.2007,(19)24-32.
15 Aderem A,et al.How do you see CG?,Cell,2000,103:993-996
16 KawaiT,Adachi O,OgawaT,etal.Unresponsiveness of MyD88-deficient mice to endotoxin[J].Immunity,1999(11):115
17 Takeuchi O,Takeda K,Hoshlno K,et al.Cellularresponses to bacterial cell wall components are mediated through MyD88-dependent signaling cascades[J].Immunol,2000,12(1):113
18 Burns K,Clatworthy J,Martin L.et al.Tollip:a new component of the IL-1 RI pathway,L inks IRAK to the IL-1 receptor[J]Nat Cell Biol,2000,2(6):346-35
19 Kerkmann M,Rothelafusser S,Homung V.et al.Activation with CpG-A and CpG-B oligonucleotides reveals two distinct regulatory pathways Of type Ⅰ IFN synthesis in human plasmacytoid dendritic dells.J Immunol,2003,170(9):4465
20 Takauji R.Iho S.Takatsuka H.et al.CpG-DNA-induced IFN-alpha production involves p38 MAPK-dependent STAT1 phosphorylation in human plasmacytoid dendritic cell precursors.J Leukoc Biol.2002,72(5):1011
21 Underhill DM.Toll-like receptors:networking for success.Eur J Immunol,2003,33(7):1767
22 蒋为薇,王良喜,丁国富等,CpG-N ODN对CpG-S ODN诱导hPBMC释放TNF-a的 抑制作用。中国抗生素杂志,2007,Mar,32(3):187-190
23 肖文珺,周育森,Toll样受体与新型佐剂-佐剂研究的新趋势。微生物学免疫学进展,2007,35(2):60-3
24 Kundu SD,Lee C,Billips BK,et al.The toll-like receptor pathway:A novel mechanism of infection-induced carcinogenesis of prostate epithelial cells.Prostate,2OO8,Feb 1;68(2):223-9
25 Lim EJ,Lee SH,Lee JG,et al.Toll-like receptor 9 dependent activation of MAPK and NF-kB is required for the CpG ODN-induced matrix metalloproteinase-9 expression.Exp Mol Med,2007,Apr 30;39(2):239-45
26.吴羽中,丁国富,周红,抗人TLR-9抗体对CpG寡核苷酸介导的人外周血单核细胞肿瘤坏死因子-α释放的影响。中国临床药理学与治疗学。2006,Mar;11(3):324-327
27.石永云,凌斌,周颖等,妊娠高血压综合症患者蜕膜中Th1Th2型细胞因子的变化。安徽医科大学学报,2006;41(4):456-8
28.Tsuda H,Sakai M,Michimata T,et al.Characterization of NKT cell in human peripheral blood and decidual lymphocytes.Am J Reprod Immunol,2001;45(5):295-302.
29 Dittmer U,Olbrich AR.Treatment of infectious diseases with immunostimulatory oligodeoxynucleotides containing CpG motifs.Curr Opin Microbiol,2003;6(5):472-7.
30.Fk Gibbons,E Israel,A Deykin,B Schaub,et al.The combined effects of zafirlukast,prednisone,and inhaled budesonide on IL-13 and IFN-gamma secretion. J Clin Immunol, 2005;25(5):437-44
31. KitagakiK, Jain VV, Businga TR, et al. Immunomodulatory effects of CPG oligodeoxynucleotides on established Th2 responses. [J] Clin Diagn Lab Immunol, 2002, 9:1260
1 Krieg AM,Yi AK,Matson S,et al.CpG motifs in bacterial DNA trigger direct B.cell activation[J].Nature,1995,374(6522):546-549.
2 Mosmann TR,Cherwinski H,Bond MW,etal.Two types of murine helper Tcell clone.I.Definition according to profiles of lymphokine activities and secreted proteins[J],J Immunol,1986,136(7):2348-2357.
3 吴健,徐军,钟南山。BC6与变应性哮喘TH1TH2平衡[J]。国外医学。呼吸系统分册。2001,21(3):151-153,156
4 拉杰。哮喘发病机理及TH1TH2比例的关系[J]。天津医科大学学报,2002,8(3):403-406
5 Krieg AM.lmmune effects and mechanisms of action of CpG motifs[J].Vaccine,2000,19(6):618-622.
6 Hartmann G,et al.Delinenation of a CpG phosphorothioate oligodexynucleotide for activating primate immune responses in vitro and in vivo,J Immunol.2000,164:1617-1624
7 Krieg AM,et al.Mechanism of action of CpG DNA,Curr Top Microbiol Immunol,2000,247:1-21
8 Kaisho T,et al.Dendritic-cell function in Toll-like receptor and MyDD-knockout mice,Trends Immunol,2001,22;78-83
9 O'Neill L,et al.Specificity in the innate response:pathogen recognition by Toll-like receptor combinations,Trends Immunol, 2001, 22:70
10 Ahmad-Nejad P, Hacker H, Rutz M, et al. Bacterial CpG DNA and lipopolysaecharides activate Toll—like receptors at distincct cellular compartments. Eur J Immunol. 2002. 32(7): 1958
11 Takeshita F, Gursel I, Ishii KJ, et al. Signal transduction pathways mediated by the interaction Of CpC DNA with Toll—like receptor 9. Semin Immunol, 2004, 16(1): 17
12 Hemmi H, et al. A Toll-like receptor recognizes bacterial DNA, Nature, 2000, 408 (7) :740-745
13 Gursel M, Gursel I, Mostowski HS, et al. Cxcl 16 influences the nature and Specificity of CpG-Induced Immune Activation. J Immunol. 2006 Augl;177 (3):1575-80
14 Aderem A, et al. How do you see CG?, Cell, 2000, 103:993-996
15 Kerkmann M, Rothelafusser S, Homung V. et al. Activation with CpG-A and CpG-B oligonucleotides reveals two distinct regulatory pathways Of type I IFN synthesis in human plasmacytoid dendritic cells. J Immunol, 2003, 170(9): 4465
16 Takauji R. Iho S. Takatsuka H. et al. CpG—DNA—induced IFN—alpha production involves p38 MAPK—dependent STAT1 phosphorylation in human plasmacytoid dendritic cell precursors. J Leukoc Biol. 2002. 72(5): 1011
17 Underhill DM. Toll—like receptors: networking for success. Eur J Immunol,2003,33(7):1767
18 Verthelyi C,Klinman DM.Immunoregulatory activity of CpG oligonucleotidesin humans and nonhuman primates[J].Clin lmmunol,2003.109(1):64-71.
19 李宁,范学工,汤参娥 等TLR9在CpG寡脱氧核苷酸所致种属特异性免疫效应中的意义[J]。中南大学学报(医学版),2005,30(5):533-535
20 Choudhury BK.Wild JS.Alam R.et al.In vivo role of p38 mitogen -activated protein kinase in mediating the anti-inflammatory effects of CpG oligodeoxynucleotide in murine asthma.J Immunol.2002.169(10):5955-5961
21 hiang DJ.Ye YI.Chen WL.et al.Ribavirin or CpG DNA sequence-modulated dendritic cells decrease the IgE level and airway inflammation.Am J Respir Crit Care Med.2003.168(5):575-580.
22 Jozefowski S,Sulahian T,Arredouani M,Koblzik L Role of scavenger receptor MARCO in macrophage responses to CpG oligodeoxynucleotides Journal of Leukocyte Biology.2006 Aug 1,volume 80
23 Greco E,De Spirito M,Papi M.et al.CpG oligodeoxynucleotides induce Ca(2+)-dependent phospholipase D activity leading to phagolysosome maturation and intracellular mycobacterial growth inhibition in monocytes.Biochem Biophys Res Commun.2006 Jul 18 2006 Sep 8;347(4):963-9
24 Redford TW,et al.Cyclosporine A enhances IL-12 production by CpG motifs in dacterial DNA and synthetic oligodeoxynucleotides, J Immunol, 1998, 161:3930-3935
25 Yi AK, Peckham DW , Ashman RF, et al. CpG DNA rescues B cells from apoptosis by activating NFkappaB and preventing mitochondrial membranepotential disruption via a chloroquine-sensitive pathway [J]. Int Immunolml, 1999. 11(12): 2015—2024.
26 Santeliz JV, Van Nest G, Traquina P, et al. Amb a l-linked CpG oligedeoxynucleotides reverse established airway hyperresponsiveness in a murine model of asthma[J]. J Allergy Clin Immunol, 2002, 109(3): 455-462.
27 Krieg AM , Wagner H. Causing a commotion in the blood: immunotherapy progresses from bacteria to bacterial DNA [J]. Immunol Today, 2000, 21(10): 521-526.
28 Bendigs S, et al. CpG-oligodeoxynucleotides co-stimulate primary T cells in the absence of antigen-presenting cells, Eur J Immunol, 1999, 29:1209-1218
29 Wild JS, Sur S. CpG oligonucleotide modulation of allergic inflammation[J]. Allergy, 2001, 56(5): 365-376
30 Yi AK, Yoon JG, Hong SC, etal. Lipopolysaccharide and CpG DNA synergize for tumor necrosis factor-alpha production through activation of NF-kappa B[J]. Int Immunol, 2001, 13(11): 1391-1404.
31 Hessel EM, Chu M, lizcano JO, etal. Immunostimulatory oligonucleotides block allergic airway inflammation by inhibiting Th2 cell activation and IgE-mediated cytokine induction. J Exp Med. 2005 Dec 5;202(11):1563-73
32 Amaral CC, Garcia IP, Fernandes GF, etal. Adjuvant effect of synthetic oligodeoxyribonucleotides(CpG-ODN) from the Paracoccidioides brasiliensis gp43 gene on the Th2-Th1 immunomodulation of experimental. Scand J Immunol. 2005 Oct:62(4):325-33
33 Sugai T, Mori M, Nakazawa M, etal. A CpG-containing oligodeoxynucleotide as an efficient adjuvant counterbalancing the Th1/Th2 immune response in diphtheria-tetanus-pertussis vaccine. Vaccine. 2005 Nov 16;23 (46-47):5450-6. Epub 2005 Jun 21
34 Risini D. W, M J. McCluskie. Yu Xu, et al. CpG DNA induces stronger immune responses with less toxicity than other adjuvant[J]. Vaccine. 2000, 18: 1755-1762.
35 Antoine F.Carpentier Cancer immunotherapy with CpG-ODN Medicine/Science 2005 Jan 7 , 17-dimethyltestoscerone
36 Wang H, Rayburn ER, etal. Immunomodulatory oligonucleotides as novel therapy for breast cancer: pharmacokinetics, in vitro and in vivo anticancer activity, and potentiation of antibody therapy. Mol Cancer Ther. 2006 Aug;5(8):2106-14
37 den Brok MH, Sutmuller RP, etal. Synergy between In situ Cryoablation and TLR-9 Stimulation Results in a highly effective in vivo Dendritic Cell Vaccine. Cancer Res. 2006 Jul 15;66(14):7285-92
38 Mo JH , Park SW, Rhee CS, Takabayashik, etal. Suppression of allergic response by CpG motif oligodeoxynucleotide-house-dust mite conjugate in animal model of allergic rhinitis. Am J Rhinol. 2006 Mar-Apr:20(2):212-8
39 Lazarczyk M, Grzela K, Grzela T. Immunostimulatory oligonucleotides in therapy of allergic diseases. Expert Opin Biol Ther. 2005 Apr:5(4):525-36
40 Ashino S, Nishimura T. The critical role of IFN-alpha/beta and IFN-gamma in the regulation of airway inflammation. Nippon Rinsho. 2006 Jul:64(7):1260-8