双链RNA和咪喹莫特联合刺激的协同作用研究  

Synergistic effect of combined double-stranded RNA and imiquimod stimulation

作　　者：李萃[1] 王慧琪[2] 王文静[2] 狄静芳[2] 曾山[2] 林羿[2] 

机构地区：[1]中南大学湘雅医院卫生部肿瘤蛋白质组学重点实验室,长沙410008 [2]暨南大学组织移植与免疫研究中心,广州510632

出　　处：《中华微生物学和免疫学杂志》2009年第4期328-335,共8页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金（30500558,30872761,30730087）;教育部新世纪优秀人才支持计划项目（2007TO）

摘　　要：目的研究双链RNA（double—stranded RNA，dsRNA）和咪喹莫特联合刺激是否具有协同效应。方法在Toll样受体3（TLR3）的特异性刺激剂dsRNA、TLR7的特异性刺激剂咪喹莫特（R837）单独刺激或二者联合刺激条件下，检测BALB／c×C57BL／6和NK细胞缺陷的非肥胖型糖尿病（NOD）×C57BL／6小鼠胚胎吸收率。采用小鼠体内注射上述刺激剂的方法，流式细胞术检测子宫CD45^＋细胞内细胞因子表达水平。为进一步鉴定CD45^＋细胞身份，在体外培养系统中采用dsRNA和咪喹莫特刺激胎盘和底蜕膜来源的子宫CD3^＋T细胞和CD49b^＋NK细胞，并检测细胞内细胞因子表达水平。采用丝裂原激活的蛋白激酶（mitogen—activated protein kinase，MAPK）抑制剂SP600125和PD98059阻断细胞因子表达水平的增加。结果dsRNA和咪喹莫特联合刺激对胚胎吸收率的增高具有协同作用，同时对CD45^＋细胞内TNF—α和IFN-γ的表达增强具有协同刺激作用。进一步细胞鉴定研究显示，虽然在BALB／c小鼠CD3^＋细胞和CD49b^＋NK细胞中均可发现这种协同效应，但是在NOD小鼠，这种细胞因子水平的增高应主要归因于CD3^＋T细胞，因为在CD49b^＋NK细胞不显示这种细胞因子增高趋势。上述刺激剂合用的协同效应可部分地被JNK（Jun N-terminal kinase）MAPK抑制剂SP600125阻断，而几乎被ERK（extracellular signal—regulated kinase）MAPK抑制剂PD98059所完全阻断。结论增强的TLR3和TLR7联合信号可能是通过NOD小鼠Th1型T细胞，而不是NK细胞所传导。ERK MAPK途径可能在TLR3和TLR7参与的细胞信息传递过程中发挥关键性作用。Objective To investigate the effect of combined double-stranded RNA （dsRNA） and imiquimod stimulation on uterine immune cells. Methods In BALB/c ×C57BL/6 mice and non-obese dia- betic （NOD） × C57BL/6 mice, embryo resorption rate was detected in the presence or absence of Toll-like receptor 3 （ TLR3 ） agonist dsRNA [ poly （I： C） ], TLR7 agonist imiquimod （ R837）, or their combination, respectively. In in vivo system, the status of intracellular cytokine production in uterine CD45 ^＋ cells was de- tected by flow cytometry. To identify the CD45^＋ cells, uterine CD3^＋ T cells and CD49b^＋ NK cells derived from placenta and decidua basalis were stimulated with dsRNA and imiquimod in in vitro systems, and the status of intracellular cytokine production was detected. Mitogen-activated protein kinase （MAPK） antagonists SP600125 and PD98059 were used to block the increase of cytokine production. Results A synergistic increase of embryo resorption was observed after the induction of dsRNA and imiquimod combination. Meanwhile, a synergistic increase of TNF-α and IFN-γ production was detected after the induction in CD45^＋ cells. Further study found that although synergistic effect can be detected in both CD3^＋ cells and CD49b^＋ cells in BALB/c mice, the status was different in NOD mice. The eytokine increase should mainly be attributed to CD3^＋ T cells, since no such increase was detected among the CD49b^＋ NK cells in the NOD mice. The synergistic effect of combined agonists was partially inhibited by Jun N-terminal kinase （JNK） MAPK inhibitor SP600125 and almost completely abrogated by extracellular signal-regulated kinase （ERK） MAPK inhibitor PD98059. Conclusion Boosted TLR3 and TLR7 signal may be transmitted via Thl-type T cells, rather than NK cells in NOD mice. ERK MAPK pathway may be critical in TLR3 and TLR7 involved signaling.

关 键 词：动物模型 免疫缺陷 免疫调节 TOLL样受体 

分 类 号：R96[医药卫生—药理学]