探讨不同TLR信号级联间相互干扰的抑炎作用机制，对寻找用于控制和治疗自身免疫性疾病的新策略具有十分重要的意义。本课题组前期发现一种无免疫刺激性CpG-c41分子，它与TLR9结合并发生"无刺激内化"状态后，能交叉干扰LPS诱导TLR4信号级联的部分通路，抑制IL-6和IFN-β释放,而不影响TNF-a释放。虽干扰位置已锁定在局部范围，但具体干扰环节、作用方式等机制尚不清楚。本课题拟采用细胞免疫、激光共聚焦定位、定量PCR、蛋白磷酸化检测等技术，针对CpG-c41诱导TLR9无刺激内化干扰LPS/TLR4信号级联模型，对比观察TLR9与TLR4受体介导内化的效能，在内体循环过程中CpG-c41与LPS的转运状况，以及信号关键信号分子的活动状况。从而阐明TLR9无刺激内化对TLR4信号级联的负调控方式与作用环节，全面揭示其窜扰机制，为未来抗炎抗过敏及自身免疫性疾病治疗提供新的理论依据和策略。

Exploring anti-inflammation mechanism caused by the cross interference in different TLR signal pathways is very important for new strategy to prevent and treat the autoimmune diseases. In previous study, we found a novel non-stimulatory CpG-c41 molecule, which is able to lead to non-stimulatory endocytosis after binding with TLR9. Under this situation, a potential crosstalk disturbed the part of the signal pathways in the LPS induced TLR4 cascade, suppressing the release of IL-6 and IFN-β，but not that of the TNF-a. Although the target region was at the range from TLR4 internalization to the binding of TRAM/TRIF, it is still obscure about the detailed mechanism of the non-stimulatory endocytosis of TLR9 cross-interfering with the TLR4 signal cascade. In this study, using the cellular immunofluorescence staining and confocal laser scanning technique, quantitative real-time PCR and phosphorylation examination by Western Blot,etc, based on the model of CpG-c41 induced TLR9 non-stimulatory endocytosis disturbing the LPS induced TLR4 signal cascade, we observe the different performances between TLR4 and TLR9 mediated endocytosis, the trafficking of LPS and CpG-c41 in the dynamic endosomal cycling, the activities of key signal molecules, in order to illustrate the negative regulatory process and the action target during the crosstalk of different TLR mediated endocytosis, and reveal the detailed mechanism of cross interference. This study would provide logical proofs and new stategy to the future therapies for inflammatory, allergic and other autoimmune diseases.

探讨不同TLR信号级联间相互干扰的抑炎作用机制，对寻找用于控制和治疗自身免疫性疾病的新策略具有十分重要的意义。本课题组前期发现一种无免疫刺激性CpG-c41分子，它与TLR9结合并发生“无刺激内化”状态后，能交叉干扰LPS诱导TLR4的部分免疫效应，而其具体干扰机制尚不清楚。本课题拟采用细胞免疫、激光共聚焦定位、定量PCR、蛋白磷酸化检测等技术，针对CpG-c41内化模型，对比观察CpG-c41对其它TLRs干扰状况。本研究发现，无论在体内外实验中，无免疫刺激分子CpG-c41均能抑制由单刺激分子或复合分子引发的内膜型TLRs活化，显著降低它们引起的各种前炎症因子释放水平，然而，对膜上型TLRs的活化没有影响。同时，CpG-c41的这种多重抑制作用与下游信号通路无关。正由于TLR4同时存在膜上和内膜两种分布，而表现出CpG-c41对其部分抑制。同时，我们发现CpG-c41能够抑制由咪喹莫特诱发的炎症因子释放，减少免疫细胞的浸润，而缓解小鼠银屑病症状。而且，这种效应机制是独立于TLR9的，这也证实CpG-c41作用于信号级联上游，可能与内化与转运有关。于是，我们进一步调查了CpG-c41的内化转运过程，发现在内化入胞过程中，与TLRs和CD14辅助分子没有关系，而是其5‘端上糖基5碳发挥了关键作用，说明CpG-c41入胞过程发生在识别之前，即入胞和识别是两个独立的过程。继而，我们搞清了CpG-c41是通过与刺激分子竞争入胞而导致对TLR3活化的干预；不同的是，CpG-c41与刺激分子争夺识别受体而导致TLR7/8活化受到干预。同时，我们发现，CpG-c41内化转运是动态过程，内体的暂时饱和不会导致这样的交叉干预。这些结果提示CpG-c41以多种方式干预了内膜性TLRs的活化，更深入地展示了固有免疫的调节机制，为未来抗炎抗过敏及自身免疫性疾病治疗提供新的理论依据和策略。