实验模式动物树鼩MyD88信号通路的初步研究

Tree shrew is a good candidate to develop as a little experimental animal model. However, lacking tree shrew's bioinformation, method and reagent kits of detection narrow its application. Our previous works have confirmed that the number of toll-like receptors (TLRs) subtypes in tree shrew are the same with that in human, both are composed of TLR1～10. And the tree shrew TLR8 can be up-expressed by R848 - the human TLR8 ligand. It is same to human's TLR8 and different with rodent (including rat and mouse) nonfunctional TLR8. Based on these previous works, in this project, we are going to study the signal pathway MyD88, the common downstream pathway of TLR7, 8, 9. We are going to study the key molecules of MyD88 pathway by using of culture system of tree shrew immunity cells. RT-PCR, RACE, qRT-PCR, ELISA,RNAi will be employed, to determine the expression of mRNA and protein of TLR7, TLR8, TLR9 and the corresponding downstream key molecules in MyD88 pathway, effective cytokines, to confirm their function and changing of Toll-like receptors expression in antiviral immunity of tree shrew. This will provide the theoretical and technical basis for research of virus recognition mechanism and control strategies in virus-infected model of tree shrew in the future.

树鼩是目前很有发展潜力的小型实验动物，但是由于其生物信息、检测方法和工具缺乏，限制了使其做为动物模型的应用。本课题组前期实验已证实树鼩Toll样受体（TLRs）的亚型数量与人相同（有TLR1～10）；并发现人TLR的配体R848能上调树鼩的TLR8的表达（与人TLR8变化相同）；而啮齿类（包括大、小鼠）的TLR8是无功能的。我们拟在这个研究基础上进一步深入研究树鼩与病毒感染相关的TLR7、8、9的功能及其下游MyD88信号通路上的关键分子及功能；并进一步通过建立树鼩的体外免疫细胞培养模型，采用RT-PCR、RACE、qRT-PCR、ELISA等技术，用人TLR7、8、9的相应配体刺激树鼩免疫细胞后，测定树鼩TLR7、8、9及MyD88通路关键分子的变化及相互调节关系。为今后应用树鼩作为模式动物开展病毒识别机制和防治策略研究提供理论基础和技术平台。

树鼩是目前很有发展潜力的小型实验动物，但是由于其生物信息、检测方法和工具缺乏，限制了使其做为动物模型的应用。本课题组前期实验已证实树鼩Toll样受体（TLRs）的亚型数量与人相同（有TLR1～10），本研究在此基础上进一步对树鼩与病毒感染相关的TLR7、8、9的蛋白结构进行了进化分析；并通过抗体芯片技术，从目前成熟的人、小鼠、大鼠抗体中筛选出了能用于树鼩检测的抗体库，以此为基础，构建了可用于树鼩检测的定制抗体芯片；继而，应用此定制芯片，初步检测了树鼩TLR7/8激活后，MyD88信号通路及其它通路的调控变化及通路间的相互作用。我们的研究结果，为短期内应用树鼩作为动物模型建立了一种可行的检测方案。