2012-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/656753摘要：先天性免疫反應可以利用TLR 及RLR 偵測病毒核酸衍生物，當配體和這些受體結合後所引發的訊號可以導致發炎激素及第一型干擾素的產生，而這些細胞激素對病毒的清除是十分重要的。第一型干擾素的訊息傳遞會活化STAT 家族中STAT1、STAT2、STAT3三個成員，由基因剔除鼠的結果知道其中STAT1 及STAT2 對干擾素引起的作用是極為重要的，然而，STAT3 在其中的角色仍然不清楚，因此我們利用缺乏STAT3 的細胞或老鼠來研究這個問題。在初期的結果中，我們發現STAT3 缺損的細胞在受到第一型干擾素刺激後，STAT1 的活化、基因的表現及抗病毒反應有明顯比正常的細胞來得增加，這個結果暗示STAT3 可能會負調控干擾素的反應。在報導基因的分析中，我們發現STAT3 的SH2而非DBD 或TAD 功能區對STAT3 的抑制作用是必需的，換句話說STAT3 的蛋白質間的交互作用而非轉譯能力是造成抑制反應所需的。經過干擾素刺激後，我們發現利用染色質免疫沉澱(ChIP)方法去濃縮ISRE 的量在STAT3 缺損的細胞中也是比較高，這個結果暗示STAT3 可能會抑制轉譯複合物的形成或結合到引子區的過程。此外，我們也發現主要引發抗病毒反應的TLR 反應在STAT3 缺損的細胞或老鼠中也有增強的現象，我們也在報導基因的分析中証實STAT3 可以抑制TLR 及一個RLR 下游分子MAVS 所激發的IFNβ 及NFκB引子的活性。有趣的是，TLR 訊息傳遞會直接造成STAT3 serine 的磷酸化。這個結果証實STAT3 的確會負調控第一型干擾素及TLR 的反應，因此我們想進一步去分別研究STAT3 抑制干擾素及TLR 的分子機制。以下是我們想進行的實驗：1. 去分析STAT3抑制干擾素反應所需的功能區2. 去研究STAT3如何抑制ISGF3引發之轉譯作用3. 去尋找和STAT3有交互作用的蛋白質而且能夠協助STAT3去抑制干擾素反應4. 去分析STAT3對TLR及RLR所引發反應的抑制效果5. 去分析STAT3如何分別抑制在TLR及RLR反應中產生干擾素及發炎激素的訊息傳遞路徑6. 去分析STAT3在病毒感染時對免疫反應調控角色<br> Abstract: Innate immune response can sense nucleic acids derived from viruses using toll-like receptors(TLRs) and RIG-I-like receptors (RLRs). Upon ligand binding, these receptors will triggersignals leading to production of proinflammatory cytokines and type I IFNs, which are criticalfor viral clearance. The signaling events of type I IFN will result in activation of STAT1,STAT2 and STAT3, three STAT family members. While essential role of STAT1 and STAT2for type I IFN-mediated response has been well demonstrated in gene-targeting mice, the roleof STAT3 in this response remains unclear. We have used cells or mice lacking STAT3 toaddress this issue. Our preliminary results show that STAT3KO cells display enhanced STAT1activation, gene induction and antiviral response following IFNα/βstimulation as opposed toWT control, suggesting that STAT3 may negatively regulate IFN response. Reporter assaysshow that SH2 rather than DNA binding domain (DBD) or transactivation domain (TAD) ofSTAT3 is required for the suppressive effect, suggesting that protein-protein interaction andnot transcriptional activity of STAT3 is sufficient for the activity. Chromatinimmunoprecipitation (ChIP) assays show that increased enrichment of IFN-stimulatedresponsible element (ISRE) is observed in STAT3KO cells following IFN stimulation,suggesting that STAT3 may down-modulate the formation or binding of IFN-stimulatedtranscriptional complex on promoters of ISGs. In addition, we also note that TLR response, amajor process triggering antiviral response, is markedly enhanced in STAT3KO cells andmice. Reporter assays further confirm that STAT3 suppresses IFNβand NFκB promoteractivity driven by TLR and MAVS, a downstream signal mediator of RLRs. Interestingly,TLR signaling can directly activate STAT3 by serine phosphorylation. Thus, we havedocumented a negative role of STAT3 in type I IFN and TLR response. Here, we propose toinvestigate the molecular mechanisms underlying the suppressive effects of STAT3 on IFNandTLR-mediated response, respectively.1. To dissect domain/regions required for suppressive effect of STAT3 on IFN response2. To study how STAT3 inhibits ISGF3-mediated transcription3. To identify STAT3-interacting protein that facilitates suppressive effect of STAT3 on IFNresponse4. To characterize the suppressive effect of STAT3 on TLR and RLR-mediated response5. To characterize the suppressive effect of STAT3 on TLR/RLR signaling pathways leadingto production of type I IFN and proinflammatory cytokines, respectively6. To characterize the effect of STAT3 on immune response during viral infection