2011-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/643516摘要：根據我們近來的研究成果發現，調控第一型細胞激素表現的主要轉錄因子，T-bet，它在臍帶血和鼻腔自然殺手細胞淋巴癌(nasal NK cell lymphoma)中的表現是受控於轉譯作用被抑制。第一型細胞激素和第二型細胞激素表現在輔助T 細胞(T-helper cell)中已經被廣泛研究。因此我們將此觀念延伸至自然殺手細胞 (NK1 和NK2)，提出一個線性模式。在此模式當中，第2 型NK 細胞(NK2)會先分化為第0 型NK 細胞(NK0) ，再分化為第1型NK 細胞(NK1)。根據這個模式，NK2 細胞分泌細胞激素IL-13 和IL-5，NK0 細胞分泌IL-13, IL-5 和IFNg，而NK1 細胞則分泌IFNg。在基因剔除鼠模式的研究當中發現，T-bet 表現缺陷的小鼠無法產生IFNg。由此推論，T-bet 在自然殺手細胞的最終分化過程以及對第一型細胞激素的趨化是非常重要的。在此計劃提案裡，我們將針對T-bet 在自然殺手細胞以及鼻腔自然殺手細胞淋巴癌中所扮演的生物上和病理上的角色，更進一步地利用下列實驗研究釐清:1) T-bet 基因標的轉殖鼠模式 (knock-in mouse model)A) T-bet 單一對偶基因置換為EGFP，藉以追蹤T-bet mRNA 在NK 細胞的分化過程裡以及在活體組織中的表現。B) T-bet 兩股對偶基因置換為EGFP，藉以研究缺乏T-bet 的NK 細胞的分化和功能。2) T-bet mRNA 環形模式 (circular model)根據我們研究已了解T-bet 的表現是受控於mRNA 的5’端和3’端非轉譯區(untranslated regions, UTRs) ，並提出一個位於5’端的RNA 偽結合模型(psudoknot model) 。然而，關於3’端非轉譯區的闡釋仍待釐清。我們將構築一系列T-bet 的突變株，以建立一個連結5’端和3’端非轉譯區的環形模式。3) microRNA 的確認我們將利用和microRNA 互補的技術，以結構為基礎來闡述此轉譯抑制區。根據環形模式上的RNA 序列，推演出具有和它互補潛能的microRNA，並且在自然殺手細胞株YT 細胞當中測試，再於NK 細胞當中證實。4) 病理上以及臨床上的意義我們將使用鼻腔自然殺手細胞淋巴癌(nasal NK cell lymphomas) 的檢體，藉以建立T-bet 在病理上和臨床上的意義。<br> Abstract: We have recently reported that translation of T-bet, the master transcription factor fortype I cytokine expression, is blocked in T-bet- NK cells from umbilical cord blood and innasal NK cell lymphomas (manuscript submitted).Type I & type II cytokine expression is usually studied in T-helper cells (Th1 & Th2).Recently the concept us extended to NK cells (NK1 & NK2), and a linear model is proposed.In this model, a type II NK (NK2) cell develops into a type 0 NK (NK0) cell, which thendevelops into a type I NK (NK1) cell. According to this model, NK2 cells secret IL-13 andIL-5, NK0 cells secret IL-13, IL-5, and IFNg, and NK1 cells secret IFNg. In a knockoutmouse model, T-bet-deficiency NK cells were unable to produce IFNg. Thus, T-bet isimportant in the terminal differentiation of NK cells, and is the master transcription factorfor type I cytokine polarization.In this proposal, we will perform experiments to further our understandings regardingthe biological & pathological roles of T-bet in NK cells development and in NK celllymphomas.1) A T-bet knock-in mouse model.A) Single-allele replacement of T-bet by EGFP to trace the expression of T-bet mRNAin developing NK cells and also the tissue distributions.B) Double-allele replacement of T-bet by EGFP to study the development & functionof T-bet- NK cells.2) A circular model for T-bet mRNA.The block is due to both 5’ & 3’ un-translated regions (UTRs). We have proposed a RNApsudoknot model for the 5’UTR. However, details of the 3’UTR remain unclear. We willperform extensive mutagenesis on the T-bet mRNA to establish a circular model thatincorporates contributions from both 5’ & 3’UTRs.3) Identifications of micro RNAsWe will launch a complementary microRNA approach to delineate the structural basis forthe block. Based on the circular model, potential microRNAs will be tested in the YTcell line of NK cell origin, and verified in NK cells. This is a continuation from aprevious NSC proposal on microRNAs in nasal NK cell lymphomas.4) Pathological & clinical significance. We will use nasal NK cell lymphomas to establishpathological & clinical significance.