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標題: | YT 細胞株中 5’ 及 3’UTR 的二級結構對 T-bet 轉譯之調控 Regulation of T-bet Translation in the YT Cell Line by Secondary Structures of the 5’ and 3’UTR |
作者: | I-An Lai 賴怡安 |
指導教授: | 林中梧 |
關鍵字: | T-bet,後轉錄調控, T-bet,post transcriptional control, |
出版年 : | 2009 |
學位: | 碩士 |
摘要: | 自然殺手細胞 (NK cell) 是一種存在於先天免疫系統 (innate immune system) 中具有毒殺細胞能力的淋巴球,它們能夠抵禦癌細胞以及被病毒感染的細胞。目前所知NK 細胞所表現的細胞激素除了TNF-α、GM-CSF 之外,也會分別表現不同的細胞激素而分類為 type II、type 0 及 type I 三個亞群,它們表現的細胞激素分別為 IL13+ / IL5+、IL13+ / IFN-γ+、IFN-γ++。另一方面,若以細胞表面分子 CD56 來定義 NK 細胞發育的成熟度,type I 是發育最成熟的族群,其細胞表面分子型態為 CD161+ / CD56 bright,而 type II 則處於未成熟階段,表現型態為 CD161+ / CD56 -,type 0 介於兩者之間,表現型態為 CD161+ / CD56 dim。因此相對於 T 輔助細胞 (Th cell) 分支狀的分化,現對於NK細胞有type II--> type 0-->type I 直線狀發育分化的假說。
T-bet (T box expressed in T cells) 屬於 T-box 轉錄因子,它會在 Th1 細胞中大量表現,因此在免疫系統當中能夠調控 Th1 細胞以及 Th2 細胞的分化,扮演了相當重要的角色。在此我們試圖探討 T-bet 與 NK 細胞發育及分化的相關性,我們以 NK92 及 YT 這兩株人類自然殺手細胞的細胞株,分別作為 type I 及 type 0 的細胞株模式。根據先前的微陣列 (microarray) 及西方墨點法 (western blotting) 之實驗結果,顯示這兩株細胞的 T-bet mRNA 表現量相差不多,但NK92 之T-bet蛋白質則有顯著的表現量。根據此研究數據,我們推測 T-bet 可能和 NK type I的分化及發育有關,並且 T-bet的表現是藉由轉譯的層次來調控。在真核細胞中,基因的表現若是藉由調控轉譯的進行,通常是藉由 mRNA的 5’或3’端非轉譯區域 (UTR) 上的二級結構的參與。 本篇論文主要是透過構築一系列於T-bet 序列上做不同的突變株質體,以電穿孔的方式送入 YT中,再利用流式細胞儀分析技術或西方墨點法來觀察這些突變對 T-bet 的轉譯表現量之影響,藉此了解mRNA二級結構的分佈對調控 T-bet 轉譯表現的影響。 Abstract Natural killer (NK) cells are a type of cytotoxic lymphocytes which constitute a major component of the innate immune system, and they can resist cancer cells and cells infected by viruses. As we known, NK cells can secrete TNF-α、GM-CSF, besides, they can express different cytokines separately so divided into three groups as following : type II, type 0, and type I, and the expression patterns of the three groups are mainly IL13+ / IL5+、IL13+ / IFN-γ+、IFN-γ++, respectively. On the other hand, the cell surface marker, CD56, is used to define the stage of development of NK cells, and type I is the maturest group which expresses CD161+ / CD56 bright on the cell surface; type II is on the immature stage and the expression pattern is CD161+ / CD56 -, and type 0 is between type I and type II, and it expresses CD161+ / CD56 dim. So on the opposite of T helper (Th) cells which differentiate divergently, there is a type II-->type 0--> type I hypothesis of linear development and differentiation for NK cells. T-bet (T box expressed in T cells) is a member of the T-box family of transcription factors, and its expression is primarily limited to the immune system, and is rapidly induced in early developing Th1 cells. T-bet therefore plays a critical role in Th1 / Th2 cell differentiation. Here we attempted to investigate the regulation of T-bet in the development and differentiation of NK cells. We used two human NK cell lines, NK92 and YT, to be the cell line models of type I and type 0, respectively. Based on the previous data of microarray and western blotting, they revealed mRNA expression levels of T-bet in both cell lines were about the same, nevertheless, the protein expression of T-bet in NK92 was obviously higher. According to these results, we proposed the expression of T-bet could promote the development of type I NK cells and the regulation of T-bet expression was on the translation level. Transcript-selective translational control of eukaryotic gene expression is often directed by structural elements in the 5’ and 3’ untranslated region (5’ and 3’ UTR) of the mRNA. In this thesis, we constructed series of plasmids contained different mutations on T-bet, and then the mutants were transfected into the YT cell line by electroporation. The effect of T-bet mutations on translation was analyzed by flow cytometry or western blotting in order to investigate the distributions of the secondary structures of mRNA on regulation of T-bet in the process of translation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44304 |
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