YT 細胞株中 5’ 及 3’UTR 的二級結構對 T-bet 轉譯之調控

Abstract

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.