臺灣博碩士論文加值系統

MicroRNAs (簡稱miRNAs) 是一群小片段的RNA，為內生性不會轉譯出蛋白質的RNA分子，長度約22個核甘酸，藉由與目標基因的未轉譯區域結合，進一步抑制mRNA的表現或終止轉譯作用。雖然miR-122是肝臟特異性的miRNA，但在小鼠胚胎發育時期，肝臟是免疫系統發育的主要器官之一，且在先前研究發現miR-122剔除鼠的肝臟有Kupffer cells上升及脾臟腫大的現象，因此我們假設miR-122可能具有調節免疫系統發育或反應的功能。本研究觀察到在miR-122剔除鼠中，脾臟與胸線有顯著的腫大，而且脾臟中macrophages比例上升，雖然B和T細胞組成比例不變，但全部的細胞總數也是上升，因此miR-122剔除可能造成免疫細胞不正常的累積。我們分析骨髓中免疫細胞發現，miR-122剔除鼠的骨髓內細胞總數倍正常小鼠增加，其中neutrophils和macrophages的發育正常，但是miR-122剔除鼠中的pre-B 細胞的比例上升。為了研究miR-122剔除是否影響骨髓中的B細胞增生，我們取出骨髓細胞後加入IL-7觀察反應，結果發現miR-122剔除鼠的proB和preB對IL-7的增生反應較強。為了瞭解脾臟腫大是否和細胞異常增生有關，我們分離出脾臟細胞，純化出B或T細胞，再加入不同刺激測量增生反應，結果發現miR-122剔除的脾臟B細胞，在LPS及Pam3CSK4刺激後細胞增生稍強，而T細胞的反應則和正常小鼠相似。進一步的結果顯示miR-122剔除的脾臟細胞以及B細胞，在加入LPS後分泌的IL-6也較正常細胞增加。我們的結果顯示miR-122剔除鼠具有免疫系統發育及反應異常的現象，因為miR-122在肝臟的表現量最高，在免疫系統只有少數的細胞有極低的表現，所以免疫細胞的異常是否由前驅細胞或是發炎的肝臟細胞所引發，仍需進一步探討。

MicroRNAs (miRNAs) are a group of endogenous small non-coding RNAs with a length of about 22 nucleotides. Once they bind to the complementary sequences of target mRNAs, they will cause target mRMA degradation or termination of translation. Although miR-122 is a liver-specific miRNA, previous studies found that miR-122 knockout mice have splenomegaly and an increase of Kupffer cells in the liver. Because fetal liver is one of the hematopoietic organs in mice, we hypothesize that miR-122 might be involved in the immune regulation. In this study, we observed splenomegaly and thymic enlargement in the miR-122 knockout mice. Although the percentages of B and T cells composition remained unchanged, total number of B and T cells was increased. The percentage of macrophages in the spleen of miR-122 konckout mice was increased. Therefore, miR-122 deficiency might cause abnormal accumulation of immune cells. In the bone marrow, both total cell number and the percentage of pre-B cells were increased in the miR-122 knockout mice, whereas the development of neutrophils and macrophages were normal. To test whether the miR-122 regulates precursor B cell proliferation, we stimulated bone marrow cells with IL-7. The result shows that cell proliferation of the Pro-B and Pre-B population was increased in the miR-122 knockout mice. Consistent with this, miR-122 null splenic B cells had a hyper-proliferation response to LPS and Pam3CSK4 stimulation. However, the proliferation response of splenic T cells was normal. Furthermore, LPS-stimulated IL-6 production by miR-122 knockout splenocytes and B cells was significantly enhanced. In summary, miR-122 knockout mice have abnormalities in the immune system development and response. Because the expression of miR-122 was low in the immune cells, investigations are required to reveal whether these abnormal phenotypes are caused by defects in the immune cells or defects in the hepatocytes.

中文摘要…………………………………………………………………4
Abstract………………………………………………………………5
緒論
MicroRNA………………………………………………………6
miR-122…………………………………………………………7
免疫細胞的發育與分化…………………………………………8
MicroRNA與免疫………………………………………………11
研究目的…………………………………………………………………13
材料與方法
材料
miR-122剔除鼠……………………………………………14
小鼠採血與分離細胞…………………………………………14
細胞培養………………………………………………………14
淋巴細胞的純化………………………………………………15
流式細胞儀分析………………………………………………16
活體外刺激淋巴細胞…………………………………………17
細胞增生試驗…………………………………………………18
酵素連結免疫吸附分析………………………………………18
方法
小鼠犧牲與採血…………………………………………………21
小鼠器官採取及分離細胞………………………………………21
細胞培養及淋巴細胞的純化……………………………………21
流式細胞儀分析…………………………………………………23
活體外淋巴細胞的增生…………………………………………23
酵素連結免疫吸附分析(ELISA) ……………………………24
統計分析…………………………………………………………25
結果
miR-122對免疫細胞發育的影響…………………………………26
miR-122對免疫細胞活化與增生發育的影響……………………28
miR-122在各組織的表現量………………………………………31
討論…………………………………………………………………………32
參考文獻……………………………………………………………………37
圖表…………………………………………………………………………43
附錄…………………………………………………………………………67

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