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研究生:康智凱
研究生(外文):Jhi-Kai Kang
論文名稱:第三號誘餌受體對小鼠B細胞的影響
論文名稱(外文):The effect of decoy receptor 3 (DcR3) on mouse B cells
指導教授:呂春敏
指導教授(外文):Chuen-Miin Leu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:69
中文關鍵詞:第三號誘餌受體免疫調控類鐸受體B細胞相關自體免疫反應
外文關鍵詞:DcR3immunomodulationToll-like receptorsB cell-related autoimmunity
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第三號誘餌受體(Decoy Receptor 3, DcR3)是腫瘤壞死因子受體家族(Tumor necrosis factor receptor superfamily)的成員,因DcR3缺少穿膜區域,是屬於分泌型蛋白質,先前的研究指出DcR3的過量表現與腫瘤生成有正相關,腫瘤細胞可能藉由表現DcR3來中和FasL、LIGHT以及TL1A的功能以躲避免疫系統的攻擊。除此之外,DcR3具有直接調節免疫細胞的能力;例如,DcR3可以結合到單核球細胞表面的glycosaminoglycans,加強單核球的貼附能力;DcR3也會抑制巨噬細胞和樹突細胞的成熟及細胞分化;在DcR3基因轉殖鼠中,DcR3會使T細胞趨向Th2的分化形態。我們實驗室先前利用DcR3與人類免疫球蛋白之融合蛋白(DcR3.Fc)的研究發現,DcR3會與人類或小鼠的B細胞結合,並抑制人類B細胞之增生及活化。為了進一步驗證並建立活體的模型,我們首先測試DcR3.Fc對小鼠B細胞在體外的作用,結果發現DcR3會抑制由金黃色葡萄球菌引發的B細胞增生,也會抑制TLR2 ligand (Pam3CSK4)、TLR4 ligand (LPS)、抗IgM抗體所引起的B細胞增生及活化,但無法抑制由TLR9 ligand (CpG)所引起的細胞增生,顯示DcR3的作用具有專一性。DcR3也可以抑制TLR2 ligand刺激後IL-6、IL-12、TNF-a、IL-10等發炎因子mRNA的表現,但是活化B細胞表現的Bcl-6和AID基因則不受影響。本論文的研究發現DcR3可抑制小鼠B細胞的活化及增生,同時觀察到發炎相關的細胞激素表現也會減少,未來希望進一步測試DcR3在活體內抑制致病B細胞過度活化的能力,進而調節因過度發炎反應所造成的自體免疫疾病。
Decoy Receptor 3 (DcR3), which is a member of the tumor necrosis factor receptor superfamily, lacks the transmembrane domain and is a secretory protein. DcR3 is overexpressed in tumor cells and promotes tumorigenesis by neutralizing the cytotoxic effects of FasL, LIGHT and TL1A. Futhermore, DcR3 has been shown to modulate cell function by triggering multiple signal cascades that are independent of its three known ligands. DcR3 has been identified to interact with the haparan sulfate proteoglycans (HSPGs) on monocyte surfaces. DcR3 suppresses the maturation and differentiation of macrophages and dendritic cells, and it can skew T cell response to the Th2 phenotype in a DcR3 transgenic mouse model. Our previous studies have found that DcR3.Fc fusion protein binds to human and mouse B cells, and has the ability to suppress the proliferation and activation of human B cells. To understand the function of DcR3 in vivo, we first investigated the effects of DcR3.Fc on mouse B cells in vitro. We found that DcR3 not only suppressed the proliferation and activation induced by Staphylococcus aureus, but also suppressed the proliferation and activation induced by TLR2 ligand (Pam3CSK4), TLR4 ligand (LPS) and anti-IgM antibodies. However, DcR3.Fc did not suppress the proliferation induced by TLR9 ligand (CpG). Futhermore, the expression of IL-6, IL-12, TNF-a and IL-10 induced by Pam3CSK4 was inhibited by DcR3, whereas the expression of Bcl-6 and AID after activation remained un-affected. In summary, we found that DcR3.Fc suppresses the activation and proliferation of mouse B cells in vitro. The expression of inflammatory cytokines induced by TLR2 ligand is also inhibited by DcR3.Fc. The results in this study suggest that DcR3 may be able to regulate B cell function in vivo, and possibly be used to alleviate B cell-related autoimmunity.
目錄……………………………………………………………………2
英文摘要………………………………………………………………4
中文摘要………………………………………………………………5
緒論……………………………………………………………………6
腫瘤壞死因子受體超家族中的死亡受體及誘餌受體………………6
第三號誘餌受體(DcR3)………………………………………………7
DcR3的免疫調節功能………………………………………………..8
B細胞的活化、增生及分化………………………………………….9
TLR in B cells and TLR signaling…………………………...10
DcR3和自體免疫疾病………………………………………………11
研究動機與目標……………………………………………………12
材料與方法…………………………………………………………14
A. 材料……………………………………………………………14
試劑與溶液…………………………………………………14
培養基與培養液……………………………………………24
菌株與細胞株………………………………………………26
抗體…………………………………………………………27
B. 方法……………………………………………………………28
細菌轉型(Transformation) ……………………………28
大量質體製備………………………………………………28
細胞培養……………………………………………………29
DcR3.Fc融合蛋白之表現………………………………….29
融合蛋白之純化……………………………………………30
蛋白質電泳…………………………………………………30
西方墨點法…………………………………………………30
蛋白質銀染…………………………………………………31
細胞貼附實驗………………………………………………31
生物素(Biotin)標定蛋白質………………………………32
細胞染色及流式細胞儀分析………………………………32
細胞純化……………………………………………………32
B細胞增生之偵測. ……………………………………...34
酵素連結免疫吸附分析(ELISA)…………………………34
mRNA定量……………………………………………………35
小鼠關節炎之誘發(collagen-induced arthritis)……37
結果…………………………………………………………………39
DcR3融合蛋白的純化……………………………………………39
DcR3.Fc可和小鼠B細胞結合…………… …………………….39
DcR3.Fc抑制由金黃色葡萄球菌引起的B細胞增生…… ……….40
DcR3.Fc抑制由Pam3CSK4引起的小鼠B細胞增生及活化…… ….41
DcR3.Fc抑制由LPS引起的小鼠B細胞增生及活化…………… …42
DcR3.Fc抑制由Pam3CSK4及LPS引起的小鼠B細胞分裂………….42
DcR3.Fc對於其他刺激物的影響…………………………… …..43
DcR3.Fc不會影響TLR ligands刺激後B細胞釋放細胞激素的能力43
DcR3.Fc抑制TLR2配體刺激後小鼠B細胞表現細胞激素的能力 .44
處理DcR3.Fc對B細胞TLR2表現量的影響…………………… ….45
DcR3.Fc對活化後B細胞基因表現的影響………………………..46
CIA (collagen-induced arthritis)小鼠實驗系統的建立…..46
討論…………………………………………… ………………….48
參考文獻……………………………… ………………………….52
圖表……………………… ……………………………………….57
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