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研究生:謝宗翰
研究生(外文):Tsung-Han Hsieh
論文名稱:FcRL1增強B細胞受體活化訊號之機制
論文名稱(外文):The mechanism by which Fc Receptor Like-1(FcRL1) enhances B cell receptor-induced activation
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:62
中文關鍵詞:B細胞受體B細胞
外文關鍵詞:FcRL1
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Fc receptor like proteins(簡稱FcRLs),是一群與Fc受體在序列上相似的受體蛋白。目前在人類發現有8種FcRL蛋白:FcRL1-6,FcRLA和FcRLB;FcRL1-6是穿膜受體,FcRLA及FcRLB則是胞內蛋白。人類的FcRL1蛋白的胞外區域由3個Ig domains組成,穿膜區段含有一個帶有負電荷的麩氨酸(Glutamic acid),而胞內區段則帶有兩個與Immunoreceptor tyrosine-based activation motif (ITAM)相似的Motifs。之前的研究發現,FcRL1為B細胞的活化受體,同時也可以增強B細胞抗原受體(B cell receptor,簡稱BCR)引起的細胞活化。為了研究FcRL1如何增強BCR活化的機制,我們首先觀察FcRL1和BCR共同受體CD19之間的交互作用。由於FcRL1在B細胞上表現的時間與CD19一致,且FcRL1的穿膜區段帶有負電荷的麩氨酸,推測可能會與CD19穿膜區段的正電荷組氨酸(Histidine)有交互作用。當我們將FcRL1及CD19共同表現在293T細胞上,利用免疫沉澱的方法,結果發現兩者會被共同沉澱下來。CD19和FcRL1的結合是具有專一性的,因為當FcRL1和CD71共同表現在293T細胞時,CD71不會跟FcRL1結合。我們發現FcRL1穿膜區段的麩氨酸或是胞內區段對於與CD19的結合是不必要的。當我們利用免疫螢光染色法標示FcRL1及CD19,在共軛焦顯微鏡下也觀察到兩者的位置有部份重疊的現象。為了進一步研究FcRL1如何增強BCR的活化機制,我們同時加入抗FcRL1抗體及抗IgM抗體刺激B細胞,結果發現CD19的磷酸化很明顯地增強,CD19磷酸化的增加也導致和其結合的2個磷酸化蛋白大增,其中一個CD19結合蛋白是PI3K的p85���nsubunit。而FcRL1對於IgM引起的ERK活化或蛋白質磷酸化並沒有明顯影響。本研究的結果顯示FcRL1會和CD19結合,並在和BCR共同連結時增加CD19的磷酸化,引發PI3K結合上CD19,FcRL1可能藉由此機制增加BCR所導致的胞內鈣離子上升及細胞活化。
Fc receptor-like proteins (FcRLs) are a group of receptors that have homologous sequence to the classical Fc receptors. Eight human FcRLs have been identified: FcRL1-6, FcRLA and FcRLB. FcRL1-6 are transmembrane proteins; FcRLA and FcRLB are expressed in the cytoplasm. All FcRLs except FcRL6 are expressed by B cells. Human FcRL1 has 3 extracellular immunoglobulin-like domains, a negative charged glutamic acid in the transmembrane domain, and 2 ITAM-like motifs in the cytoplasmic region. Previous study has demonstrated that human FcRL1 is an activation receptor on B cells, and it can enhance BCR-induced activation. To study the mechanism by which human FcRL1 enhances BCR-induced activation, we first examined whether FcRL1 associates with BCR coreceptor CD19 because of the co-expression FcRL1 and CD19 in B cells. Furthermore, a glutamic acid in the transmembrane domain of FcRL1 may associate with the positive charged histidine in the transmembrane domain of CD19. Our biochemical results demonstrated that HA-FcRL1 could be specifically co-immunopreciptated with CD19, but not with an non-related surface receptor CD71 in a 293T overexpression system. In addition, neither the glutamic acid in transmembrane domain nor the intracellular region of FcRL1 was essential for the association. The partial co-localization of FcRL1 and CD19 was seen on the surface of transfected 293T cells by immunofluorescence microscopy. Our signaling analysis in B cells showed that tyrosine phosphorylation of CD19 was dramatically increased after FcRL1 co-ligation with surface IgM. Consistent with this finding, the recruitment and phosphorylation of phosphatidylinositol 3’-kinase (PI3K) was significantly enhanced by FcRL1 co-ligation. However, the total protein tyrosine phosphorylation and ERK activation were not affected. Collectively, our result suggest that FcRL1 co-ligation upregulated CD19 phosphorylation, followed by the activation of PI3K. FcRL1 may act as a co-receptor in the BCR signaling.
中文摘要---------------------------------------------------------------------------------------- 1
Abstract------------------------------------------------------------------------------------------ 2
序論---------------------------------------------------------------------------------------------- 3
Fc受體------------------------------------------------------------------------------------- 3
Fc Receptor Like molecules(FcRLs)的發現----------------------------------------- 3
人類Fc Receptor Like molecules(FcRLs)------------------------------------------- 4
人類FcRL1 ------------------------------------------------------------------------------ 5
B 淋巴球專一性細胞標記:CD19--------------------------------------------------- 5
B 淋巴球訊息傳遞---------------------------------------------------------------------- 6
RNA 干擾--------------------------------------------------------------------------------- 8
研究動機與目的------------------------------------------------------------------------- 8
材料與方法------------------------------------------------------------------------------------- 10
材料---------------------------------------------------------------------------------------- 10
方法---------------------------------------------------------------------------------------- 22
RNA 抽取--------------------------------------------------------------------------- 22
反轉錄反應(Reverse Transcription,簡稱RT)------------------------------- 22
聚合酶鏈式反應(Polymerase Chain Reaction,簡稱PCR)---------------- 23
質體建構---------------------------------------------------------------------------- 23
大腸桿菌轉型作用(Transformation)------------------------------------------- 23
小量質體DNA 的抽取----------------------------------------------------------- 23
大量質體DNA 的抽取----------------------------------------------------------- 24
細胞轉染作用(Transfection)----------------------------------------------------- 24
免疫沉澱分析(Immunoprecipitation)-------------------------------------------25
西方墨點法(Western Blot)--------------------------------------------------------25
蛋白質生物素標定(Protein Biotinylation)-------------------------------------------------25
B 淋巴球活化(B Lymphocyte Activation)--------------------------------------26
流式細胞儀分析(Flow Cytometry)----------------------------------------------26
免疫螢光染色分析(ImmunoFluorescence analysis)--------------------------26
含shRNA 之Lentivirus 的生產-------------------------------------------------27
測Lentivirus 病毒效價(titer)-----------------------------------------------------27
利用Lentivirus 感染B 淋巴球送入表現載體-------------------------------- 28
結果---------------------------------------------------------------------------------------------- 29
轉染hFcRL1 及CD19 在293T 細胞上--------------------------------------------- 29
hFcRL1 與CD19 會結合--------------------------------------------------------------- 29
hFcRL1 與CD71 不會結合------------------------------------------------------------ 30
hFcRL1 穿膜區段的負電荷麩氨酸及胞內區段對於與CD19 結合為非必要的
---------------------------------------------------------------------------------------------- 30
hFcRL1 會增強anti-IgM 所刺激細胞內鈣離子濃度的上升-------------------- 31
hFcRL1 不會增強所有蛋白的酪氨酸磷酸化-------------------------------------- 32
hFcRL1 會增強CD19 的磷酸化------------------------------------------------------ 32
hFcRL1 不會增強CD19 酪氨酸531 的磷酸化------------------------------------ 33
hFcRL1 增加PI3K 對磷酸化的CD19 結合----------------------------------------33
hFcRL1 不會增強IgM 下游ERK 的活化-------------------------------------------34
討論---------------------------------------------------------------------------------------------- 35
參考文獻---------------------------------------------------------------------------------------- 40
圖表---------------------------------------------------------------------------------------------- 44
附錄---------------------------------------------------------------------------------------------- 58
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