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研究生:章茜文
研究生(外文):Chien-Wen Chang
論文名稱:小鼠FcRLl訊息傳遞之研究
論文名稱(外文):The study of mouse Fc receptor like-1 (mFcRL1) signaling
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:2010
畢業學年度:98
語文別:中文
論文頁數:59
中文關鍵詞:FcRLB細胞訊息傳遞
外文關鍵詞:FcRLB cellsignaling
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FcRLs (Fc receptor-like molecules)是一與 Fc受體基因具同源性的家族,在人類中有 8 個成員,而在小鼠中則有 6 個成員。在人類與小鼠的 FcRL 家族當中,人類 FcRL1(hFcRL1)與小鼠 FcRL1(mFcRL1)的序列相似性最為顯著。mFcRL1為一穿膜蛋白,胞外區域由兩個 Ig-like domains 所構成,胞內區域則帶有一個負責傳遞活化訊號的 ITAM (immunoreceptor tyrosine-based activation motif )及在一個可扮演活化或抑制性角色的 ITSM(immunoreceptor tyrosine-based switch motif )。我們之前的研究發現,hFcRL1 可作為 B細胞抗原受體 (B cell receptor,簡稱 BCR)的協同受體(coreceptor),加強由 BCR 所引發的活化作用以及細胞增生;hFcRL1 在單獨刺激後則會活化下游 Grb2-ERK 以及 PI3K-Akt 等途徑,最後造成 B 細胞的增生與活化。由於 mFcRL1 之胞內區域也帶有一 ITAM,另帶有一 ITSM,所以我們推測 mFcRL1 可以藉由胞內區域中的 motifs 來傳遞訊息並促進 B 細胞的活化及增生。為了研究 mFcRL1 的作用,我們構築了五個mFcRL1 突變株,分別將 ITAM 及 ITSM 中的三個酪氨酸置換成苯丙氨酸(Tyr→Phe)。由於目前並無 mFcRL1 的配體(ligand)或 agonist 抗體,我們將 CD32 (FcγRIIB)的胞外及穿膜區域連結至mFcRL1的胞內區域,形成融合受體(chimeric receptor),之後再利用抗體刺激來誘發反應。結果發現到受到刺激後,mFcRL1中的酪氨酸會有磷酸化的現象,而所有突變株中當 Y281 被置換時磷酸化的情形則有明顯的減少,因此 Y281 應是活化後最主要會被磷酸化的位置。CD32-mFcRL1 融合受體在刺激後,也會引起其他蛋白的磷酸化,我們觀察到一個 110kD 大小的蛋白磷酸化增加,而這個現象在 Y281F 突變株中有減少的情形。我們也同時測試 mFcRL1 是否可以作為 BCR 協同受體,目前觀察到在兩者共同刺激之下會加強部分的蛋白磷酸化,但對 ERK活化的影響並不顯著。然而,在共同連結之下反而會減弱 Akt 的活化。總結本篇研究可以得知,mFcRL1胞內區域具有傳遞訊息的能力,同時也有調控BCR 訊息傳遞的潛力。
FcRLs (Fc receptor-like molecules) are a newly found receptor family that shares homology with the classical Fc receptors. There are 8 members in humans and 6 in mice. Among these family members, hFcRL1 and mFcRL1 contain most
significant homology, so we speculated that mFcRL1 may be the ortholog to hFcRL1. mFcRL1 is a transmembrane protein, and it possess two Ig-like domains in the extracellular region and one ITSM (immunoreceptor tyrosine-based switch motif) and one ITAM (immunoreceptor tyrosine-based activation motif) in the cytoplasmic region, so mFcRL1 may deliver activation signal. Our previous study showed that
hFcRL1 can act as a BCR coreceptor which enhances B cell activation and proliferation. In addition, hFcRL1 can activate Grb2-ERK and PI3K-Akt signal pathways. We wanted to know whether mFcRL1 induces signaling transduction and
B cell activation. To test this idea, we first converted the 3 conserved tyrosines of mFcRL1 cytoplasmic domain to phenylalanines and fused the sequence with CD32 extracellularand transmembrane domains to generate chimeric receptors. After we introduced the chimeric receptors into IIA1.6 cell line and selected stable lines, these cells were used to perform experiments. We found that the chimeric receptor was tyrosine phosphorylated after ligation, and phosphorylation was significantly disappeared in the Y281F mutant, so Y281 is the major tyrosine phosphorylation site in mFcRL1. Furthermore, chimeric receptor induced some protein phosphorylation, and a protein about the 110kDa showed obvious phosphorylation after stimulation. We next examined the effect on chimeric receptor on BCR signaling. When coligation chimeric receptor with BCR, it enhanced some of BCR-induced protein phosphorylation, but had no effects to ERK activation. In addition, chimeric receptor reduced Akt activation. In conclusion, our study suggests that mFcRL1 can induce
signal transduction and it may regulate BCR signaling.
目錄........................................1
摘要........................................3
Abstract....................................4
序論........................................6
傳統Fc受體..................................6
Fc受體相似蛋白 (Fc Receptor-like molecules, FcRLs)
的發現......................................7
FcRLs 的結構與分布..........................7
Fc Receptor-like molecules
(FcRLs)的功能...............................8
小鼠 FcRL1 (mFcRL1).........................9
ITAM、ITIM 和 ITSM 序列.....................9
B細胞受體與B細胞的活化......................11
研究動機....................................12
材料與方法..................................13
A.材料......................................13
試劑與溶液..................................13
培養基與培養液..............................18
菌株與細胞株................................20
抗體........................................20
B.方法......................................22
質體構築....................................22
細菌轉型(Transformation)....................24
質體抽取....................................24
DNA洋菜膠電泳
(DNA gel electrophoresis)...................25
細胞株的建立................................25
流式細胞儀分析(flow cytometry)..............25
B淋巴球的活化
(B lymphocytes activation)..................26
免疫沉澱(immunoprecipitation)...............26
蛋白質電泳與西方墨點法
(SDS-PAGE and western blot).................26
蛋白質生物素標定
(protein biotinylation).....................27
抗體純化(antibody purification).............27
小鼠脾臟B細胞的感染.........................27
結果........................................28
融合受體質體的建構以及細胞株的建立..........28
CD32-mFcRL1融合受體受到刺激後會磷酸化.......29
CD32-mFcRL1融合受體會引起其他蛋白的磷酸化...30
CD32-mFcRL1融合受體並不會引起ERK的活化......30
CD32-mFcRL1 融合受體並不會與 Syk、Grb2 結合.31
CD32-mFcRL1融合受體會增強IgG引發的所有蛋白磷酸化31
CD32-mFcRL1融合受體不會加強IgG所引發的ERK活化32
CD32-mFcRL1融合受體減弱IgG所引發的Akt活化...33
小鼠脾臟B細胞轉染系統的建立.................33
討論........................................34
參考文獻....................................40
圖表........................................44
附錄........................................58

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