臺灣博碩士論文加值系統

Deltex (DTX)在果蠅中負責調控Notch訊息的傳遞，然而對於DTX在哺乳類動物中的作用機制以及生理功能目前尚未明瞭。哺乳類動物中共發現有四種DTX，分別為DTX1、DTX2、DTX3和DTX4。DTX1已知能透過降解MEKK1(C)蛋白質的表現，進而抑制T細胞的活化。DTX3因缺少WWE domains故其結構異於其他DTX成員，並且無法和Notch結合。去除WWE domains後的DTX1發現仍具有部分抑制T細胞活化的能力，顯示DTX3可能也參與調控T細胞的活化。故在本研究中我們藉由大量表現DTX3和降低Dtx3的表現，探討DTX3在T細胞活化中扮演的角色。我們發現Dtx3在DO11.10及EL4這兩種T細胞細胞株，以及在老鼠脾臟的CD4+ T細胞中有表現。進一步利用反轉錄病毒感染的方式，建立能穩定分別表現DTX1和DTX3的DO11.10細胞株。結果發現大量表現DTX3，能部分抑制DO11.10細胞活化時IL-2的產生、JNK的活化和p38 MAPK的磷酸化。同時大量表現DTX3也能部分降解MEKK1(C)蛋白質的表現。我們進一步利用Dtx3 miR RNAi降低DO11.10細胞中Dtx3的表現。結果發現降低Dtx3的表現能增加DO11.10細胞活化時IL-2的產生和JNK、p38 MAPK及ERK的活化。同時降低Dtx3的表現也能增加MEKK1(C)蛋白質的表現。因此本實驗的結果顯示DTX3在T細胞活化時，同樣扮演負向調節的角色。為了探討DTX3的生理功能，我們同時著手建立DTX3專一性抗體以及Dtx3基因剔除老鼠。我們從基因轉殖中心獲得329個胚胎幹細胞基因組DNA，並利用四種不同的PCR引子組進行基因型鑑定。最後總共篩選出5個positive clones，同時也利用南方點墨法證實這5個clones確實被Dtx3剔除載體正確標的。在進行囊胚顯微注射後目前已產生數隻嵌合鼠，將被用來進一步產生Dtx3基因剔除老鼠。

Deltex (DTX) is a regulator of Notch signals in Drosophila, but its action mechanism and physiological functions are poorly understood in mammals. There are four DTX homologues (DTX1, DTX2, DTX3 and DTX4) in mammals. DTX1 has been identified as a negative regulator of T cell activation through degradation of active MEKK1 protein. DTX3 is unique among the DTX family for the absence of WWE domains, and its inability to bind Notch. DTX1 with both WWE domains deleted retains the partial capacity to inhibit T cell activation, suggesting DTX3 may also involved in T cell activation. In this study, we aim to elucidate whether DTX3 participates in T cell activation by DTX3 overexpression and Dtx3 downregulation. We found that Dtx3 was expressed in T cell lines including DO11.10 and EL4, as well as in primary T cells. We established DO11.10 clones transduced with DTX1 or DTX3. Overexpression of DTX3 in DO11.10 cells partially suppressed T-cell receptor (TCR)-stimulated IL-2 production, JNK activation, and p38 MAPK phosphorylation. The protein level of C-terminal catalytic domain of MEKK1 [MEKK1(C)] was also partially downregulated by DTX3. The physiological role of DTX3 in T cells was further examined by Dtx3 knockdown. We found that TCR-stimulated production of IL-2 and activation of JNK, p38 MAPK, and ERK were enhanced when Dtx3 was downregulated. The protein level of MEKK1(C) was also enhanced when Dtx3 was downregulated. Together, these data suggest that DTX3 also act as a negative regulator of T cell activation. For analysis on the physiological functions of DTX3, we are in the progress to generate DTX3-specific antibodies, as well as to produce Dtx3-KO mice. We obtained 329 ES cell genomic DNA from the Transgenic Core Facility and screened it by different PCR primers. After the PCR genotyping, 5 positive clones were identidied and the insertion of targeting construct was also confirmed by Sothern blotting. Blastocyst microinjection has generated a number of chimeric mice that will be used to produce Dtx3-/- mice in near future.

摘要..........................................................................................................け
Abstract.....................................................................................................げ
目錄..........................................................................................................ご
圖目錄......................................................................................................し
第一章 簡介........................................................................................1
一、 T細胞活化(T cell activation).......................................................................1
二、 Deltex.............................................................................................................2
1. Deltex之簡介.........................................................................................................................2
2. Deltex的結構……………………………………………………………….........……..…..3
3. Deltex的功能……………………………………………………………..……..….......…..3
4. Deltex與淋巴細胞發育…………………………………………………..…….......……....4
5. Deltex與T細胞活化…………………………………………………..………..….......….4
三、 MEKK1..........................................................................................................5
1. MEKK1之簡介…………………………………………………………….….…...…...…..5
2. MEKK1參與之訊息調控……………………………………………….…...……......……5
3. MEKK1的生理功能……………………………………….………………......……...……6
四、 研究方向與目的............................................................................................6
第二章 材料與方法............................................................................8
一、 細胞株與細胞培養.......................................................................................8
1. 細胞株....................................................................................................................................8
2. 細胞培養................................................................................................................................8
二、 藥品與試劑...................................................................................................8
三、 抗體...............................................................................................................9
四、 質體構築.......................................................................................................9
1. pGC-IRES-DTX3-YFP的構築..............................................................................................9
2. 微小型干擾RNA單股DNA寡核酸設計(miR RNAi single-stranded DNA oligos).........10
3. pcDNA��6.2-EmGFP-Dtx3-miR質體構築.........................................................................10
4. 鏈接前體miRNA (chaining pre-miRNAs)..........................................................................11
五、 質體DNA的轉染(Transfection).................................................................11
1. 磷酸鈣轉染法(Calcium phosphate transfection).................................................................11
2. 反轉錄病毒感染法(Retroviral infection)............................................................................11
3. 電穿孔法(Electroporation)...................................................................................................12
六、 Dtx3-miR穩定細胞株(stable cell lines)之建立.........................................12
七、 IL-2產量分析(IL-2 production assay)........................................................13
1. T細胞活化..........................................................................................................................13
2. IL-2產量分析……………………………………………………………...………...........13
八、 RNA的純化...............................................................................................14
九、 RNA反轉錄與聚合�○s鎖反應(RT-PCR)...............................................14
1. RNA反轉錄.........................................................................................................................14
2. 聚合�○s鎖反應..................................................................................................................15
十、 西方點墨法(Western blot)..........................................................................15
十一、 Dtx3基因剔除鼠(Dtx3-KO mice)之建立..................................................16
1. 胚胎幹細胞基因標的(ES cell gene targeting)…………………………………......…..….16
2. 置備胚胎幹細胞基因組DNA(ES cell genomic DNA)………………………..…..……..16
3. 基因型鑑定(Genotyping) ─ PCR........................................................................................16
十二、 南方點墨法(Southern blot)………………………………….........………17
1. Dtx3探針(probe)之製備……………………………………...…………….......…..……17
2. 前雜交反應(Prehybridization).............................................................................................18
3. 雜交反應(Hybridization).....................................................................................................18
第三章 結果......................................................................................20
一、 Dtx3在T細胞細胞株及老鼠CD4+ T細胞中均有表現............................20
二、 大量表現DTX3抑制T細胞活化...............................................................20
1. DTX3蛋白質在DO11.10細胞中穩定表現……………………………….……………20
2. DTX3部分抑制DO11.10細胞活化後IL-2的產生.........................................................21
三、 大量表現DTX3對於T細胞活化中訊息傳遞的影響...............................21
1. DTX3抑制DO11.10細胞活化時JNK的活化...................................................................22
2. DTX3抑制DO11.10細胞活化時p38 MAPK的活化.......................................................22
3. DTX3不影響DO11.10細胞活化時ERK的活化..............................................................23
4. DTX3部分抑制DO11.10細胞中MEKK1(C)蛋白質的表現...........................................23
四、 降低Dtx3表現促進T細胞活化.................................................................23
1. Dtx3 miR RNAi降低DO11.10細胞中Dtx3的表現……………………………………..23
2. 降低Dtx3表現增加DO11.10細胞活化時IL-2的產生.....................................................24
3. Dtx3的表現量在Dtx3-miR穩定細胞中降低………………………………………..…..24
4. Dtx3-miR穩定細胞活化時產生較多的IL-2……………………………………..………25
五、 降低Dtx3表現對於T細胞活化中訊息傳遞的影響.................................25
1. 降低Dtx3表現增加DO11.10細胞活化時JNK的活化………………………….………25
2. 降低Dtx3表現增加DO11.10細胞活化時p38 MAPK的活化…………………….……26
3. 降低Dtx3表現增加DO11.10細胞活化時ERK的活化……………….....……......……..26
4. 降低Dtx3表現增加DO11.10細胞中MEKK1(C)蛋白質的表現......................................26
六、 建立Dtx3基因剔除鼠.................................................................................27
1. 利用PCR引子Dtx3/ Gli1及Dtx3/ Lrig3進行基因型鑑定的結果……..……......………27
2. 利用PCR引子Dtx3/ neo及Dtx3/ LacZ進行基因型鑑定的結果………..…….......…….27
3. 南方點墨法分析結果..........................................................................................................28
第四章 討論......................................................................................29
圖表..........................................................................................................33
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