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DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 李財坤(Tsai-Kun Li) | |
dc.contributor.author | Wan-Ning Leu | en |
dc.contributor.author | 呂婉寧 | zh_TW |
dc.date.accessioned | 2021-06-16T02:31:44Z | - |
dc.date.available | 2023-07-29 | |
dc.date.copyright | 2015-09-25 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-07-30 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/53863 | - |
dc.description.abstract | 在轉錄延伸(transcription elongation)的過程中,由於DNA的雙股結構,RNA Polymerase(RNAP)複合體的前方會形成緊密的正向超螺旋(positive supercoil),而後方形成鬆散的負向超螺旋(negative supercoil);而負超螺旋鬆散的DNA容易被新合成的RNA入侵而形成一個DNA與RNA雜合的結構稱為R-loop;R-loop結構中暴露出的ssDNA被認為transcription-associated mutagenesis (TAM)以及transcription-associated Recombination(TAR)有關。根據實驗室前期研究,RNase H 與 topoisomerases可以調控R-loop結構的形成。DNA topoisomerases是一種可以直接作用在DNA上的酵素,藉由切割及黏合DNA的方式來改變DNA的拓樸結構,藉由舒緩RNAP complex後方鬆散的負超螺旋進而抑制R-loop的形成。因為R-loop是一種open DNA chromatin的狀態,被認為與細胞內許多作用機制相關,其中在B細胞抗體歧異性(antibody diversification)上被認為與class switch recombination(CSR)的發生有關,CSR是一種轉錄相關的基因重組作用,由activation-induced cytidine deaminase(AID)主導,可能藉由在S region中R-loop結構暴露出的ssDNA上產生U:G mispair而使修復蛋白在DNA上產生切割,接著由遠端序列的重組進而使B細胞isotypes的switching發生。根據以上論述,我們認為topoisomerases可能藉由調控R-loop 形成的方式參與在CSR之中。首先,小鼠脾臟B細胞因受到cytokines刺激後因為細胞內部再進行旺盛的基因表現,型態上會有細胞脹大的現象。顯微鏡底下計數的結果可以看到在刺激的細胞脹大的比例逐漸上升,並且在36小時時達到高峰 (~ 10%)。為了要偵測topoisomerases是否參與在CSR的過程中,所以先看topoisomerases在CSR的過程中是否有蛋白表現量上的變化?結果看到在刺激後的B細胞中,隨著AID的上升,Top1跟Top2β的蛋白量在24小時時上升,而36小時則降得比原本還低,Top2α以及Top3蛋白則看不到顯著的變化。接下來使用CH12F3細胞來做後續的實驗,第一個先看上述的現象是否也會在此細胞株發生,在刺激後的CH12F3細胞中可以看到跟小鼠脾臟B 細胞相似的趨勢:隨著AID的上升;Top1跟Top2β的蛋白量先上升,而後下降,這些細胞的暗示Top1跟Top2β可能參與在CSR的調控之中。接著問Top1跟Top2β在CSR中的重要性,我們利用lentivirus感染的方式knockdown Top1或Top2β的表現後,藉由流式細胞儀(flow cytometry)來偵測CSR的頻率是否有減少?為了有效的觀察CSR的變化,我們亦使用了一個加入SCI質體的CH12F3,稱為HTT112。經過72小時的刺激,HTT112細胞以帶有PE螢光的anti-IgA抗體染色,最後由flow的偵測來確認HTT112細胞的CSR的發生頻率。由結果可以看到HTT112細胞的CSR發生頻率比沒有SCI質體的CH12F3還要高出許多。接著生產了用於knockdown topoisomerases的病毒,並且測試其knockdown的效能並當作選擇knockdown序列的依據。接著用lentivirus knockdown HTT112細胞的Top1,經由72小時的刺激以及後續的染色,發現Top1 knockdown的HTT112細胞的surface-IgA有明顯的比對照組低出許多,代表CSR頻率有明顯的下降。 | zh_TW |
dc.description.abstract | During transcription, unwinding of the DNA double helix and tracking process lead to positive and negative supercoil arising in front and behind of the advancing RNA polymerase. Nascent RNβA strand invades into the DNA duplex and hybridize with the complementary template strand occasionally and creating the transient three-stranded structure called R-loop. R-loop is sensitive to chemical agents leading to higher mutagenesis and is believed to link to transcription-associated mutation (TAM) and recombination (TAR), such as somatic hypermutation (SHM) and class switch recombination (CSR). Our previous studies suggested that DNA topoisomerases, through resolving DNA topological problems during transcription, can regulate the formation of R-loop. Our aim is to dissect the potential involvement of topoisomerases in the process of CSR by regulating the formation of R-loop structure. Here, splenic B cells were first isolated from WT mice and screened for candidate topoisomerases by monitoring protein expression in a time course after treatment of switching cytokines, interleuckine-4 (IL-4) and lipopolysaccharide (LPS). The Top1 and Top2 | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T02:31:44Z (GMT). No. of bitstreams: 1 ntu-104-R02445127-1.pdf: 3449250 bytes, checksum: 83f0b735741ab0ffc3ac55def4cb59c8 (MD5) Previous issue date: 2015 | en |
dc.description.tableofcontents | 口試委員審定書 …………………………………………………………………....I 致謝………………………………………………………………….………………..II 中文摘要………………………………………………………………….…...…….III ABSTRACT ……………………………………………………………………….V CONTENTS………………………………………………………..……….…….VII INTRODUCTION……………………………………………………………..…….1 1. Transcription-associated mutation (TAM) and transcription-associated recmbination (TAR)……………………………………………………………….…1 1.1 Class switch recombination (CSR)…………………..……………….……………2 1.2 CSR activation, stimuli and R-loop formation……….……………………………3 1.3 Switch regions (S regions)…….……………………….…………………………..4 2. R-loop formation and regulation………………………………………...……….5 2.1 Features of R-loop and forming prerequisites …………………………...……….7 2.2 Regulation of R-loop………………………………………………………………7 2.3 R-loop formation and potential regulation in the immunoglobulin S regions..…...8 3. Activation induced cytidine deaminase (AID)…………………………………..9 3.1 Structure of AID………………………………………………………………...10 4. DNA Topoisomerases…………..………………………………………………...11 4.1 Topoisomerase I (Top1)………..………………………………………………...11 4.2 Topoisomerase II (Top2β)………..………………………………………………13 4.3 Topoisomerses and R-loop formation……………………………………………14 SPECIFIC AIMS………..…………………………………………………………..16 MATERIALS AND METHODS………………………………………...…………17 - Cell lines and plasmid………………………………..………………………...17 - Cell culture……………………………………………………………………...17 - CSR induction and stimulation…………..……………………………...………18 - Cell enlargement analysis………………………………………………………18 - Antibodies and primers…………………………………………………………18 - Immunoblotting analysis………………………………………………………..19 - Quantitative measurement and statistic analysis……………………………….21 - shRNA-mediated knockdown……………………………………………………21 - Flow cytometry………………………………………………………………….22 - S9.6 antibody production………………………………………………………..22 - S9.6 antibody purification and dialysis…………………...……………………23 RESULTS……………………………………………………………………………24 - After the co-treatment of lipopolysaccharide with interleukine-4, splenic B cells underwent proliferation……………………………………..………………………...24 - Protein levels of Top1 and Top2βwere changed during CSR in splenic B cell………………………………………………………………………………………25 - Protein levels of Top1 and Top2β were changed during CSR in CH12F3 cell line………………………………………………………………………….…..25 - The CSR frequency of HTT112 is higher than CH12F3….……………….……26 - Efficiency of topoisomerases knockdown………...……………………………..27 - Knockdown Top1 leads to impairment in HTT112 cells…...……………..……..28 - The CSR frequency is no significant difference between shLuc and shTop2β HTT112 cells…………………….………………………………………….29 - Knockdown AID leads to CSR impairment in HTT112 cells expectedly………..30 DISCUSSION……………………………………………………………………….32 FIGURES AND TABLE……………………………………………………………37 - Table 1: The list of the lentiviral vector (pLKO.1)-based clones from of RNAi Core Facility……………………………………………..……………..………38 - Figure 1. Enlargement of splenic B cell after cytokine stimulation…………….39 - Figure 2. Top1 and Top2β protein but not Top3 or Top2α proteins were regulated after stimulation in Splenic B cells…………………………………40 - Figure 3. Top1 and Top2β proteins were also regulated after stimulation in HTT112………………………………………………………………...………..43 - Figure 4. The CSR frequency of HTT112 is significantly higher than CH12F3…………………………………………………………..……………..46 - Figure 5. Knockdown efficiency test of shTop1, shTop2α, shTop2β, shTop3α, and shTop3 plasmid on a MEF cell line……………………………………………48 - Figure 6. Knockdown Top1 leads to CSR impairment in HTT112 cells……..…51 - Figure 7. There is no significant different between shLuc and shTop2β HTT112 cells………………………………………………………………..…………….54 - Figure 8. Knockdown AID leads to CSR impairment in HTT112 cells…………57 REFERENCES……………………………………………………………………...60 | |
dc.language.iso | en | |
dc.title | 探討DNA拓樸異構酶是否參與在免疫球蛋白(抗體)轉換的過程 | zh_TW |
dc.title | Investigate on the involvement of DNA topoisomerases in immunoglobulin class switch recombination | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 鄧述諄(Shu-Chun Teng),李建國(Chien-Kuo Lee) | |
dc.subject.keyword | 拓樸異構?,抗體型別轉換,誘發活化性胞嘧啶核?脫胺?,R圈, | zh_TW |
dc.subject.keyword | Topoisomerase,Class switch recombination,Activation induce-cytidine deaminase,R-loop, | en |
dc.relation.page | 66 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2015-07-30 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 微生物學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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