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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 黃楓婷(Feng-Ting Huang) | |
dc.contributor.author | Hsin-Yu Chen | en |
dc.contributor.author | 陳欣宇 | zh_TW |
dc.date.accessioned | 2021-06-07T17:54:39Z | - |
dc.date.copyright | 2012-08-28 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-08-16 | |
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Cutting Edge: DGYW/WRCH Is a Better Predictor of Mutability at G:C Bases in Ig Hypermutation Than the Widely Accepted RGYW/WRCY Motif and Probably Reflects a Two-Step Activation-Induced Cytidine Deaminase-Triggered Process. The Journal of Immunology, 172(6), 3382-3384. Rumbaugh, J. A., Murante, R. S., Shi, S., & Bambara, R. A. (1997). Creation and Removal of Embedded Ribonucleotides in Chromosomal DNA during Mammalian Okazaki Fragment Processing. Journal of Biological Chemistry, 272(36), 22591-22599. Sohail, A., Klapacz, J., Samaranayake, M., Ullah, A., & Bhagwat, A. S. (2003). Human activation-induced cytidine deaminase causes transcription-dependent, strand-biased C to U deaminations. Nucleic Acids Research, 31(12), 2990-2994. Stavnezer, J. (1996). Immunoglobulin class switching. Current Opinion in Immunology, 8(2), 199-205. Tonegawa, S. (1983). Somatic generation of antibody diversity. Nature, 302(5909), 575-581. Turchi, J. J., Huang, L., Murante, R. S., Kim, Y., & Bambara, R. A. 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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/15891 | - |
dc.description.abstract | 在外來抗原刺激之下,B cell進行抗體類別轉換重組 (class switch recombination, CSR) 以分泌不同抗體型式,由原本分泌IgM轉換為IgA、IgG、IgE、IgD,有關於CSR如何完成詳細步驟還不是那麼清楚,因此實驗想探討誘發活化性胞嘧啶核苷脫胺酶 (activation-induced cytidine deaminase, AID) 如何參與標的步驟,其中之一假說為R-loops参與作為標的。為了探討R-loops在CSR機制所扮演之角色,本實驗藉由之方法為改變細胞內RNase H表現量。在in vitro情況,RNase H會降解RNA/DNA hybrid的RNA,因此會破壞R-loop結構。因此實驗過量表現E. coli RNase HI或是murine RNase H1於小鼠B細胞株 (CH12F3-2A),另一方面建立穩定表現RNase H2 shRNA的細胞株,降低細胞中RNase H2表現,進而觀察細胞中CSR是否受到影響。
結果顯示過量表現RNase HI (1) 的CH12F3-2A中,R-loops模式 (pattern) 與出現的頻率並無差異,或許可解釋為何CSR頻率在過量表現RNase HI (1) 不受影響。RNase H2 knock down使RNase H2任一subunit表現降低時,CSR皆有不同程度增加,有可能因R-loops出現頻率增加而使CSR隨之增加,但仍需更多實驗分析詳細機制才可更加確認。 | zh_TW |
dc.description.abstract | Upon antigen stimulation, B cells undergo class switch recombination (CSR) to switch from secreting IgM to other isotypes, such as IgA, IgG, IgE, IgD. However, the detailed mechanism of many steps involved in the recombination was unclear yet. This study focused on the activation-induced cytidine deaminase (AID) targeting step. One of the hypotheses for this is R-loops. In order to find out the role of R-loops in CSR, in this study, we altered the expression of RNase H in cells. RNase H degrades RNA in an RNA/DNA hybrid in vitro, thus RNase H could destroy R-loops in cells. Therefore, we would either over-express E.coli or murine RNase H1 in murine B cells (CH12F3-2A) or knock down RNase H2 expression in cells by shRNA. We would investigate the pattern and percentage of R-loops in these cells. In addition, the level of CSR is checked in these cells as well. Results indicated that in RNase HI(1)-overexpression cells, the pattern and the frequency of R-loops were not affected. It might explain the reason of the level of CSR was unchanged in cells. For knocking down expression of any subunit of RNase H2 in cells, the level of CSR increased. One of the possible reasons was that R-loops increased in cells and enhanced CSR. However, the detail mechanism for this needed further experiments to confirm. | en |
dc.description.provenance | Made available in DSpace on 2021-06-07T17:54:39Z (GMT). No. of bitstreams: 1 ntu-101-R99b22026-1.pdf: 1117233 bytes, checksum: e0213eeacb8e4fe98276382736597dd0 (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | 目錄
口試委員會審定書 i 誌謝 ii 摘要 iii Abstrat iv 縮寫表 vi 目錄 vii 第一章、 背景介紹 1 1.1抗體 (Antibody) 1 1.2抗體多樣性 (Antibody diversity) 2 1.3抗體類型轉換重組 (Class switch recombination, CSR) 3 1.4誘發活化性胞嘧啶核苷脫胺酶 (Activation-induced cytidine deaminase, AID) 4 1.5鹼基切除修復 (Base excision repair, BER) 與核酸配對錯誤修復 (Mismatch repair, MMR) 4 1.6 AID targeting 5 1.7 R-loops 6 1.8核糖核酸酶H (Ribonuclease H, RNase H) 7 1.9研究動機與目的 9 第二章、 材料與方法 10 2.1藥品與試劑 10 2.2細胞培養與刺激 12 2.3 RNase HI (1) 過量表現系統 12 2.4流式細胞儀分析 13 2.5核酸萃取 (酚-氯仿萃取法) 13 2.6 Bam HI作用 14 2.7亞硫酸氫鈉處理DNA (Sodium bisulfite treatment) 14 2.8聚合酶鏈鎖反應 (Polymerase chain reaction, PCR) 15 2.9洋菜膠電泳 (Agarose gel electrophoresis) 18 2.10 TA cloning (TOPO TA Cloning® Kits, Invitrogen) 18 2.11大腸桿菌勝任細胞 (Competent cell) 製備 18 2.12電轉型作用 (Electroporation Transformation) 19 2.13質體DNA萃取 (Mini-preparation&Midi-preparation) 19 2.14 DNA定序分析 20 2.15 R-loops訊號頻率分析 20 2.16製備帶有特定基因之shRNA序列的質體 20 2.17 ScaI作用 22 2.18轉染作用 (transfection) 22 2.19嘌呤霉素 (puromycin) 篩選 23 2.20抽取total RNA 23 2.21製備互補DNA (Complementary DNA, cDNA) 23 2.22即時定量聚合酶鏈反應 (Real time PCR) 24 2.23相對定量 (Relative Quantization, △△Ct method) 26 第三章、 實驗結果 27 3.1過量表現RNase HI (1) 28 3.1.1在CH12F3-2細胞株過量表現RNase HI (1) 對CSR之影響 28 3.1.2過量表現RNase HI (1) 對R-loops訊號頻率之影響 28 3.2降低rnase h2的表現 (RNase H2 knock down) 29 3.2.1刺激一至三天對CH12F3-2A之rnase h2表現量影響 29 3.2.2 RNase H2a knock down對CSR之影響 30 3.2.3 RNase H2b knock down對CSR之影響 31 3.2.4 RNase H2c knock down對CSR之影響 32 第四章、 討論 33 第五章、 實驗圖表 36 圖一、過量表現RNaseHI (1) 對CSR之影響 37 圖二、細胞株中於Sμ及Sα之R-loops pattern 40 圖三、過量表現RNase HI (1) 情形下Sμ及Sα中R-loops訊號頻率 41 圖四、CH12F3-2A刺激一至三天後CSR比例變化 42 圖五、CH12F3-2A細胞受刺激一至三天後RNase H2表現量 44 圖六、降低rnaseh2a表現量對CSR之影響 45 圖七、穩定表現shRNA之細胞株中rnase h2a表現量 46 圖八、降低rnase h2b表現量對CSR之影響 47 圖九、穩定表現shRNA之細胞株中rnase h2b表現量 48 圖十、降低rnase h2c表現量對CSR之影響 49 圖十一、穩定表現shRNA之細胞株中rnase h2c表現量變化 50 參考文獻 51 附圖 56 附圖一、RNase HI-IRES-EGFP建構質體示意圖 56 附圖二、過量表現外來E. coli之RNase HI 56 | |
dc.language.iso | zh-TW | |
dc.title | 影響核醣核酸酶H表現以探討其於R-loops與抗體類型轉換重組之相關聯性 | zh_TW |
dc.title | Effects of altering the expression of RNase H on R-loops and class switch recombination | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 常怡雍(Yee-yung Charng),廖憶純(Yi-Chun Liao),楊健志(Chien-Chih Yang),張世宗(Shih-Chung Chang) | |
dc.subject.keyword | 抗體類別轉換重組,誘發活化性胞嘧啶核苷,脫氨酶,R環,核醣核酸酶,H,轉換區, | zh_TW |
dc.subject.keyword | class switch recombination,activation-induced cytidine deaminase,R-loops,RNase H,switch region, | en |
dc.relation.page | 57 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2012-08-16 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科技學系 | zh_TW |
顯示於系所單位: | 生化科技學系 |
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