2010-01-012024-05-16https://scholars.lib.ntu.edu.tw/handle/123456789/670038摘要：類轉換重組 (CSR) 是一種脫氧核糖核酸重組 (DNA recombination)，類轉換重組可藉由改變抗體的恆定區 (constant region) 區域 (domain)，而產生具有不同效應作用 (effector function) 的 B 細胞。體細胞突變 (somatic hypermutation) 利用突變 V(D)J片段 (segment) 的方式以增加抗體的抗原決定區的變異性。激活誘導脫氨&#37238; (activation induced deaminase, AID) 在類轉換重組及體細胞突變中非常重要，負責於此二作用中定位 (targeting) 的功能。激活誘導脫氨&#37238;與脫氧核糖核酸修復系統 (DNA repair system) 相結合，於體細胞突變作用中，會在V(D)J片段上製造基因突變。而於類轉換重組作用中，會在開關區域 (switch regions) 產生脫氧核糖核酸雙鏈斷裂 (DNA double strand break)。然而，目前為止，有關激活誘導脫氨&#37238;如何達到基因定位及DNA裂解的詳細機制仍不清楚。 由之前的研究發現，R-環 (R-loops) 可於小鼠 B細胞中的活化的開關區域中偵測到，包含有S，S3，及S2b等區域。除此之外，研究結果指出在類轉換重組作用中，R-環是激活誘導脫氨&#37238;的基因定位機制中的目標。為進一步確定此一發現，老鼠B細胞株，CH12F3將被用來探討R-環於類轉換重組作用中所扮演的角色。CH12F3為IgM陽性細胞，於細胞培養液中加入轉化生長因子-β (TGF-)、白細胞介素4 (IL-4) 及CD40抗體，可以體外刺激細胞轉變成IgA陽性細胞。CH12F3細胞株易於培養及用於基因操作方面的實驗。與基因剃除老鼠及轉植基因鼠相比，產生基因剃除細胞株及轉植基因細胞株所需花費的時間較短及費用也較低。於此研究計畫中首一目標是了解CH12F3細胞中R-環的資訊，至目前為止，仍無CH12F3中R-環相關的研究。在確定CH12F3中有R-環的存在後，第二目標是影響細胞株中R-環的形成。於生物體內，有許多的基因會影響R-環的形成，例如核糖核酸&#37238;H (RNase H) 及其他基因等。於此計畫中，會先從核糖核酸&#37238;H2開始著手。藉由遺傳工程方法，於CH12F3細胞中過度表達核糖核酸&#37238;H2或剔除核糖核酸&#37238;H2基因，以達到影響R-環的形成。之後利用流式細胞儀分析細胞株類別轉換的能力，即可得知R-環於類轉換重組作用中的重要性。 <br> Abstract: Class switch recombination (CSR) is a DNA recombination responsible for changing constant domains of antibodies to produce different effector functions of B cells. Somatic hypermutation (SHM) mutates the V(D)J segment to enhance the antigen-binding affinity of antibodies. Activation induced deaminase (AID) is required and responsible for targeting in both SHM and CSR. With the DNA repair system, AID causes mutations at the V(D)J segment and double strand breaks at switch regions in SHM and CSR, respectively. However, the targeting and cleavage mechanisms of AID are still not clear yet. From previous studies, R-loops are detected at stimulated switch regions in primary murine B cells, including S, S3, and S2b. In addition, R-loops are likely the target for AID in class switch recombination. To further confirm the result, the B cell line, CH12F3 is used to investigate the role of R-loops in class switch recombination. CH12F3 cells are IgM positive cells, after the in vitro stimulation with TGF-, IL-4 and -CD40 antibody, cells switch to IgA positive cells. CH12F3 cells are easy to maintain and suitable for the genetic manipulation. Comparing to knockout mice or transgenic mice, knocking out or over-express genes in cell lines is cheaper and faster. The first step of this study is to gain more knowledge about R-loops in CH12F3 cells. To date, there is no report about the existence of R-loops in the S region in CH12F3 cells. The second step is perturbing the R-loop formation in CH12F3 cells. In cells, many genes or factors would affect R-loop formation, including endogenous RNase H and other genes etc. In this study, we will start with RNase H2 gene. Later, we will also study other genes related to R-loop formation. By using genetic methods, either over-express RNase H2 expression or disrupt the RNase H2 expression and followed by the FACS analysis, the importance of R-loop in class switch recombination can be addressed.抗體類轉換重組體細胞突變開關區域CH12F3細胞株R-環antibodyclass switch recombinationsomatic hypermutationswitch regionsCH12F3 cellsR-loops