核酸內切酶Slx1對抗體類型轉換重組之影響及其功能探討

Studies on the role of Slx1 in class switch recombination

謝文娟(Wen-Chuang Hsieh)

指導教授 ： 黃楓婷

國立臺灣大學/生命科學院/生化科技學系/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10438

摘要

抗體類型轉換重組 (class switch recombination, CSR) 是B細胞為了有效抵抗外來抗原所進行的分子機制，可使B細胞原分泌IgM轉換成分泌不同類型的抗體。因CSR分子機制相當複雜，以至於目前研究對CSR各階段所參與之因子尚不清楚。故本論文欲探討參與CSR機制中尚未被發掘之潛在重要分子。藉由微陣列 (microarray) 分析小鼠B細胞於刺激進行CSR後表現量上升的基因，挑選出目標基因Slx1。Slx1與Slx4形成複合體，為一結構特異性內切酶 (structure specific endonuclease)，能專一性截切基因重組過程中的數種DNA二級結構，依功能及表現量上預測，Slx1有可能參與於CSR反應。 本篇研究目的即是於小鼠B細胞株 (CH12F3) 中，以基因抑制和基因剔除之方式探討Slx1是否作用於CSR。CH12F3細胞經刺激後會進行CSR，自表現IgM轉變為表現IgA的抗體類型。本研究發現，受刺激兩天之細胞其Slx1基因表現量約為未受刺激細胞的兩倍。為驗證Slx1對CSR的重要性，本研究以具有Slx1 shRNA的質體轉染入細胞中，得到一株Slx1基因抑制效率高達80% 之轉染株，其CSR發生頻率為控制組的1.6倍。為更加確定此結果，本論文嘗詴於細胞中剔除Slx1基因。目前已得到Slx1+/puro及Slx1+/-之細胞株，進行分析後發現其CSR發生頻率與控制組相比為顯著性的降低，此表現型與Slx1基因抑制之實驗截然不同。由此研究結果推測Slx1可能參與CSR機制中，但未來仍需更多實驗結果證實Slx1於CSR所扮演的角色。

關鍵字

抗體類型轉換重組 ； Slx1 ； Slx4 ； 基因抑制 ； 基因剔除

並列摘要

To efficiently defend various antigens, B cells secrete many different isotypes of antibodies. Upon stimulation, B cells switch from secreting IgM to other isotypes of antibodies. The isotype switching is through class switch recombination (CSR). Until now, the detailed molecular mechanism of CSR remains unclear. Therefore, this study aimed to gain insights into CSR by analyzing the differential gene expression pattern from microarray data of the murine B cell line. The up-regulated genes during CSR were further confirmed by qRT-PCR. From literature researching, we selected Slx1 as the potential candidate gene. Slx1 forms the complex with Slx4 and the complex is known as a structure-specific endonuclease. Previous studies indicate that the Slx1-Slx4 complex processes various DNA secondary structures formed during DNA repair. Hence, Slx1 might be involved in CSR.
In this study, through knocking down Slx1 or knocking out Slx1 gene in CH12F3 cells, the CSR model cell line, the role of Slx1 in CSR was investigated. First, Slx1 mRNA level in stimulated CH12F3 cells was about two-fold of unstimulated cells by qRT-PCR assay. The result was similar with the microarray analysis. Moreover, CSR frequency in Slx1 knockdown cells increased comparing to wild type. As for Slx1 knockout in CH12F3 cells (Slx1+/-), surprisingly, the CSR was dramatically reduced comparing to wild type, which was totally opposite to the results of Slx1 knockdown experiments. The results suggest that Slx1 might be involved in CSR. The requirement and the role of Slx1 in CSR need further experiments to confirm.

並列關鍵字

class switch recombination (CSR) ； Slx1 ； Slx4 ； knockdown ； knockout

參考文獻

Aoufouchi, S., Faili, A., Zober, C., D'Orlando, O., Weller, S., Weill, J. C., & Reynaud, C. A. (2008). Proteasomal degradation restricts the nuclear lifespan of AID. J Exp Med, 205(6), 1357-1368.

Aravind, L., & Koonin, E. V. (2001). Prokaryotic homologs of the eukaryotic DNA-end-binding protein Ku, novel domains in the Ku protein and prediction of a prokaryotic double-strand break repair system. Genome Res, 11(8), 1365-1374.

Auerbach, A. D. (2009). Fanconi anemia and its diagnosis. Mutat Res, 668(1-2), 4-10.

Bachrati, C. Z., & Hickson, I. D. (2008). RecQ helicases: guardian angels of the DNA replication fork. Chromosoma, 117(3), 219-233.

Barreto, V., Reina-San-Martin, B., Ramiro, A. R., McBride, K. M., & Nussenzweig, M. C. (2003). C-terminal deletion of AID uncouples class switch recombination from somatic hypermutation and gene conversion. Mol Cell, 12(2), 501-508.