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標題: | Srsf1於抗體類型轉換重組機制中扮演之角色探討 Investigation of the Role of Srsf1 in Class Switch Recombination |
作者: | Jia-Yu Lin 林家伃 |
指導教授: | 黃楓婷 |
關鍵字: | 抗體類型轉換重組,Srsf1,Srsf1異構型,核質分離,基因剔除, class switch recombination (CSR),Srsf1,Srsf1 isoform,subcellular fractionation,gene knockout, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 為能有效保護宿主不受到不同的外來抗原入侵,B細胞必須具備製造多樣化抗體的能力,而其中之一便是透過抗體類型轉換重組機制 (Class switch recombination, CSR) 使細胞表面從表現抗體IgM轉變為其他類型抗體。Srsf1能藉由阻止R-loop結構生成以穩定基因體,且其異構型Srsf1-3可能參與於SHM中。由於R-loop結構被認為存在於CSR中,且SHM和CSR兩機制有許多相似處,我們推測Srsf1亦有可能參與在CSR中。在論文中我們透過兩部分的實驗探討Srsf1於CSR中的可能角色:觀察CSR進行時的Srsf1特性變化包含srsf1基因表現及其於細胞中分佈情形,以及於小鼠B細胞株 (CH12F3) 中進行srsf1基因剔除。由實驗結果發現,細胞經刺激後,Srsf1-1的mRNA含量於刺激兩天組別無顯著變化,於刺激三天組別下降至九成左右,其蛋白質表現量則於刺激兩天及三天後下降至未受刺激組別的八成左右,此外,Srsf1-3的mRNA含量亦於刺激兩天及三天後下降至未受刺激組別的八成左右。透過核質分離觀察Srsf1-1於細胞中的分佈,發現於未受刺激及受刺激組別中,大部份Srsf1-1皆位於細胞核中,且細胞經刺激後,細胞質中Srsf1-1下降至未受刺激組別之六成左右,而細胞核部分則有待內部控制蛋白質 (internal control) 決定後才能進一步分析。接著第二部分實驗於CH12F3細胞中進行srsf1 基因剔除,欲確認其對CSR的重要性,目前針對srsf1+/-細胞株經刺激後初步檢測其CSR發生頻率,發現和wild type組別相比並無顯著變化,而srsf1 基因可能對於細胞生存具重要性,致使目前尚未得到srsf1-/-細胞株。綜合目前的實驗結果,Srsf1於CSR中的功能仍需更多實驗證據進行推論。 To effectively protect the host against different kinds of pathogens, B cells are capable to secret various isotypes of antibodies. Through class switch recombination (CSR), the immunoglobulin isotype of B cells switch from IgM to other isotypes. Srsf1 was known to maintain genome stability by preventing R-loops formation, and its isoform Srsf1-3 might be involved in somatic hypermutation (SHM). Since R-loop structures exist in the CSR process and SHM shares similar mechanisms with CSR, we speculated that Srsf1 might also participate in CSR. The aim of the thesis is to investigate the role of Srsf1 in CSR from two aspects. First, the characteristics changes of Srsf1 during CSR were addressed, including the expression level and subcellular location of Srsf1. Second, the srsf1 gene was knocked out in CH12F3 cells, a murine B cell line as the CSR model. After CSR stimulation, the mRNA amounts of srsf1-1 remained unchanged in two-day-stimulated group, and decrease by 10% in three-day-stimulated group, and the protein amount of Srsf1-1 decreased slightly by about 20% after stimulation for two days and three days. The mRNA amounts of srsf1-3 decreased slightly by about 20% after stimulation for two days and three days. Next, Srsf1-1 was found mostly located in the nuclear fraction in both unstimulated and stimulated cells. Moreover, the Srsf1-1 amount in the cytoplasm fraction decreased apparently by about 40%. However, the change of the Srsf1 amount in the nuclear fraction was not determined yet due to the uncertainty of the suitable internal control. Finally, srsf1+/- cell clones were generated by knocking out the srsf1 gene in CH12F3 cells. Preliminary results of srsf1+/- cells showed no significant influence on CSR. Nevertheless, srsf1-/- cells failed to be obtained possibly due to the important role of Srsf1 in cell survival. In conclusion, the role of Srsf1 in CSR needs more experiments to be confirmed. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55893 |
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顯示於系所單位: | 生化科技學系 |
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