以蛋白質體學法尋找抗體類型轉換重組機制中潛在重要的蛋白質

Abstract

抗體類型轉換重組 (class switch recombination, CSR) 使B細胞從表現IgM轉換成表現其他類型的抗體而能有效因應外來抗原。本論文旨在尋找並探討尚未被發掘參與CSR機制中的蛋白質，藉由解析這些蛋白質的功能，能對CSR機制有更進一步的了解。研究材料為小鼠B細胞株CH12F3，此細胞株在培養液中添加α-CD40抗體、IL-4和TGF-β刺激之後會進行CSR，從表現抗體IgM轉變成表現抗體IgA，藉由二維電泳分離未受刺激和受刺激3天的CH12F3全蛋白質樣品，將有表現量差異的蛋白質以質譜儀鑑定，鑑定得到的蛋白質有RanBP1、SRSF1和RhoGDI2。然而免疫染色結果顯示這三個蛋白質表現量在刺激3天的細胞中沒有上升的情形，與二維膠片結果不符，推測問題是內部系統控制組選擇不當，目前仍在尋找合適的內部系統控制組。 文獻報導指出DT40細胞中SRSF1會抑制R-loops的生成，而SRSF1的異構型 (isoform) SRSF1-3對於SHM的進行十分重要。目前已知R-loops會參與CSR且SHM和CSR機制上有許多相似的部份，所以本論文對於SRSF1是否參與在CSR做更深入的研究。由SRSF1表現量和細胞受刺激時間關係的實驗結果，發現在受刺激2天或3天細胞中，SRSF1蛋白質表現量沒有顯著改變，但Srsf1 mRNA在受刺激2天細胞中表現量上升約3倍，推測SRSF1可能在後轉錄層次被調控，此外二維免疫染色亦觀察到SRSF1有磷酸化程度改變的可能。最後嘗試建立穩定表現Srsf1 shRNA的細胞株，觀察是否會影響CSR頻率，但目前尚未得到有效抑制SRSF1表現的細胞株。未來實驗設計可以朝Srsf1基因剔除 (gene knockout)、SRSF1在細胞被刺激前後是否有磷酸化程度和細胞內區域化分布 (localization) 改變等方向進行，才能進一步了解SRSF1是否為CSR的參與因子。

To efficiently defend various antigens, B cells switch immunoglobulin isotype from IgM to others by class switch recombination (CSR). Previous studies indicate that various proteins are involved in CSR, but the detailed mechanism remains unclear. Therefore, in the thesis, we planned to identify more potential and unknown proteins involved in CSR. The murine B cell line, CH12F3 cells, switch from IgM-positive cells to IgA-positive cells after stimulation with α-CD40 antibody, interleukin 4 (IL-4) and transforming growth factor beta (TGF-β). Total cell lysates of unstimulated and stimulated CH12F3 were collected and analyzed by the two-dimensional electrophoresis (2DE), and the up-regulated or down-regulated proteins between these two samples were identified by the mass spectrometry. RanBP1, SRSF1 and RhoGDI2 were found up-regulated in stimulated cells. However, with the 1DE followed by western blot with the corresponding antibody, the protein level of these proteins had no difference in unstimulated and stimulated cells. We speculated the problem was the internal control used in 2DE might not be suitable for quantification. Previous studies show that SRSF1 influence the formation of R-loops and frequency of SHM in the chicken DT40 cell, so we are interested if SRSF1 is involved in CSR. After stimulation, Srsf1 mRNA expression increased about 3-fold comparing to unstimulated cells, but the protein level of SRSF1 remained unchanged. Interestingly, on the 2DE followed by the western blotting with α-SRSF1 antibody, several protein spots with the same molecular weight have different isoelectric points were shown on the membrane. We inferred that these protein spots might due to the different phosphorylation status of SRSF1. Therefore, we speculated when cells undergo CSR, SRSF1 altered its phosphorylation status but not protein expression level. Next, in order to know if SRSF1 is involved in CSR, the SRSF1 expression was knockdown in CH12F3 by shRNA. However, the stable Srsf1 knockdown cell has not been obtained yet. The role of SRSF1 in CSR needs further experiments to confirm, such as Srsf1 gene knockout, the phosphorylation status and subcellular localization of SRSF1 during CSR.