- 學位論文
探討Vhl在巨噬細胞中調控細胞極化之角色
The role of Vhl (von Hippel–Lindau tumor suppressor) in the polarization of macrophage
摘要
第一章 巨噬細胞是先天免疫中重要的防禦角色，除了主導生理上之發炎反應，經不同刺激可以將巨噬細胞極化為M1和M2兩類族群，分別促進發炎及調控組織修復。過去對巨噬細胞在發炎反應的研究已趨於成熟，但是對巨噬細胞極化成M1和M2的相關機制仍不完整。Von Hippel-Lindau (VHL)基因是一抑制腫瘤基因，該基因的突變在人體會造成一系列之腫瘤的發生。VHL基因所編碼的蛋白為一E3泛素連接酶，對於調控蛋白的分解為一重要的角色。VHL的影響相當廣泛，例如會辨識低氧誘發因子-1α (HIF-1α)並調控細胞面對低氧環境的反應。過去研究已知Vhl基因剔除的巨噬細胞在小鼠發炎模式下有更強的發炎反應，但VHL調控巨噬細胞的極化現象仍不清楚，因此本研究主要針對VHL調控巨噬細胞的極化進行探討。在本研究中我們經siRNA轉染以降低小鼠骨髓分化巨噬細胞中Vhl基因及蛋白的表現，且在此系統下比較巨噬細胞極化現象，發現降低Vhl表現之巨噬細胞在M1有較低之Tnfa、Il1b的基因表現，而在M2則有較高之Arg1、Ym1、Fizz1的基因表現。而經流式細胞術分析可發現降低Vhl表現之巨噬細胞在M1有較低之MHCII、CD86的表現，而在M2則有較高之PD-L2表現。為探討Vhl調控巨噬細胞極化的機制，根據過去研究我們探討Vhl、Hif1α、p53之間的調控，卻發現在我們培養之骨髓分化巨噬細胞的p53蛋白表現與過去研究相互矛盾，故降低Vhl表現之巨噬細胞比較p53、Hif1α、HIF2α的基因表現，結果並無觀察到顯著差異。在本研究中我們發現降低Vhl表現之巨噬細胞有相對較低之M1極化能力與相對較高之M2極化能力，但其機制與功能性探討仍須被探討和驗證。 第二章 真菌免疫調節蛋白為一群具有相似胺基酸序列、結構與免疫調節功能的真菌蛋白。過去研究顯示真菌免疫調節蛋白可促使免疫細胞增殖、分泌細胞激素，和抑制過敏及腫瘤。真菌免疫調節蛋白的物種來源多樣性，使不同來源的真菌免疫調節蛋白之特性仍待驗證。樹突細胞為主要負責抗原呈現及調控發炎反應的免疫細胞。樹突細胞在接觸抗原後會活化並將抗原呈現給輔助型T細胞以啟動後天免疫反應，故樹突細胞在調節免疫中為一重要的角色。過去研究指出真菌免疫調節蛋白如LZ-8、FIP-Fve可以活化樹突細胞，並且可作為疫苗之佐劑增強疫苗的功效。不過真菌免疫調節蛋白中的草菇免疫調節蛋白 (FIP-Vvo) 在樹突細胞的影響仍未被探討。本研究遂利用Saccharomyces cerevisiae生產之重組FIP-Vvo在小鼠骨髓分化樹突細胞上之影響進行探討。我們發現經FIP-Vvo刺激可促使骨髓分化樹突細胞之第二型主要組織相容性複合體表現增加，且可促進其分泌促發炎激素-介白素-6、介白素-12，與可活化T細胞之介白素-2。最後FIP-Vvo可增強骨髓分化樹突細胞促使抗原專一性的CD4+ T細胞增生之能力。本研究顯示S. cerevisiae生產之重組FIP-Vvo可部分活化小鼠骨髓分化樹突細胞，卻可增加其活化抗原專一性的CD4+ T細胞增生的功能，使重組FIP-Vvo可成為疫苗佐劑的一潛在目標。
關鍵字
Von Hippel-Lindau ； 巨噬細胞 ； 極化 ； 低氧誘發因子-1α ； 低氧誘發因子-2α ； p53 ； 真菌免疫調節蛋白 ； Volvariella volvacea ； 樹突細胞 ； 成熟並列摘要
Chapter 1 Macrophages play a crucial role in killing pathogens and regulating the inflammatory response in innate immunity. Depending on different stimuli and cytokines in microenvironment, macrophages can polarize towards two opposite functional states M1 and M2 types. In this study we explore the role of Von Hippel-Lindau (VHL) in macrophage polarization. Vhl gene is a tumor suppressor gene. Moreover, inheritance of a mutated Vhl gene in human causes VHL disease, an autosomal-dominant cancer syndrome. VHL gene encodes a E3 ubiquitin ligase which serves as a chaperon and promotes protein degradation. Previous study has shown that macrophage-specific Vhl-/- possessed stronger inflammatory ability. However, the role of VHL in M2 macrophage polarization remains unknown. In our study, we found that knockdown Vhl in bone marrow-derived macrophages (BMDM) by siRNA transfection could suppress Tnfa and Il1b mRNA expression in M1 subset. Furthermore, it could promote Arg1, Ym1 and Fizz1 mRNA expression in M2 subset. Moreover, by flow cytometry analysis, we found that knockdown Vhl in BMDM had lower MHCII and CD86 expression in M1 cells, whereas it had higher PD-L2 expression in M2 cells. To study the mechanism mediated by VHL in macrophage polarization, we focus on the expression of Hif1α and p53, both of which have been reported to interact with Vhl and have impact on macrophage polarization. However, our result of p53 expression in macrophage polarization was contradictory to previous study. Then we examined p53, Hif1α and Hif2α mRNA expression, and found that no significant difference between knockdown Vhl and control macrophages. In our study, we found that knockdown Vhl in BMDM suppress the capability of M1 polarization, whereas enhance the capability of M2 polarization. Nevertheless, the mechanism and functional test of polarization need to be further studied. Chapter 2 Fungal immunomodulatory protein (FIP) is a group of proteins isolated from fungi, and which has similar amino acid sequence and immune modulatory capability. Previous studies indicated that FIPs could promote proliferation and cytokine production of immune cells. FIPs have been shown to have the immunomodulatory effect on allergy and immunotherapy to tumor. Dendritic cell is the most potent antigen-presenting cell. Furthermore, induction of mature dendritic cell is critical for directing immune response, including inflammation and adaptive immunity. Previous studies have shown that LZ-8 and FIP-Fve, the typical members in FIP family, activate dendritic cell, and may be a potential adjuvant to enhance the efficacy of vaccine. However, the immunomodulatory effect of FIP-Vvo, the FIP from Volvariella volvacea, on dendritic cell remain unknown. Here, we employ mouse bone marrow-derived dendritic cell (BMDC) to investigate the immunomodulatory effect of the recombinant FIP-Vvo, which is produced by cloned Saccharomyces cerevisiae expression system. We found that treatment of BMDC with recombinant FIP-Vvo could induce the expression of MHC II, as well as the production of IL-2 and IL-6. Furthermore, recombinant FIP-Vvo enhanced the ability of BMDC to induce antigen specific CD4 T cell proliferation. These results suggest that the recombinant FIP-Vvo is able to partially activate BMDC. Furthermore, it can increase the ability of BMDC to induce antigen the proliferation of specific CD4 T cell. Thus, recombinant FIP-Vvo could be a potential adjuvant for vaccination.