探討脾酪氨酸激酶在替代性活化巨噬細胞中的角色

The role of spleen tyrosine kinase in alternatively activated macrophages

吳若寧(Jo-Ning Wu)

指導教授 ： 朱清良

共同指導教授 ： 許秉寧

國立臺灣大學/醫學院/免疫學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201901669

摘要

脾酪氨酸激酶(Spleen Tyronsine kinase,Syk)為非受體酪氨酸激酶家族的一員，早期研究指出Syk 具有調控造血幹細胞增生、發育、分化等功能。後續研究則發現Syk不僅在造血幹細胞中為重要調控因子，同時也在免疫細胞中擔任重要的訊息傳遞蛋白。先前的研究發現Sykfl/fl LysM-Cre小鼠中骨髓細胞分化噬骨細胞數量銳減，同時巨噬細胞之免疫反應能力缺失，揭示Syk在巨噬細胞的發育和活化中為重要的調控因子。迄今，許多文獻發現巨噬細胞具有高度異質性，可分別藉由Th1以及Th2分泌的細胞激素極化為M1 和 M2 兩大族群 ，而其中於M1族群中Syk已在多篇文獻中被探討，並得知其參與在TLR4以及IFNα/β下游訊息傳遞; 然而，M2 族群Syk所扮演的角色尚未確立，因此我們欲藉由Sykfl/fl 以及 Syk∆LysM ，再進一步極化為M2中不同族群，對於發育、分化以及極化能力進行觀察，了解Syk 在M2族群中如何調控相關免疫反應。利用即時聚合酶連鎖反應(qPCR)以及流式細胞術(FCS)進行分析，不同刺激物下巨噬細胞極化為M1、M2a以及M2c之能力差異；針對功能性檢測則利用酵素免疫分析法(ELISA)和T淋巴球共培養實驗，觀察Syk 在細胞激素以及抑制T細胞增生之能力。本篇研究結果顯示Syk 缺失加劇巨噬細胞極化為M2，在M2a相關基因表達顯著上升，M1相關細胞表面標的蛋白-CD86則顯著下降，同時MHCII在M2a族群中高度表達。功能性檢測-T細胞共培養實驗下，M2a抑制T 細胞增生機制主要需藉由直接接觸，Syk 缺失之巨噬細胞抑制T細胞增生之能力有下降之趨勢，目前可能機制是Syk影響PD-L2表現量下降所導致。後續研究中，我們更進一步探討其中機制，發現 Syk 在IL-4刺激下活化，並且主導下游MAPK 以及Akt之活化狀態

關鍵字

Sykfl/fl LysM-Cre小鼠 ； 典型活化巨噬細胞 ； 替代性活化巨噬細胞 ； 介白素-4 ； 介白素-10

並列摘要

Spleen Tyrosine kinase (Syk), a non-receptor tyrosine kinase, has been recognized as a pivotal factor in various signal transductions and involved in the development, proliferation, and differentiation of hematopoietic stem cells. Many studies have shown that Syk played an important role in macrophage activation and function. Concomitantly, macrophages have been identified as heterogenous population with high plasticity and ability to polarized into diverse subsets in response to different stimulators respectively. Intense studies of Syk were focusing on M1 macropahges; whereas the role of Syk in M2 macrophages was still elusive. To dissect the role of Syk in M2 macropahges both polarization and activation, we have isolated bone marrow cells from Sykfl/fl and Syk∆LysM mice and developed into macrophages in vitro, and further polarized macrophages into different subsets. Real time polychain reaction(qPCR) and flow cytometry (FCS) have been applied to analyze differences between Sykfl/fl and Syk∆LysM macrophages polarization ability. Subsequently, ELISA and T cells co-culturing were conducted as functional assay to evaluate M2 macrophages activation state. According to our results, Syk deficient macrophages are less capable to polarize into M1 status and skews to M2a spectrum. Besides, we find CD86 expression is significantly lower in M1 subsets with MHCII significantly increase in M2a subsets at 24 hr. In functional analysis, we found that Syk deficient M2a compromise to suppress T cells proliferation. So far, we’ve hypothesized through lower PD-L2 expression in Syk deficient macrophages, and concomitantly we have also verified that direct interaction is necessary for M2a suppression ability. We took deeper look into IL-4 stimulated BMDMs unveiling the role of Syk in mediation of MAPK and Akt activation state.

並列關鍵字

Sykfl/fl LysM-Cre ； Classically activated macrophage ； Alternatively activated macrophage ； Interleukin-4 ； Interleukin-10

參考文獻

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