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研究生： 楊立瑄
Yang, Li-Shiuan
論文名稱： 建立老鼠髓樣巨噬細胞之訓練免疫模型
Establishing an in vitro trained immunity model in murine bone marrow-derived macrophages
指導教授： 鄭世進
Cheng, Shih-Chin
口試委員: 徐嘉琳
Hsu, Chia-lin
劉卜慈
Liu, Pu-Ste
學位類別： 碩士
Master
系所名稱： 生命科學暨醫學院 - 分子醫學研究所
Institute of Molecular Medicine
論文出版年： 2019
畢業學年度： 107
語文別： 中文
論文頁數： 63
中文關鍵詞： 訓練免疫 、老鼠 、巨噬細胞 、模型建立 、先天免疫
外文關鍵詞： Trained immunity, murine, macrophage, model establishing, innate immunity
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    • 訓練免疫或先天免疫記憶是近幾年發現的概念，不同於後天免疫記憶，先天免疫記憶是一非專一性的防護，對於第一次感染或第二次感染宿主的病原體啟動更強與更快的免疫反應，給予宿主更好的免疫防護。為更深入了解先天免疫記憶產生機制，科學家們已建立起人類訓練免疫的細胞模型。然而，為了進一步探討訓練免疫反應之分子作用機制，老鼠模型是一較好調控的工具。在建立老鼠模型之前，第一步要於老鼠先天免疫細胞中建立訓練免疫模型。我們的目標是建立一個老鼠訓練免疫細胞模型，能再現人類訓練免疫細胞模型的實驗結果並利用其探討訓練免疫的產生機制。於人類訓練免疫細胞模型觀察到的細胞激素分泌上調、代謝物的重新分配與表觀遺傳學的修飾，在我們建立的老鼠細胞模型皆能再現這些現象。此外我們還偵測了細胞分泌一氧化氮的能力、毒殺大腸桿菌的能力與抗原呈現能力等未在人類細胞模型驗證的細胞功能。有趣的是，經訓練的巨噬細胞分泌較高量的一氧化氮，但毒殺大腸桿菌的能力卻沒有隨之提高，而抗原呈現的能力在細胞經訓練後有明顯提高的現象。總結，訓練免疫尚有許多未知的內容值得探索，作為訓練免疫的研究模型，我們建立的老鼠訓練免疫細胞模型與人類訓練免疫細胞模型具有高度相似性。

    Trained immunity or the innate memory is a novel discovery from the past few years, which facilitates a faster and enhanced immune response of the innate immune system. Different from the adaptive memory, the innate memory is a non-specific protection which can protect host from infections caused by non-related pathogens. To explore the underlying mechanism of trained immunity, the in vitro human training model had been demonstrated previously. Based on the observations in the in vitro human model revealing which pathway and metabolite play an essential role in inducting trained immunity. To regulate and manipulate cells with target pathway or metabolite, murine is an available tool to do the gene modification, metabolite depletion, and etc. Therefore, we want to demonstrate an in vitro murine model to reproduce the results of the in vitro human model. Our in vitro murine model showed the similar results of up-regulated cytokine production, reprogrammed metabolic profiling, and modified epigenetic landscape compared to that of human in vitro model. In addition, we further measured the NO production, E. coli-killing capacity, C. albicans phagocytosis capacity, ROS production, and MHCII expression. The NO production and antigen presenting capacity are up-regulated by training obviously, however, the E. coli-killing, C. albicans phagocytosis, and ROS production are lower than control. In conclusion, we have successfully established an in vitro trained immunity model in murine BMDMs.

    摘要……………………………………………………….……………..2 Abstract...................................................................................................3 Contents………………………………………………………………...4 Introduction Innate immunity is the first line defense of the host…………….…….7 Adaptive-like immunological memory in macrophages…………….....8 Metabolic reprogramming and epigenetic change of trained immunity.9 Aims and approaches…………………………………………....…11 Materials and methods Bone marrow isolation and differentiation to bone marrow-derived macrophages (BMDMs)………………………………………………12 Trained immunity experimental design……………………….............13 Cytokine measurements………………………………………………14 mRNA extraction and RT-qPCR……………………………………...14 Lactate production measurements…………………………………….15 Western blot…………………………………………………………...15 Griess assay…………………………………………………………...16 FITC-labeled C. albicans preparation and phagocytosis measurements…………………………………………………..……..17 ROS production measurements……………………………………….18 E. coli-killing measurements…………………………………….……18 Chromatin immunoprecipitation……………………………………...19 Flow cytometry……………………………………………………….20 Results Trained macrophages with bigger cell size……………..…………….21 Training accelerates and promotes the cytokine production entirely…21 Training up-regulates the pro-inflammatory gene transcription……...22 Training induces higher lactate production and up-regulates glycolysis and mTOR pathway……………………………………….………….22 Training induces higher nitric oxide production……………………...23 Training does not boost the ROS production, E.coli-killing capacity, and phagocytosis of E. coli and C. albicans……………………………....24 Training induces H3K4 tri-methylation at the promoter region of TNF and IL6 gene…………………………………………………………..25 Major histocompatibility complex II molecules I-A/I-E expression is increased after training………………………………….……………26 Discussion…………………………………………………………...27 Reference…………………………………………………...……….33 Figure…………………………………………………………...…….35 Table…………………………………………………..………………55 Supplementary figures………………………………………....56

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