臺灣博碩士論文加值系統

巨噬細胞在免疫調控中扮演著相當重要的角色，其在接受周遭多樣化的刺激之下，會有廣泛的活化型態。典型活化型態 (M1) 及替代型活化型態 (M2) 被用以描述其活化狀態中的兩個極端，而他們分別有促進發炎反應及抑制發炎反應的功能，以達到組織的平衡狀態。Regnase-1是一個核糖核酸酶，藉由調控促進發炎相關的激素之信使核糖核酸的降解，在免疫反應中扮演關鍵角色。並且，報導已知它在巨噬細胞替代型活化的過程中極為重要，而這個調控的過程包含了内質網壓力、活性氧分子、及自噬作用的參與。然而，關於這條路徑中較細節的調控機制仍尚未釐清。我們研究的目的是藉由CRISPRi-dCas9基因編輯系進行全基因範圍的篩選，以探索藉由Regnase-1所調控巨噬細胞替代型活化的過程中之新調控者。藉由流式細胞儀的偵測，我們將找出經過CRISPRi抑制其表現並Regnase-1過度表現處理下，會造成M2標記螢光表現量下降之細胞的基因，它們即為參與在Regnase-1所調控下游之替代型巨噬細胞活化中潛在的調控者。我們已測試並比較四種的老鼠巨噬細胞株，並藉由流式細胞儀檢測幾種M2標記區分M1/M2表現型的效果。我們的結果顯示，利用流式細胞儀共同偵測Egr2及CD206的表現，在老鼠骨髓源性巨噬細胞 (BMDMs) 及經不死處理的骨髓源性巨噬細胞 (immortalized BMDMs) 能夠達到區分出M2表現型的效果。我們也建立了CRISPRi-Regnase-1及誘發型Regnase-1過度表現的系統，以利於接下來需進行的原則驗證試驗。我們的結果也推論出内質網壓力相關蛋白及巨噬細胞替代型活化之潛在相關性，接續的實驗中也將進一步闡明此關聯性。

Macrophages are crucial players in immune regulation. They have a wide spectrum of activation states depend on the diverse surrounding stimuli they receive. Classical activation (M1) and alternative activation (M2) are described as two extremes of their polarized states, which elicit pro-inflammatory responses and anti-inflammatory responses respectively to maintain tissue homeostasis. Regnase-1 is a ribonuclease essential in controlling immune responses by regulating mRNA decay of proinflammatory cytokines, and it is reported to be important in promoting macrophage M2 polarization in which ER stress, ROS and autophagy are involved. However, detailed regulatory mechanism of this pathway is remained unclear. The goal of our study is to perform a genome-scale CRISPRi-dCas9 screening to explore new regulators in Regnase-1 mediated M2 polarization. By flow cytometry detection of M2 markers expression, we can identify genes that after CRISPRi disruption and Regnase-1 overexpression lead to decreased M2 expression, as potential regulators in this pathway. We have tested and compared the M2 phenotypes of four mice macrophage cell lines and examined the M1/M2 discrimination of several M2 markers by flow cytometry analysis. Our results demonstrated the M2 discriminating ability of Egr2 and CD206, which by flow cytometry detection can together be used to distinguish M2 phenotypes in both BMDMs and immortalized BMDMs. We have also established CRISPRi-Regnase-1 and inducible Regnase-1 overexpression system for further proof-of-principle screening and the preparations of the large-scale screening. Our data also infer a potential relation between ER stress related protein and M2 polarization, which is to be further investigated in the future works.

Table of Contents
Acknowledgements………………………………………………………………...........i
中文摘要………………………………………………………………………………..ii
Abstract……………………………………………………………………………....…iii
Chapter 1 Introduction…………………………………………………………………1
1.1 Macrophage polarization……………………………………………...…………..1
1.1.1 Overview of macrophage polarization………………………………………1
1.1.2 Regulation of macrophage polarization……………………………………..3
1.1.3 M2-like tumor-associated macrophages (TAMs)……………………………4
1.2 Regnase-1－a ribonuclease that is prominent in immune regulation……………..4
1.2.1 Characterization of Regnase-1………………………………………………5
1.2.2 Dynamic regulation of Regnase-1 in immunological processes…………….7
1.2.3 Regulatory roles of Regnase-1 in macrophage alternative polarization…….8
1.2.4 Other reported roles of Regnase-1…………………………………………10
1.3 Summary………………………………………………………………………...11
Chapter 2 Materials and Methods………………………………………………………13
2.1 Mice………………………………………………………………………….…13
2.2 Bone marrow-derived macrophages (BMDMs)…………………………….….13
2.3 Immortalized bone marrow-derived macrophages (iBMDMs)………….……..14
2.4 Cell culture conditions…………………………………………………..…..….14
2.5 Western blot………………………………………………………………….…15
2.6 RNA extraction and quantitative real-time PCR…………………………..…...16
2.7 Flow cytometry…………………………………………………………………17
Chapter 3 Results……………………………………………………………………….19
3.1 Macrophage cell lines RAW264.7 and J774A.1 showed poor discrimination of M0/M2 phenotypes examined by flow cytometry……………………………..…….19
3.2 Examination of the ability of M2 markers in discriminating M2 BMDMs……...20
3.3 Combinatory use of M2 markers CD206 and Egr2 exhibited remarkable discrimination in M2 immortalized BMDMs iBMDMs)……………………………22
3.4 Establishing inducible Tet-on system……………………………………………24
3.5 Test runs of all-in-one CRISPRi system targeting for Regnase-1 in BMDMs…..25
3.6 Potential correlation between ER stress related protein and M2 polarization…...27
Chapter 4 Discussion………………………………………………………………...…29
4.1 Possible limitations of the reported M2 markers and alternative ways to distinguish M2 macrophages……………………………………………………...…29
4.2 Possible ways to improve the CRISPRi-mediated gene silencing………………30
Chapter 5 Figures……………………………………………………………………….32
Figure 3-1 Flow cytometry analysis of Egr2 expression in RAW264.7 cells……….32
Figure 3-2 qPCR analysis and flow cytometry analysis of Egr2 expression in J774a.1 cells…………………………………………………………………………..………35
Figure 3-3 Flow cytometry analysis of mice macrophage surface marker F4/80 of BMDMs………………………………………………………………...……………37
Figure 3-4 Flow cytometry analysis of IL-4 induced BMDMs……………………...38
Figure 3-5 Flow cytometry detection of M2 markers Egr2 and IL-10 in BMDMs….40
Figure 3-6 IL-4 and IL-10 co-stimulation didn’t show a better discriminating ability in BMDMs……………………………………………………………………...……42
Figure 3-7 Western blot and qPCR analysis of M2 markers of IL-4-stimulated BMDMs…………………………………………………………………...…………44
Figure 3-8 Western blot and qPCR analysis of M2 markers in IL-4-stimulated iBMDMs……………………………………………………………………………..45
Figure 3-9 Flow cytometry results showing the discriminating ability of CD206, Egr2 but not PD-L2 in M2-polarized iBMDMs ………………………………………......46
Figure 3-10 Validation of the inducible protein expression of Regnase-1 in Tet-on system …………………………………………………………………….………....49
Figure 3-11 Schematic description of the two selected sgRNAs targeting for Regnase-1 gene……………………………………………………………………….………..50
Figure 3-12 qPCR and flow cytometry results of CRISPRi-Regnase1 (sgRNA#2)- mediated knockdown in BMDMs……………………………………………………51
Figure 3-13 qPCR, western blot, and flow cytometry results of CRISPRi-Regnase1 (sgRNA#1)- mediated knockdown in BMDMs………………………………...……53
Figure 3-14 mRNA expression of different forms of XBP1 analyzed by qPCR...…..55
Table……………………………………………………………………………………57
List of primers used for quantitative PCR analysis:…………………………………57
References…………………………………………………………………………...…58

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