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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蔡欣祐(Hsin-Yue Tsai) | |
| dc.contributor.author | Wei-Yu Chen | en |
| dc.contributor.author | 陳威羽 | zh_TW |
| dc.date.accessioned | 2022-11-25T06:35:04Z | - |
| dc.date.copyright | 2021-11-09 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-09-27 | |
| dc.identifier.citation | Watanabe, S., et al., The role of macrophages in the resolution of inflammation. J Clin Invest, 2019. 129(7): p. 2619-2628. Varol, C., A. Mildner, and S. Jung, Macrophages: development and tissue specialization. Annu Rev Immunol, 2015. 33: p. 643-75. Fan, X., et al., Double Roles of Macrophages in Human Neuroimmune Diseases and Their Animal Models. Mediators Inflamm, 2016. 2016: p. 8489251. Pei, Y. and Y. Yeo, Drug delivery to macrophages: Challenges and opportunities. J Control Release, 2016. 240: p. 202-211. Orecchioni, M., et al., Macrophage Polarization: Different Gene Signatures in M1(LPS+) vs. Classically and M2(LPS-) vs. Alternatively Activated Macrophages. Front Immunol, 2019. 10: p. 1084. Martinez, F.O. and S. Gordon, The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000Prime Reports, 2014. 6. Atri, C., F.Z. Guerfali, and D. Laouini, Role of Human Macrophage Polarization in Inflammation during Infectious Diseases. Int J Mol Sci, 2018. 19(6). De Santa, F., et al., The Role of Metabolic Remodeling in Macrophage Polarization and Its Effect on Skeletal Muscle Regeneration. Antioxid Redox Signal, 2019. 30(12): p. 1553-1598. Abdelaziz, M.H., et al., Alternatively activated macrophages; a double-edged sword in allergic asthma. J Transl Med, 2020. 18(1): p. 58. Anders, C., et al., Integrated Metabolomics, Transcriptomics, and Signal Protein Profile Define the Effector Function and Associated Metabotype of Five Polarized Macrophage Phenotypes. bioRxiv, 2020. Chanmee, T., et al., Tumor-associated macrophages as major players in the tumor microenvironment. Cancers (Basel), 2014. 6(3): p. 1670-90. Benoit, M., B. Desnues, and J.L. Mege, Macrophage polarization in bacterial infections. J Immunol, 2008. 181(6): p. 3733-9. Roszer, T., Understanding the Mysterious M2 Macrophage through Activation Markers and Effector Mechanisms. Mediators Inflamm, 2015. 2015: p. 816460. Lopes, I.d.A. Macrophages in M1/M2 states and exposed to amyloid-beta: a RNA-seq analysis in human cells. 2017. Cassetta, L. and J.W. Pollard, Targeting macrophages: therapeutic approaches in cancer. Nat Rev Drug Discov, 2018. 17(12): p. 887-904. Han, S., et al., Tumor microenvironment remodeling and tumor therapy based on M2-like tumor associated macrophage-targeting nano-complexes. Theranostics, 2021. 11(6): p. 2892-2916. Mantovani, A., et al., Tumour-associated macrophages as treatment targets in oncology. Nat Rev Clin Oncol, 2017. 14(7): p. 399-416. Anfray, C., et al., Current Strategies to Target Tumor-Associated-Macrophages to Improve Anti-Tumor Immune Responses. Cells, 2019. 9(1). Yang, Q., et al., The role of tumor-associated macrophages (TAMs) in tumor progression and relevant advance in targeted therapy. Acta Pharm Sin B, 2020. 10(11): p. 2156-2170. DeNardo, D.G., et al., CD4(+) T cells regulate pulmonary metastasis of mammary carcinomas by enhancing protumor properties of macrophages. Cancer Cell, 2009. 16(2): p. 91-102. Gocheva, V., et al., IL-4 induces cathepsin protease activity in tumor-associated macrophages to promote cancer growth and invasion. Genes Dev, 2010. 24(3): p. 241-55. Sica, A. and A. Mantovani, Macrophage plasticity and polarization: in vivo veritas. J Clin Invest, 2012. 122(3): p. 787-95. Tugal, D., X. Liao, and M.K. Jain, Transcriptional control of macrophage polarization. Arterioscler Thromb Vasc Biol, 2013. 33(6): p. 1135-44. Kapoor, N., et al., Transcription factors STAT6 and KLF4 implement macrophage polarization via the dual catalytic powers of MCPIP. J Immunol, 2015. 194(12): p. 6011-23. Zhou, L., et al., Monocyte chemoattractant protein-1 induces a novel transcription factor that causes cardiac myocyte apoptosis and ventricular dysfunction. Circ Res, 2006. 98(9): p. 1177-85. Liang, J., et al., A novel CCCH-zinc finger protein family regulates proinflammatory activation of macrophages. J Biol Chem, 2008. 283(10): p. 6337-46. Akira, S., Regnase-1, a ribonuclease involved in the regulation of immune responses. Cold Spring Harb Symp Quant Biol, 2013. 78: p. 51-60. Xu, J., et al., Structural study of MCPIP1 N-terminal conserved domain reveals a PIN-like RNase. Nucleic Acids Res, 2012. 40(14): p. 6957-65. Habacher, C. and R. Ciosk, ZC3H12A/MCPIP1/Regnase-1-related endonucleases: An evolutionary perspective on molecular mechanisms and biological functions. Bioessays, 2017. 39(9). Yokogawa, M., et al., Structural basis for the regulation of enzymatic activity of Regnase-1 by domain-domain interactions. Sci Rep, 2016. 6: p. 22324. Matsushita, K., et al., Zc3h12a is an RNase essential for controlling immune responses by regulating mRNA decay. Nature, 2009. 458(7242): p. 1185-90. Mizgalska, D., et al., Interleukin-1-inducible MCPIP protein has structural and functional properties of RNase and participates in degradation of IL-1beta mRNA. FEBS J, 2009. 276(24): p. 7386-99. Li, M., et al., MCPIP1 down-regulates IL-2 expression through an ARE-independent pathway. PLoS One, 2012. 7(11): p. e49841. Wilamowski, M., et al., Substrate specificity of human MCPIP1 endoribonuclease. Sci Rep, 2018. 8(1): p. 7381. Mino, T., et al., Regnase-1 and Roquin Regulate a Common Element in Inflammatory mRNAs by Spatiotemporally Distinct Mechanisms. Cell, 2015. 161(5): p. 1058-1073. Iwasaki, H., et al., The IkappaB kinase complex regulates the stability of cytokine-encoding mRNA induced by TLR-IL-1R by controlling degradation of regnase-1. Nat Immunol, 2011. 12(12): p. 1167-75. Liang, J., et al., MCP-induced protein 1 deubiquitinates TRAF proteins and negatively regulates JNK and NF-kappaB signaling. J Exp Med, 2010. 207(13): p. 2959-73. Xu, R., et al., CCL2 promotes macrophages-associated chemoresistance via MCPIP1 dual catalytic activities in multiple myeloma. Cell Death Dis, 2019. 10(10): p. 781. Xiaoming, A., et al., Macrophage Regnase-1 Deletion Deteriorates Liver Ischemia/Reperfusion Injury Through Regulation of Macrophage Polarization. Front Physiol, 2020. 11: p. 582347. Zhang, B., et al., M2-polarized tumor-associated macrophages are associated with poor prognoses resulting from accelerated lymphangiogenesis in lung adenocarcinoma. Clinics (Sao Paulo), 2011. 66(11): p. 1879-86. Tariq, M., et al., Gefitinib inhibits M2-like polarization of tumor-associated macrophages in Lewis lung cancer by targeting the STAT6 signaling pathway. Acta Pharmacol Sin, 2017. 38(11): p. 1501-1511. Vadevoo, S.M.P., et al., IL4 Receptor-Targeted Proapoptotic Peptide Blocks Tumor Growth and Metastasis by Enhancing Antitumor Immunity. Mol Cancer Ther, 2017. 16(12): p. 2803-2816. Surdziel, E., et al., Multidimensional pooled shRNA screens in human THP-1 cells identify candidate modulators of macrophage polarization. PLoS One, 2017. 12(8): p. e0183679. Li, X., et al., O-GlcNAc Transferase Suppresses Inflammation and Necroptosis by Targeting Receptor-Interacting Serine/Threonine-Protein Kinase 3. Immunity, 2019. 50(3): p. 576-590 e6. Qi, L.S., et al., Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 2013. 152(5): p. 1173-83. Gilbert, L.A., et al., CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell, 2013. 154(2): p. 442-51. Adamson, B., et al., A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response. Cell, 2016. 167(7): p. 1867-1882 e21. Liang, J.R., et al., A Genome-wide ER-phagy Screen Highlights Key Roles of Mitochondrial Metabolism and ER-Resident UFMylation. Cell, 2020. 180(6): p. 1160-1177 e20. Horlbeck, M.A., et al., Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. Elife, 2016. 5. Wang, G.G., et al., Quantitative production of macrophages or neutrophils ex vivo using conditional Hoxb8. Nat Methods, 2006. 3(4): p. 287-93. Van den Bossche, J., et al., Mitochondrial Dysfunction Prevents Repolarization of Inflammatory Macrophages. Cell Rep, 2016. 17(3): p. 684-696. Kiguchi, N., et al., Peripheral interleukin-4 ameliorates inflammatory macrophage-dependent neuropathic pain. Pain, 2015. 156(4): p. 684-693. Enam, S.F., et al., Evaluation of M2-like macrophage enrichment after diffuse traumatic brain injury through transient interleukin-4 expression from engineered mesenchymal stromal cells. J Neuroinflammation, 2020. 17(1): p. 197. Li, M., et al., Optimal promoter usage for lentiviral vector-mediated transduction of cultured central nervous system cells. J Neurosci Methods, 2010. 189(1): p. 56-64. Qin, J., et al., An efficient strategy for generation of transgenic mice by lentiviral transduction of male germline stem cells in vivo. J Anim Sci Biotechnol, 2015. 6: p. 59. Kreiss P, C.B., Rangara R, et al., Plasmid DNA size does not affect the physicochemical properties of lipoplexes but modulates gene transfer efficiency. Nucleic Acids Res., 1999. 27(19): p. 3792-3798. Counsell, J.R., et al., Lentiviral vectors can be used for full-length dystrophin gene therapy. Sci Rep, 2017. 7(1): p. 79. Sweeney, N.P. and C.A. Vink, The impact of lentiviral vector genome size and producer cell genomic to gag-pol mRNA ratios on packaging efficiency and titre. Mol Ther Methods Clin Dev, 2021. 21: p. 574-584. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/82288 | - |
| dc.description.abstract | 巨噬細胞是一群對於先天性免疫以及組織恆定性相當重要的免疫細胞，透過接受環境中不同的刺激物，而有不同的活化型態。極化的巨噬細胞可分為典型活化型態(M1)與另類活化型態(M2)兩個極端型態，前者具有促進發炎反應的功能，而後者則與抗發炎反應息息相關。目前的文獻證實在哺乳動物的惡性腫瘤中，有一群與另類活化型態(M2)相似的巨噬細胞，他們受CD4+T細胞分泌的白介素-4（Interleukin-4，IL-4）所誘導並參與癌症轉移(metastasis)的過程。MCPIP1是一個具有核醣核酸酶活性的蛋白，曾被報導在IL-4所誘導之M2巨噬細胞極化過程中扮演著不可或缺的角色。儘管受IL-4所誘導的M2巨噬細胞對於癌症的了解極其重要，但是詳細的機制，像是參與在訊號傳導途徑中的基因卻尚未清楚。因此，我們的研究目的為確認MCPIP1是否確實參與在巨噬細胞另類極化過程，並且建立CRISPR干擾(CRISPR interference，CRISPRi)的全基因篩選平台，找出IL-4所誘導之M2巨噬細胞極化過程中的調控基因。期望透過流式細胞儀(flow cytometry)以及次世代定序(next generation sequencing，NGS)，我們能夠篩選出受CRISPRi所干擾，導致在IL-4誘導下無法成功進行M2極化的基因，即為潛在的M2極化的調控者。 在本篇研究中，首先我們製備野生型(wild-type)與MCPIP1突變型之永生化老鼠骨髓源性巨噬細胞 (immortalized bone marrow-derived macrophages，iBMDMs) 還有可誘導過量表現MCPIP1基因之iBMDMs，來確認MCPIP1對於M2巨噬細胞極化過程的重要性，不過結果顯示其與先前研究並不完全一致。為了利用全基因篩選找出參與在M2巨噬細胞極化過程的重要基因，我們測試iBMDMs的M2表現型並優化能夠區分M2及未誘導(M0)巨噬細胞的條件，發現當細胞在IL-4誘導48小時下，透過流式細胞儀共同偵測M2巨噬細胞表面標誌CD206與CD301b， 能夠最佳區分M2及M0巨噬細胞。同時，我們也測試了其他的巨噬細胞株，像是J774A.1與Raw264.7。目前我們成功製備穩定表現dCas9-KRAB的iBMDMs，也測試了在iBMDMs中sgRNA文庫的多樣性。簡而言之，我們製備了不同的細胞株; 測試了適合的巨噬細胞株; 優化了能區分M2/M0巨噬細胞的條件，來提供CRISPRi全基因篩選的重要材料。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-25T06:35:04Z (GMT). No. of bitstreams: 1 U0001-2409202122532600.pdf: 5614544 bytes, checksum: 1b67f1e3b714f701ee08b9c97968a8c3 (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | "論文口試委員審定書 i Acknowledgements ii 摘要 iii Abstract v Chapter 1 Introduction 1 1.1 Overview of macrophages 1 1.1.1 Development of macrophages 1 1.1.2 Macrophage polarization 1 1.1.3 Hallmarks for M1 and M2 macrophages 3 1.2 Macrophage polarization is critical for cancer cell survival 4 1.3 The mechanism of macrophage polarization 5 1.4 Overview of Monocyte chemotactic protein-1-induced protein-1 (MCPIP1) 6 1.5 MCPIP1 regulates the immune response 7 1.6 MCPIP1 in M2 polarization failure 8 1.7 M2 macrophage polarization signaling pathway is still perplexity 9 1.8 Identify the required gene in M2 macrophage polarization signaling process may open a new way for cancer treatment 10 1.9 The whole-genome screen to identify involved genes for alternative (M2) macrophage polarization 10 1.10 Aim of the study 11 Chapter 2 Materials and Methods 14 2.1 Mice 14 2.2 Cells and Culture conditions 14 2.3 Isolation of bone marrow-derived macrophages (BMDMs) 15 2.4 Wild-type and Mcpip1mt/mt immortalized bone marrow-derived macrophages (iBMDMs) generation 16 2.5 IL-4 mediated M2 macrophage polarization 17 2.6 LPS induction in iBMDMs 17 2.7 Lentivirus production for generation of dCas9-KRAB-expressing iBM cell line, Tet-on 3xFLAG MCPIP1 overexpression iBM cell line, Slc40a1-sgRNA- and Ago4- sgRNA-expressing iBM cell line 18 2.8 Mouse sgRNA library amplification, sgRNA library-containing lentivirus production and cells transduction 19 2.9 Genomic DNA isolation, single guide RNA containing sequence amplification, transformation and sequencing 20 2.10 RT-qPCR analysis 21 2.11 Flow cytometry 22 2.12 Western blot 23 2.13 Immunoprecipitation 24 2.14 Statistical analysis 25 Chapter 3 Results 26 3.1 Establishing both wild-type and Mcpip1mt/mt immortalized bone marrow progenitor cells (iBMs) and confirming the importance of MCPIP1 in M2 macrophage polarization 26 3.1.1 Establishing both wild-type and MCPIP1 mutant iBMs 26 3.1.2 Testing M2 polarization in both wild-type and Mcpip1mt/mt iBMDMs 28 3.1.2.1 The expression of M2 markers 28 3.1.2.2 The expression of M2 surface markers 29 3.2 Generating the inducible MCPIP1 overexpression iBM cell line to confirm the MCPIP1-mediated M2 macrophage polarization pathway 30 3.3 A whole-genome screen to identify required genes for IL-4 mediated alternative macrophage polarization 31 3.3.1 Rationale 31 3.3.2 The primary cell mouse bone marrow derived macrophages (BMDMs) showed M2 polarization capacity while IL-4 treatment for 24 h 32 3.3.3 Optimizing separating condition between M2(IL-4)/M0(uninduced) iBMDMs 33 3.3.3.1 CD206 M2 (IL-4) iBMDM surface marker 33 3.3.3.2 CD301b M2 (IL-4) iBMDM surface marker 35 3.3.3.3 In combination with PE-Cy7-conjugated CD206 antibody and APC-conjugated CD301b antibody in iBMDMs treated with IL-4 for 48 h could increase the distinction between M2 (IL-4)/M0 (uninduced) macrophages 36 3.3.4 Establishing other macrophage cell lines for backup 37 3.3.4.1 Obtaining optimal M2(IL-4) cell surface marker detection condition in macrophage-like cell line J774a.1 37 3.3.4.2 Testing macrophage-like cell line Raw264.7 41 3.3.5 Summary 43 3.4 Introducing CRISPR interference system into macrophage cell lines 44 3.4.1 Establishing dCas9-KRAB expressed iBMDMs 44 3.4.2 Introducing sgRNA into dCas9-KRAB iBMDMs for CRISPRi functional test 46 3.4.3 Establishing effective transduction of sgRNA library in iBMs 47 Chapter 4 Discussion 49 4.1 The irreproducible importance of MCPIP1 in M2 polarization 49 4.2 Combining CRISPRi screening assay and Tet-on inducible MCPIP1 overexpression iBMDM cell line to discover novel genes in MCPIP1-regulated M2 polarization signaling pathway 50 4.3 The malfunction of CD206 in the double-staining analysis in J774A.1 cells 51 4.4 The difficulty encountered in the iBM cell lines 52 Chapter 5 Figures 55 Figure 1. Observation of cell morphology by microscopy 55 Figure 2. Flow cytometry analysis of macrophage-specific surface marker CD11b and F4/80 on iBM progenitor cells and iBM progenitor cells treated with 7-day L929 conditional medium 57 Figure 3. qPCR analysis of Mcpip1 and M2/M1 marker gene expression in wild-type and Mcpip1mt/mt iBMDMs 59 Figure 4. Flow cytometry analysis of CD206 surface expression on wild-type and Mcpip1mt/mt iBMDMs with 20 ng/ml IL-4 treatment for 24 h 62 Figure 5. qPCR analysis of M2 marker gene expression in Tet-on inducible MCPIP1 overexpression iBMDMs 64 Figure 6. qPCR analysis of M2 marker gene expression in BMDMs with 20 ng/ml IL-4 treatment for 24 h 66 Figure 7. Flow cytometry analysis of M2 surface marker CD206 on uninduced BMDMs and BMDMs induced with 20 ng/ml IL-4 for 24 h 67 Figure 8. Flow cytometry analysis of CD206 surface expression on uninduced iBMDMs and iBMDMs induced with 20 ng/ml IL-4 for 24 h 69 Figure 9. Flow cytometry analysis of CD206 surface expression on uninduced iBMDMs and iBMDMs induced with 20 ng/ml time-course IL-4 71 Figure 10. Flow cytometry analysis of CD301b surface expression on uninduced iBMDMs and iBMDMs induced with 20 ng/ml time-course IL-4 73 Figure 11. Double-staining flow cytometry analysis of CD206 and CD301b on uninduced iBMDMs and iBMDMs induced with 20 ng/ml IL-4 for 48 h 75 Figure 12. qPCR analysis of M2 marker gene expression in J774a.1 cells with 20 ng/ml IL-4 treatment for 24 h 78 Figure 13. Flow cytometry analysis of CD206 surface expression on uninduced J774a.1 cells and J774a.1 cells induced with 20 ng/ml IL-4 for 24 h 79 Figure 14. Flow cytometry analysis of CD206 surface expression on uninduced J774a.1 cells and J774a.1 cells induced with 20 ng/ml IL-4 for 48 h 81 Figure 15. Flow cytometry analysis of CD301b surface expression on uninduced J774A.1 cells and J774A.1 cells induced with 20 ng/ml IL-4 for 48 h 83 Figure 16. Double-staining flow cytometry analysis of CD206 and CD301b on uninduced J774A.1 and J774A.1 induced with 20 ng/ml IL-4 for 48h 85 Figure 17. qPCR analysis of M2 marker gene expression in Raw264.7 cells with 20 ng/ml IL-4 treatment for 24 h 88 Figure 18. Flow cytometry analysis of CD206 surface expression on uninduced Raw264.7 cells and Raw264.7 cells induced with 20 ng/ml IL-4 for 24 h 89 Figure 19. Flow cytometry analysis of CD206 expression o on uninduced Raw264.7 cells and Raw264.7 cells induced with 20 ng/ml IL-4 for 48 h 91 Figure 20. Comparison of M2 marker gene and M2 surface marker expression in BMDMs, iBMDMs, Raw264.7 cells and J774A.1 cells with IL-4 induction 93 Figure 21. qPCR and western blot analysis of dCas9 expression in dCas9-KRAB iBM cell lines 95 Figure 22. Immunoprecipitation of HA-tag in dCas9-KRAB iBMDMs (1206-1) 97 Figure 23. Functional test of dCas9-KRAB iBMDMs (1206-1) by CRISPRi 98 Figure 24. Analysis of sgRNA diversity in sgRNA iBM library 99 Tables 103 Table 1. The PCR primers used for Mcpip1 genotyping 103 Table 2. The primers used for qPCR were following 103 Table 3. The sgRNA used for CRISPRi knockdown 105 Table 4. The PCR primers used for sgRNA-containing sequence amplification 105 Table 5. The primers used for DNA sequencing 105 Reference 106" | |
| dc.language.iso | en | |
| dc.subject | CRISPR干擾 | zh_TW |
| dc.subject | MCPIP1 | zh_TW |
| dc.subject | 介白素-4 | zh_TW |
| dc.subject | 另類活化型態巨噬細胞 | zh_TW |
| dc.subject | sgRNA文庫 | zh_TW |
| dc.subject | 全基因篩選 | zh_TW |
| dc.subject | macrophage alternative polarization | en |
| dc.subject | interleukin-4 | en |
| dc.subject | MCPIP1 | en |
| dc.subject | genome-wide screening assay | en |
| dc.subject | CRISPR interference | en |
| dc.subject | sgRNA library | en |
| dc.title | 建立全基因組干擾型CRISPR系統篩選參與白介素-4所誘導巨噬細胞M2型極化途徑之調控基因 | zh_TW |
| dc.title | To establish CRISPR interference-based whole-genome screening platform for identification of novel genes in IL4-mediated macrophage alternative polarization | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 徐立中(Hsin-Tsai Liu),陳佑宗(Chih-Yang Tseng) | |
| dc.subject.keyword | 另類活化型態巨噬細胞,介白素-4,MCPIP1,CRISPR干擾,sgRNA文庫,全基因篩選, | zh_TW |
| dc.subject.keyword | macrophage alternative polarization,interleukin-4,MCPIP1,CRISPR interference,sgRNA library,genome-wide screening assay, | en |
| dc.relation.page | 113 | |
| dc.identifier.doi | 10.6342/NTU202103353 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2021-09-27 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 分子醫學研究所 | zh_TW |
| dc.date.embargo-lift | 2026-09-26 | - |
| 顯示於系所單位： | 分子醫學研究所 | |
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