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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蔡欣祐 | zh_TW |
| dc.contributor.advisor | Hsin-Yue Tsai | en |
| dc.contributor.author | 李淑婷 | zh_TW |
| dc.contributor.author | Shu-Ting Lee | en |
| dc.date.accessioned | 2023-09-13T16:07:05Z | - |
| dc.date.available | 2023-11-09 | - |
| dc.date.copyright | 2023-09-13 | - |
| dc.date.issued | 2023 | - |
| dc.date.submitted | 2023-08-08 | - |
| dc.identifier.citation | 1. 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.
2. Martinez, F.O. and S. Gordon, The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000Prime Rep, 2014. 6: p. 13. 3. Abdelaziz, M.H., et al., Alternatively activated macrophages; a double-edged sword in allergic asthma. J Transl Med, 2020. 18(1): p. 58. 4. Anders, C.B., et al., Use of integrated metabolomics, transcriptomics, and signal protein profile to characterize the effector function and associated metabotype of polarized macrophage phenotypes. J Leukoc Biol, 2022. 111(3): p. 667-693. 5. Anfray, C., et al., Current Strategies to Target Tumor-Associated-Macrophages to Improve Anti-Tumor Immune Responses. Cells, 2019. 9(1). 6. Cassetta, L. and J.W. Pollard, Targeting macrophages: therapeutic approaches in cancer. Nat Rev Drug Discov, 2018. 17(12): p. 887-904. 7. 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. 8. Mantovani, A., et al., Tumour-associated macrophages as treatment targets in oncology. Nat Rev Clin Oncol, 2017. 14(7): p. 399-416. 9. 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. 10. Hu, Y., et al., Tumor-associated macrophages correlate with the clinicopathological features and poor outcomes via inducing epithelial to mesenchymal transition in oral squamous cell carcinoma. J Exp Clin Cancer Res, 2016. 35: p. 12. 11. Sica, A. and A. Mantovani, Macrophage plasticity and polarization: in vivo veritas. J Clin Invest, 2012. 122(3): p. 787-95. 12. Tugal, D., X. Liao, and M.K. Jain, Transcriptional control of macrophage polarization. Arterioscler Thromb Vasc Biol, 2013. 33(6): p. 1135-44. 13. Barrangou, R., et al., CRISPR provides acquired resistance against viruses in prokaryotes. Science, 2007. 315(5819): p. 1709-12. 14. Le Rhun, A., et al., CRISPR-Cas in Streptococcus pyogenes. RNA Biol, 2019. 16(4): p. 380-389. 15. Heler, R., et al., Cas9 specifies functional viral targets during CRISPR-Cas adaptation. Nature, 2015. 519(7542): p. 199-202. 16. Miyaoka, Y., et al., Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing. Sci Rep, 2016. 6: p. 23549. 17. Jensen, T.I., et al., Targeted regulation of transcription in primary cells using CRISPRa and CRISPRi. Genome Res, 2021. 31(11): p. 2120-2130. 18. 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. 19. Chavez, A., et al., Highly efficient Cas9-mediated transcriptional programming. Nat Methods, 2015. 12(4): p. 326-8. 20. Gilbert, L.A., et al., Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell, 2014. 159(3): p. 647-61. 21. Gilbert, L.A., et al., CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell, 2013. 154(2): p. 442-51. 22. Moore, C.B., et al., Short hairpin RNA (shRNA): design, delivery, and assessment of gene knockdown. Methods Mol Biol, 2010. 629: p. 141-58. 23. Goel, K. and J.E. Ploski, RISC-y Business: Limitations of Short Hairpin RNA-Mediated Gene Silencing in the Brain and a Discussion of CRISPR/Cas-Based Alternatives. Front Mol Neurosci, 2022. 15: p. 914430. 24. 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. 25. 陳威羽, 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, in 分子醫學研究所. 2021, 國立臺灣大學. p. 1-113. 26. De Angioletti, M., et al., Beta+45 G --> C: a novel silent beta-thalassaemia mutation, the first in the Kozak sequence. Br J Haematol, 2004. 124(2): p. 224-31. 27. Wang, Q., et al., CCL22-Polarized TAMs to M2a Macrophages in Cervical Cancer In Vitro Model. Cells, 2022. 11(13). | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/89623 | - |
| dc.description.abstract | 巨噬細胞是先天免疫系統中的一種白血球，可以根據微環境中的刺激極化為促炎性的M1巨噬細胞或抗炎性的M2巨噬細胞。M1巨噬細胞在清除病原體方面發揮著至關重要的作用，而M2巨噬細胞主要參與組織修復。腫瘤微環境中存在許多巨噬細胞，它們對腫瘤的進展具有顯著的影響。值得注意的是，腫瘤微環境中的M2巨噬細胞促進腫瘤生長，並與癌症的不良預後有關。然而，M2巨噬細胞極化的詳細機制仍然大部分未知。我們最終的目標是建立一個利用CRISPR干擾（CRISPR interference，CRISPRi）的基因組範圍篩選系統，以識別參與M2巨噬細胞極化的新因子。然而，由於巨噬細胞中轉導和轉染效率較低，本研究專注於解決建立CRISPRi篩選系統時，主要遇到的兩個困難：1）在巨噬細胞系中穩定表達功能性的dCas9-KRAB蛋白；2）在巨噬細胞系中轉導足夠多樣性的sgRNA。在這裡，我們首先在NIH-3T3和RAW264.7細胞中建立了dCas9-KRAB介導的基因抑制系統，然後檢測了之前建立的dCas9-KRAB在骨髓細胞中的功能性（immortalized bone marrow cell，iBM）。此外，我們做了一個小型sgRNA庫，其中包含15個不同的sgRNA，並利用它檢測了iBM中sgRNA的多樣性和均勻性。我們的研究結果使得在iBM中建立CRISPRi篩選系統有進一步的進展。 | zh_TW |
| dc.description.abstract | Macrophages, a type of leukocytes in innate immune system, can be polarized to pro-inflammatory M1 macrophages or anti-inflammatory M2 macrophages according to the stimuli in the microenvironment. M1 macrophages play a vital role in pathogen clearance, while M2 macrophages predominantly participate in tissue repair. Macrophages are enriched within the tumor microenvironment and significantly influence tumor progression. Notably, M2 macrophages in tumor microenvironment promote tumor growth, and have been implicated in poor prognosis of cancer. However, the detailed mechanism of the M2 macrophage polarization are still largely unknown.
Though our eventual goal is to establish a genome-wide screening system using CRISPR-interference (CRISPRi) to identify novel factors that participate M2 macrophage polarization. However, due to the poor transduction and transfection efficiency in macrophages, this study focuses on addressing two major difficulties that are encountered in establishing the CRISPRi screening system, 1) stably expressing functional dCas9-KRAB protein in macrophage cell line. 2) Transduce sufficient diversity of sgRNAs in macrophage cell line. Here, we first established the dCas9-KRAB mediated gene knockdown system in both NIH-3T3 and RAW264.7 cells, and followed by examining the functionality of the previously established dCas9-KRAB in immortalized bone marrow cells (iBM). Furthermore, the diversity and evenness of integrated sgRNAs in iBM was examined by a newly generated mini-library containing 15 different sgRNAs. Our results provide a further advance in establishing CRISPRi screening system in iBM. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2023-09-13T16:07:05Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2023-09-13T16:07:05Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 摘要................................................................................................................................. i
Abstract ........................................................................................................................ iii Chapter 1 Introduction ................................................................................................ 1 1.1 Macrophage polarization ........................................................................ 1 1.2 Tumor-associated macrophages (TAMs) and cancer ............................ 2 1.3 M2 macrophage polarization mechanism .............................................. 2 1.4 Approaches of gene silencing .................................................................. 3 1.5 Large-scale screens on macrophages ...................................................... 6 1.6 The aim of our study ................................................................................ 6 Chapter 2 Materials and Methods .............................................................................. 8 2.1 Plasmids .................................................................................................... 8 2.2 Cell culture conditions ........................................................................... 10 2.3 Transfection ............................................................................................ 11 2.4 Lentivirus production and transduction .............................................. 12 2.5 RNA extraction and quantitative real-time PCR ................................ 13 2.6 Genomic DNA isolation ......................................................................... 13 2.7 Western blot ............................................................................................ 14 2.8 Flow cytometry ....................................................................................... 15 2.9 Statistical analysis .................................................................................. 16 Chapter 3 Results ....................................................................................................... 17 3.1 Expression Analysis of Previously Generated Stable dCas9-KRAB iBM Line ............................................................................................................. 17 3.2 Functional evaluation of the previous dCas9-KRAB iBMDM 1206-1 19 3.2.1 CRISPRi with sgRNA targeting Stat6 in dCas9-KRAB iBMDM 1206-1 .......................................................................................................... 19 3.2.2 Identify functional sgRNAs to assess the functionality of dCas9- KRAB iBM 1206-1 ..................................................................................... 21 3.2.3 CRISPRi with sgRNA targeting Mrc1 in NIH-3T3 cells ............... 23 3.3 Establishment of CRISPRi system ....................................................... 24 3.3.1 Generation of new dCas9-KRAB-expressiog iBM, J774A.1 and RAW 264.7 cell lines using pLAS-CRISPRi (KRAB-MeCP2) ............... 24 3.3.2 Establishment of CRISPRi in NIH-3T3 using split-dCas9 plasmids ...................................................................................................................... 26 3.4 Examination of sgRNA transduction diversity and evenness in iBMs 28 Chapter 4 Discussion ................................................................................................. 30 4.1 The non-functional dCas9-KRAB in previously-selected iBM clones 30 4.2 The alternative of CRISPRi screening in iBM .................................... 31 4.3 The diversity and evenness of sgRNA library in iBM after transduction ........................................................................................................ 31 Chapter 5 Figures ....................................................................................................... 33 Figure 1. Expression of dCas9 protein in dCas9-KRAB iBMDMs ............... 35 Figure 2. CD206+ population in STAT6_1 dCas9-KRAB iBMDMs ............. 37 Figure 3. CRISPRi functional tests in NIH-3T3 cells and iBMs .................... 39 Figure 4. dCas9-KRAB expression in dCsa9-KRAB stably expressing cell lines transduced with pLAS-CRISPRi (KRAB-MeCP2) ............................... 41 Figure 5. Generation of dCas9-KRAB stably expressing iBM cell line with split-dCas9 plasmids .......................................................................................... 45 Figure 6. sgRNA transduction diversity in iBMs ............................................ 46 Tables ........................................................................................................................... 47 Table 1. Primers for constructing sgRNA plasmids ........................................ 47 Table 2. Primers for constructing LV-C-CRISPRi-GFP-1-10........................ 49 Table 3. Primers for RT-qPCR ......................................................................... 49 Table 4. Primers for PCR .................................................................................. 50 Table 5. Primers for the examination of sgRNA diversity and evenness ...... 50 References ..................................................................................................................51 | - |
| dc.language.iso | en | - |
| dc.subject | 替代型活化型態巨噬細胞 | zh_TW |
| dc.subject | 全基因篩選 | zh_TW |
| dc.subject | CRISPR干擾 | zh_TW |
| dc.subject | 介白素-4 | zh_TW |
| dc.subject | sgRNA文庫 | zh_TW |
| dc.subject | CRISPR interference | en |
| dc.subject | genome-wide screening assay | en |
| dc.subject | interleukin-4 | en |
| dc.subject | macrophage alternative polarization | en |
| dc.subject | sgRNA library | en |
| dc.title | 建立干擾型CRISPR篩選平台以研究替代型活化巨噬細胞 | zh_TW |
| dc.title | Establishment of CRISPR Interference-Based Screening Platform for the Investigation of Alternative Polarized Macrophage | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 111-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 徐立中;陳佑宗 | zh_TW |
| dc.contributor.oralexamcommittee | Li-Chung Hsu;You-Tzung Chen | en |
| dc.subject.keyword | 替代型活化型態巨噬細胞,CRISPR干擾,全基因篩選,介白素-4,sgRNA文庫, | zh_TW |
| dc.subject.keyword | macrophage alternative polarization,CRISPR interference,genome-wide screening assay,interleukin-4,sgRNA library, | en |
| dc.relation.page | 54 | - |
| dc.identifier.doi | 10.6342/NTU202303462 | - |
| dc.rights.note | 同意授權(限校園內公開) | - |
| dc.date.accepted | 2023-08-08 | - |
| dc.contributor.author-college | 醫學院 | - |
| dc.contributor.author-dept | 分子醫學研究所 | - |
| dc.date.embargo-lift | 2028-08-07 | - |
| 顯示於系所單位： | 分子醫學研究所 | |
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