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研究生:李健榕
研究生(外文):Chien-Jung Li
論文名稱:藉由PGLYRP1刺激表現TREM-1的免疫細胞探討TREM-1在腫瘤微環境中的角色
論文名稱(外文):Investigating the role of TREM-1 in Tumor-associated immune cells by Stimulation of PGLYRP1
指導教授:陳念榮
指導教授(外文):Nien-Jung Chen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:英文
論文頁數:62
中文關鍵詞:髓細胞觸發受體-1肽聚醣識別蛋白-1腫瘤相關巨噬細胞
外文關鍵詞:TREM-1PGLYRP1TAM
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在原發性的腫瘤微環境中有許多細胞會與腫瘤細胞交互作用。除了一些基質細胞釋放腫瘤所需的生長因子、血管上皮細胞提供養分,更有大量免疫細胞的浸潤,這些免疫細胞與腫瘤相互拮抗,產生促進腫瘤生長或抗腫瘤的效果。其中,在原發性腫瘤微環境中,有大量骨髓來源細胞的浸潤,包括腫瘤相關的巨噬細胞 (TAM)、骨髓來源的抑制性细胞 (MDSC)以及樹突狀細胞 (DC)。在本篇研究中發現,這些細胞的外膜表面會表現高量的髓細胞觸發受體-1 (Triggering receptor expressed on myeloid cells, TREM-1),且藉由髓細胞觸發受體-1剔除的小鼠模式中發現,髓細胞觸發受體-1具有抗腫瘤生長的效果,然而髓細胞觸發受體-1在腫瘤微環境中最主要的功能仍須要進一步去釐清。我們嘗試在腫瘤細胞中,表現髓細胞觸發受體-1的配體-肽聚醣識別蛋白-1 (Peptidoglycan recognition protein 1, PGLYRP1),並希望藉由在活體腫瘤微環境中,刺激這些表現髓細胞觸發受體-1的免疫細胞,並藉此探討刺激髓細胞觸發受體-1是否可成為抗腫瘤的策略,以及肽聚醣識別蛋白-1用來當作抗腫瘤藥物發展的可能性。
Many cells interact with tumor cells in the primary tumor microenvironment (TME). In addition to some stromal cells and vascular epithelial cells which release the growth factors and provide nutrients for tumor cells, a lot of immune cells infiltrating in the TME could be observed. These immune cells and tumors interact each other by producing effects of promoting tumor growth or anti-tumor. Among them, in the primary tumor microenvironment, various bone marrow-derived cells, including some polymorphonuclear leukocytes (PMN), tumor-associated macrophages (TAM), myeloid-derived suppressor cells (MDSC), and dendritic cells (DC) were found. In this study, we revealed that a high amount of Triggering receptor expressed on myeloid cells (TREM-1) were expressed on the surface of these infiltrated cells in the tumor environment but not on the unstimulated circulating myeloid cells or splenic myeloid cells. In the mouse tumor-engrafting model, it was found that TREM-1 has anti-tumor growth effect. However, the details of TREM-1 functions in the tumor microenvironment remain to be further clarified. We attempted to express Peptidoglycan recognition protein-1 (PGLYRP1), a ligand of TREM-1, on tumor cells, and aimed to stimulate TREM-1 expressing cells in the tumor microenvironment. Whether stimulating TREM-1 could be an effective anti-tumor strategy, as well as the PGLYRP1-derived reagents could be used as anti-tumor drugs remained to be further investigated in future.
摘要 i
Abstract ii
Contents iii
Introduction 1
Tumor immune microenvironment (TIME) 1
Triggering receptor expressed on myeloid cells (TREM-1) 1
The role of TREM-1 in tumorigenesis 3
Putative ligands of TREM-1 4
Peptidoglycan recognition protein 1 (PGLYRP1) 5
Hypothesis and Specific Aim 7
Material 8
1. Animals 8
2. Cell lines 8
3. Bone marrow cell isolation and differentiation 9
4. Cell culture mediums 9
4-1. Roswell Park Memorial Institute 1640, RPMI 1640 9
4-2. Dulbecco's Modified Eagle Medium, DMEM high 9
4-3. Ham's F-12 10
4-4. Other reagents 10
4-5. Stimuli for cell treatment 11
5. DNA plasmid preparation 11
6. DNA construction 12
6-1. Bacteria culture and transformation 12
6-2. Mini-DNA preparation 12
6-3. PCR amplification 12
6-4. DNA electrophoresis and gel extraction kit 13
7. Transfection reagent 14
7-1. Polyethylenimine (PEI) 14
7-2. Lipofectamine2000 14
8. Western blotting 14
SDS-PAGE(sodium dodecyl sulfate-protein gel electrophoresis) 14
9. Flow cytometry 16
Method 17
1. Cell culture 17
2. Plasmid construction 17
3. Generation of cell line 19
4. Production of mouse GM-BMMs 20
5. Heterotopic graft tumor models 20
6. Tumor-infiltrated immune cell analysis 21
7. Flow cytometry analysis 21
8. Western blotting analysis 22
Result 23
1. TREM-1 expressed myeloid cells are specifically higher in tumor environment than other tissues 23
2. Expressing a chimeric transmembrane-bound PGLYRP1 on LLC 24
3. Preliminary dissect tumor progression after inoculation of PGLYRP1-TM expressed LLC 25
4. Generating a soluble form of polymer chimeric PGLYRP1-IgM 26
5. Co-stimulation of LPS and PGLYRP1-IgM is sufficient to induce iNOS expression in a TREM-1 dependent manner 27
6. Co-stimulation of LPS and PGN with PGLYRP1-IgM seems to enhance phosphorylation of ERK via TREM-1 27
Discussion 29
Figure 32
Figure 1. 32
Figure 2. 34
Figure 3. 37
Figure 4. 41
Figure 5. 43
Figure 6. 44
Figure 7. 46
Figure 8. 47
Figure 9. 49
Figure10. 50
Figure11 52
Figure 12. 54
Figure 13. 56
Figure 14. 57
Figure 15. 58
Reference 59
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