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研究生:陳昱勻
研究生(外文):Yu-Yun Chen
論文名稱:腫瘤巨噬細胞在非小細胞肺癌之功能性分析
論文名稱(外文):Functional characterization of tumor associated macrophage in Non-small cell lung cancer
指導教授:陳惠文陳惠文引用關係
指導教授(外文):Huei-Wen Chen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:藥理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:73
中文關鍵詞:腫瘤巨噬細胞惡性胸水TREM-1環氧酵素二型前列腺素
外文關鍵詞:Tumor-associated macrophagepleural effusionTREM-1COX2PGE2
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在腫瘤微環境中巨噬細胞是發炎細胞中之多數成員又稱之為腫瘤巨噬細胞。根據近期研究發現,他們能夠刺激癌細胞生長以及促進癌症病程發展的作用。最近之證據顯示腫瘤巨噬細胞可被微環境的刺激而有不同的表現型,M1及M2 ,M1(典型活化巨噬細胞)及M2(另類活化巨噬細胞)。 M1具有引發發炎反應作用殺菌及毒殺細胞等作用;然而M2卻有免疫抑制及促腫瘤形成等作用。然而,腫瘤巨噬細胞在腫瘤微環境中的角色仍不清楚。且對於癌症的腫瘤形成、侵略轉移等行為之確切影響及功能以及對於癌症病患預後發展之影響仍舊不明。本研究發現從肺癌病患惡性胸水中分離出的巨噬細胞具有促進癌細胞移動之作用。進一步在動物實驗發現,在肺癌細胞株與巨噬細胞混合一同打入免疫缺陷鼠之皮下。我們發現病患胸水中分離之巨噬細胞似乎具有促進癌細胞生長的作用。本研究期望能釐清腫瘤巨噬細胞在腫瘤微環境中的角色,能針對腫瘤巨噬細胞在微環境中的功能,發展有利於未來應用在癌症治療、減少癌症轉移之輔助治療。

由於癌症與發炎的關係已在多方面的研究發現具有密切的相關性。除了在實驗室的研究,還有在臨床方面的觀察都能說明兩者之間有許多關聯性。科學家發現,在腫瘤形成區域有許多巨噬細胞的浸潤。近期的研究發現,他們具有刺激癌細胞生長以及促進癌症病程發展的作用。而表現在巨噬細胞表面的Triggering Receptor Expressed on Myeloid Cells-1 (TREM-1)也已被研究發現可能在癌症病程發展中扮演調控的角色。首先,我們發現在THP-1以及THP-1分化的巨噬細胞處理不同濃度的PGE2,其TREM-1的表現量會隨PGE2的濃度增加而上升。在THP-1分化的巨噬細胞與癌細胞共培養的情況下會增加TREM-1的表現量。且利用抗發炎藥物的處理後能降低癌細胞PGE2的表現量,並且在抗發炎藥物存在下的共培養,巨噬細胞的TREM-1表現量也同樣能被抑制。接著在處理抗發炎藥物之後,能抑制癌細胞在巨噬細胞存在下增加的移動能力。除此之外,我們也想探討巨噬細胞是否能藉由增加癌細胞的增生來促進癌症發展。另外,我們也分析在巨噬細胞與癌細胞共同培養之後會究竟會分化成M1(典型活化巨噬細胞),或是 M2(另類活化巨噬細胞)。基於以上,我們非常期望能夠針對腫瘤巨噬細胞在癌症微環境中的角色發展出有利於未來應用在癌症治療、減少癌症轉移之輔助治療藥物。
Infiltrated macrophages surrounding the tumor part, so called tumor-associated macrophage (TAM) are a major component of the infiltrated cells in the tumor microenvironment, which may contribute to cancer growth and promote cancer progression. The TAM has been divided into two subtypes, M1 and M2, with different functions in anti-tumorigenesis (M1), and pro-tumorigenesis (M2). The possible function of TAM on progression and invasion of human cancer, and the significance of TAM in patients’ prognosis are the topics under intensive investigation recently. In this study, the in vitro results indicated that the macrophage isolated from patients’ pleural effusion could promote cancer cell migration in the transwell migration assay. In the in vivo model, the mixture of lung cancer cell line (A549/GFP-Luc) and macrophage were co-injected to SCID mice subcutaneously. The pleural effusion macrophage showed the ability to promote tumor growth as comparing to the lung cancer cell alone group. Base on this study, the mechanisms of the TAM in the microenvironment of tumorigenesis need to be examined and developing the new strategy targets on TAM might have the potential to improve cancer treatment should be the important issue in cancer research.

Previous evidences indicated that the origin of cancer is highly correlated with chronic inflammation. Researchers discovered that, macrophages are contributed as the major component of the infiltrate cells, and have been defined as the tumor-associated macrophage (TAM). Our recent findings showed that the Triggering Receptor Expressed on Myeloid Cells (TREM) protein, which belongs to immunoglobulin super-families, could play some roles in regulating tumorigenesis. In this study, we found that TREM-1 expression in THP-1 cells or THP-1-derived macrophage could be up-regulate by PGE2 in a dose-dependent manner. Also, TREM-1 could also be up-regulated when co-culturing with COX2 highly expressed lung cancer cell line, A549. Anti-inflammatory drugs could reduce PGE2 production in A549 cells, and decrease the TREM-1 expression which up-regulate by cancer cell. In the transwell migration assay, the effects of anti-inflammatory drugs on the expression level of PGE2 and TREM-1 were correlated with cancer cell migration ability. Furthermore, we would like to clarify whether macrophage could promote cancer progression by stimulate cancer cell proliferation. Besides, we also identified the macrophage which stimulated by cancer cell, were they been stimulated to M1 orM2 subtype. Based on the present information, we are looking forward to discover some drugs as new strategy for treating cancer through regulating the TAM and related inflammatory microenviroment.
Abstract i
中文摘要 ii
Contents iii
Figure content iv
Chapter 1 Introduction 1
I. The relationship between cancer and chronic inflammation 1
II. Tumor-associated macrophage (TAM) 2
III. Metastasis 3
III. Hypothesis and strategy 4
IV. Experimental profile 5
V. Specific aims 6
Chapter 2 Materials and Methods 8
2.1 Cell line and culture condition 8
2.2 Isolation of CD 14+ from peripheral blood and pleural effusion 8
2.3 In vitro cell migration assay 9
2.4 Animal model 10
IVIS image system protocol 10
In vivo drug treatment 11
2.5 Immunohistochemistry (IHC) 12
2.6 Statistical analysis 12
Chapter 3 Results 13
3.1 Peripheral blood monocyte and pleural effusion macrophage could promote cancer cell migration ability 13
3.2 Pleural effusion macrophage could promote cancer cell proliferation in vivo 14
3.3 CD68 positive macrophages in the tumor 14
3.4 Oral treatment of anti-inflammation drugs could retard the tumor proliferation rate 15
Chapter 4 Discussion 17
Chapter 5 20
Chapter 6 References 27


Abstract i
中文摘要 ii
Content iii
Figure content v
Chapter1 Introduction 1
I. Triggering Receptor Expressed on Myeloid Cells-1 (TREM-1) is an inflammation amplifier in innate response 1
II. COX2, PGE2 involve in neoplasm development 2
III. TREM-1 expression on macrophage can be regulated by PGE2 3
IV. Hypothesis and strategy 3
V. Experimental profile 5
VI. Specific aims 6
Chapter 2 Materials and Methods 8
2.1 Cell line and culture condition 8
2.2 Collection of condition medium 8
2.3 Real-time quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) 9
2.4 Plasmid constructs and selection of stable clones 10
2.5 Western blot 10
2.6 Cell Viability 11
2.7 Enzyme-linked immunoassay 12
2.8 In vitro cell migration assay 12
2.9 Statistical analysis 13
Chapter 3 Result 14
3.1 PGE2 treatment could up-regulate TREM-1 mRNA expression level in THP-1 and THP-1 derived macrophage 14
3.2 TREM-1 expression level which up-regulate by cancer cell was reduced after anti-inflammation drugs treatment in direct and indirect co-culture 14
3.3 The anti-inflammation drugs treatment could decrease A549 migration ability in the co-culture system 16
3.4 Co-culture macrophage with COX2 over-expressed H1299 could slightly up-regulate TREM-1 expression 17
3.5 H1299 over express COX2 could have more migration ability than parental cell, but co-culture with macrophage might reduce the migration ability of H1299 17
Chapter 4 Discussion 19
Chapter 5 22
Chapter 6 References 32
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