臺灣博碩士論文加值系統

大腸直腸癌，又可稱為大腸癌、結腸直腸癌，是一種為源自結腸或直腸的癌症。大腸直腸癌的症狀包括糞便中帶血、排便習慣改變、體重減輕、以及疲倦感，症狀診斷可藉由大便潛血反應進行初篩，再藉由肛門指診、乙狀直腸鏡及大腸鏡進行確診。大腸直腸癌起因為有很多，大部分是因生活習慣及老化造成，遺傳性大腸直腸癌在所有病例中的比例不到5%。風險因子包括飲食、肥胖、抽菸、運動量不足，最重要的風險因子包含發炎性腸道疾病（分為克隆氏症和潰瘍性大腸炎）以及特定的腸道致病菌。慢性發炎在癌症形成中扮演一個極為重要的角色，慢性發炎使得促發炎的激素和活性氧化物質的堆積造成上皮細胞持續受損，腸道上皮細胞不斷活化修復訊號，形成上皮細胞的突變促使癌症形成。 髓系細胞觸發受體(Triggering receptor expressed on myeloid cells; TREM)在先天免疫中扮演了重要的調控角色，可能增強或減弱發炎反應，參與在許多感染症或是非感染性疾病之中，但至今其參與之調控機制仍然有待釐清。第一型髓系細胞觸發受體(TREM-1) 在免疫調節中扮演促發炎反應角色，然而，在我們實驗室先前的研究中發現在急性腸炎動物模式中，帶有TREM-1 基因缺陷的基因剔除小鼠有較為嚴重的發炎現象。本篇論文，欲利用TREM-1 基因缺陷的基因剔除小鼠，探討其於發炎相關癌症形成中之角色。利用偶氮甲烷及葡聚糖硫酸鈉誘發的大腸直腸癌動物模式，我們發現剔除TREM-1 能夠導引M2 型巨噬細胞的極化。此外，剔除TREM-1同時也大量產生促發炎激素，白细胞介素-6 (IL-6)，進而使信號轉導及轉錄激活蛋白3 (pSTAT3)活化，促進細胞增生、抑制細胞凋亡，皆會促進癌症的進程。綜言之，我們發現了TREM-1 在慢性發炎導致的癌症中調控新機轉，相信能在探討發炎反應調節與相關疾病的檢驗及治療上提供新的方向。

Colorectal cancer (CRC), also known as bowel cancer and colon cancer, is the development of cancer from the rectum. Colorectal cancer is the third most common cause of cancer deaths worldwide. Risk factors include family history, environmental mutagens and the most important risk factors are specific intestinal microbes and chronic intestinal inflammation. Inflammation is a critical component for tumor progression. Chronic inflammation caused epithelial regeneration signal to activate, increased oxidative stress and pro-inflammatory cytokines (such as IL-6 and TNF) which could damage intestinal barrier, and further lead to epithelial cell mutation, sequentially initiate the dysplasia. The triggering receptor expressed on myeloid cells (TREM)-1, as an amplifier of acute inflammation, could contribute to amplify LPS/TLR4-induced pro-inflammatory cytokines expression including TNF, IL-6, IL-1β, and MCP-1. TREM-1 is found mainly expressing on monocytes/macrophages and neutrophils. Moreover, regarding to the colitis, TREM-1 was significantly increased in affected tissues of inflammatory bowel disease (IBD) patients, as well as in the colons of colitis mice. Our preliminary results showed TREM-1 deficiency increased the disease severity and amplified inflammation of DSS-induced acute colitis. Thus, TREM-1 was an important regulator for acute colon inflammation, whether TREM-1 plays a role in CAC experimental murine model remains to be investigated. In this study, we aimed to determine the role of TREM-1 in colorectal cancer by adopting an azoxymethane (AOM) and dextran sulfate sodium (DSS) mouse CAC model in TREM-1 deficient mice. In AOM/ DSS model of colon cancer, TREM-1 deficiency (TREM-1-/-) contributed to higher disease activity index (DAI) in AOM/DSS-induced CAC. We also found that TREM-1 deficiency (TREM-1-/-) caused more epithelial barrier impairment, higher tumor burden and bigger-sized colonic polyps compared to AOM/DSS-treated wild-type counterparts. The TREM-1-/- colons exhibited significant increased proliferation cells and nuclear β-catenin, both associate with a pro-tumorigenesis phenomenon. Furthermore, TREM-1-/- mice exhibited a significant increase of type 2 macrophages (M2) in tumor area, simultaneously also had increased IL-6/Stat3 signaling as tumor-promoting factors to enhance chronic inflammation and tumorigenesis. In conclusion, our results demonstrate a protective role for TREM-1 in defensing AOM/DSS induced colitis associated cancer in mice.

中文摘要 i
Abstract ii
Table of Content iv
List of Figures vi
Abbreviations vii
Introduction 1
1. Colorectal cancer 1
2. Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS) model 1
3. Triggering receptor expressed on myeloid cells1, TREM-1 2
4. TREM-1 in CAC 3
5. Macrophage polarization in CAC 4
6. IL-6 and STAT3 are required for development of CAC 5
Materials 7
1. Animals 7
2. ELISA for cytokine expression 7
3. RNA purification and reverse transcription 8
4. Real-time PCR for mRNA 8
5. Immunohistochemical staining 9
6. Immunofluorescence staining 9
7. Protein quantification 10
8. Western blotting 10
9. Murine macrophage isolation 12
10. Antibody 13
Methods 15
1. AOM/DSS animal model 15
2. RNA extraction and real-time quantitative polymerase chain reaction (qRT-PCR) 15
3. IL-6 ELISA 16
4. Methylene blue whole mount staining 16
5. Immunohistochemical staining 16
6. Immunofluorescence staining 17
7. Protein extraction from colon tissue 17
8. Western blotting 18
9. Murine macrophage isolation 18
10. Flow cytometry 20
11. Statistical analysis 20
Results 21
1. TREM-1 deficiency increases the susceptibility to chemically-induced colitis and inflammation-associated CAC tumorigenesis 21
2. TREM-1 deficiency induced increasing tumor burden and disease activity index score 22
3. TREM-1 deficient colon observed more epithelial barrier impairment and tumor burden in AOM/DSS-induced CAC 22
4. Increased expression of nuclear β-catenin and Ki-67 in AOM/DSS TREM-1-/- mice 23
5. TREM-1 deficiency had more type-2 macrophage infiltration 24
6. TREM-1 deficiency increased IL-6/Stat3 to enhance chronic inflammation and tumorigenesis 25
Discussion 27
Figures 35

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