研究生: |
王之璇 Wang, Chih-Husan |
---|---|
論文名稱: |
Aspirin抑制肥胖相關發炎因子促進乳癌細胞生長之探討 The effect of aspirin inhibits obesity-related inflammatory mediators promoting the growth of breast cancer cell |
指導教授: |
謝佳倩
Hsieh, Chia-Chien |
學位類別: |
碩士 Master |
系所名稱: |
人類發展與家庭學系 Department of Human Development and Family Studies |
論文出版年: | 2017 |
畢業學年度: | 105 |
語文別: | 中文 |
論文頁數: | 118 |
中文關鍵詞: | Aspirin 、肥胖 、乳癌 、發炎反應 |
英文關鍵詞: | macrophages |
DOI URL: | https://doi.org/10.6345/NTNU202203069 |
論文種類: | 學術論文 |
相關次數: | 點閱:136 下載:7 |
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肥胖為一全球性的健康議題,流行病學研究顯示肥胖會增加女性乳癌的發生率且具有較差的預後。肥胖者的脂肪組織中有多種免疫細胞浸潤,其中以巨噬細胞數量較多,形成低度慢性發炎反應,增加發炎性細胞激素的分泌,並伴隨血管新生因子的增加及脂肪激素的失調,皆有利於腫瘤的生長。而在腫瘤的微環境中,乳癌細胞透過誘導腫瘤相關巨噬細胞來達到促進腫瘤生長及轉移的效果。Aspirin為目前廣泛使用的非固醇類抗發炎藥物,也被證實具有癌症預防的作用,然而aspirin對脂肪、乳癌及免疫細胞間之交互作用的影響仍不明確。本研究目的為探討aspirin是否能抑制脂肪細胞的發炎現象,並調節巨噬細胞的免疫反應,進而抑制乳癌細胞的生長。
第一部份,建立以TNF-α、LPS或RAW264.7巨噬細胞條件培養液 (RAW 264.7 conditioned meium, RAW-CM)誘發3T3-L1脂肪細胞發炎的實驗模式,結果顯示aspirin能顯著降低MCP-1、IL-6、IL-1β及PAI-1的分泌。第二部分建立肥胖模式下4T1細胞生長與移行能力的實驗,結果顯示3T3-L1脂肪細胞條件培養液 (Adipocyte conditioned medium, Ad-CM)會促進乳癌細胞的生長和移行,而aspirin能發揮抑制的功效。另外在Ad-CM的代謝體分析中發現3T3-L1分化為脂肪細胞後對胺基酸的利用及蛋白質合成的需求增加,脂質代謝及氧化壓力隨之上升,而在分化過程給予aspirin的處理可顯著降低Ad-CM中脂質代謝相關的2-Hydroxycaproic acid與增加氧化壓力的hydroxyphenyllactic acid含量,表示aspirin能阻斷3T3-L1脂肪細胞分化的代謝變化。在Ad-CM脂肪酸相對定量分析中3T3-L1在分化過程中給予aspirin的處理,可顯著降低C16:1、C18:1、C18:2、C20:4和C24:1,顯示aspirin減少CM中游離脂肪酸的含量。第三部分,探討巨噬細胞與乳癌細胞間的關係,在LPS刺激RAW264.7所收集的CM培養4T1細胞可抑制細胞生長,反之沒有刺激下所收集的CM則會促進4T1細胞的生長與移行、VEGF、PAI-1、TNF-α和IL-6的分泌,且aspirin處理下具有抑制的作用。接著在RAW-CM的代謝體分析,發現無LPS刺激的CM中2-ketohexanoic acid/ketoleucine、lactate和arginine等可能和肥胖相關發炎及促進癌症生長的物質顯著上升,而LPS刺激後整體的能量代謝增加,而aspirin的處理對LPS刺激之RAW-CM影響仍不明確。第四部分,在4T1細胞與RAW264.7細胞共培養模式中,與4T1細胞共培養後的RAW264.7細胞傾向免疫抑制的M2型,且aspirin處理能增加CD11c (M1型)並降低CD206 (M2型)的表現。
綜合而論,aspirin能降低脂肪細胞的發炎反應,及抑制脂肪細胞和巨噬細胞促進乳癌細胞生長的作用,尤其在腫瘤微環境下,aspirin可降低有利於癌細胞生長的細胞激素並調節RAW264.7巨噬細胞之表型趨向,aspirin由影響數種細胞來調節肥胖相關的炎症反應以達到抗腫瘤的功效。
Epidemiological studies have shown that obesity increases the incidence of breast cancer and leads the worse prognosis. The adipose tissue of obese individuals generally accompanies macrophages infiltration and inflammatory cytokines secretion. The microenviroment with a chronic, low-grade inflammation, angiogenesis and adipokine dysregulation provides an ideal condition for tumor development, growth and migration. Moreover, cancer cells secret mediators attract macrophages and other immune cells to support tumor development and metastasis. Aspirin is a non-steroidal anti-inflammatory drug (NSAID), has been known as a chemopreventive agent against several types of cancer. However, the effect of aspirin on the interaction among adipocyte, macrophage and breast cancer cell is still elusive. The aim of this work is to investigate whether aspirin can inhibit inflammatory respond of 3T3-L1 adipocyte, regulate immune respond of RAW 264.7 macrophage, and then inhibit growth of 4T1 breast cancer cell.
First, we established an inflammatory model of 3T3-L1 adipocyte by TNF-α, LPS and RAW 264.7 macrophage conditioned meium (RAW-CM) stimulation. The results had shown that aspirin significantly inhibited MCP-1、IL-6、IL-1β and PAI-1 productions in 3T3-L1 adipocytes. Second, in the obesity-associated model of 4T1 breast cancer cells, 3T3-L1 adipocyte conditioned meium (Ad-CM) treatment significantly promoted 4T1 cell growth and migration. Futhermore, the Ad-CM was collected from aspirin treatment during 3T3-L1 differentication, resulted to inhibit the cell viability of 4T1 cell. In addition, in the result of metabolic analysis of Ad-CM, protein synthesis and oxidative stress were increased in mature 3T3-L1 adipocytes. Aspirin treatment reduced oxidative stress in Ad-CM and reversed the metabolic chage of adipocytes. In the relative fatty acid quantitation analysis of Ad-CM, aspirin diminished free fatty acid C16:1, C18:1, C18:2, C20:4 and C24:1. Third, in the model of 4T1 cell cultured in RAW-CM, 4T1 cells were treated with RAW-CM with LPS stimulation significantly inhibited cell growth and migration. On the contrary, 4T1 cells were cultured in RAW-CM without stimulation resulted to promote cell growth, migration and cytokine VEGF, PAI-1, TNF- and IL-6 secretions, while aspirin treatment exerted an inhibitory effects. Additionally, molecules related to obesity-associatd inflammation and tumor development were found in RAW-CM without stimulation by the metabolic analysis, while the effect of aspirin was not clear in the CM with LPS stimulation. Finally, aspirin significantly decreased CD206 (M2) of macrophages while CD11c (M1) was increased in co-culture of 4T1 and RAW264.7 cells, suggested that aspirin blunt tumor suppressed environment through regulating macrophages M1/M2 subtypes.
In conclusion, aspirin inhibited the inflammatory respond of adipocyte and the tumor-promoting effects of adipocytes and macrophages. This study indicated that aspirin breaks the crosstalk among 3T3-L1 adipocytes, RAW264.7 macrophages and 4T1 breast cancer cells, exerted anti-tumor effects by ameliorating obesity-associated inflammation.
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