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研究生:陳建名
研究生(外文):Chien-Ming Chen
論文名稱:M2巨噬細胞對小鼠乳腺癌細胞生長之影響
論文名稱(外文):The role of M2 macrophage in modulation of breast cancer growth
指導教授:李宏謨李宏謨引用關係
指導教授(外文):Horng-Mo Lee
口試委員:劉兆蓮趙祖怡何景良
口試委員(外文):Chao-Lien LiuTsu-Yi ChaoChing-Liang Ho
口試日期:2016-12-14
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學檢驗暨生物技術學系所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2016
畢業學年度:105
語文別:中文
論文頁數:39
中文關鍵詞:腫瘤相關巨噬細胞白血球浸潤抗酒石酸性磷酸酶環境微環境癌症相關發炎反應先天性免疫反應後天性免疫反應細胞激素化學激素巨噬細胞
外文關鍵詞:tumor-associated macrophagesTAMleukocyte infiltrationtartrate-resistant acid phosphataseTRAcPnichemicroenvironmentcancer-related inflammationinnate immunityacquired immunitycytokineschemokinesmacrophage
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背景與目的:減緩甚至消除腫瘤生長和轉移一直是癌症治療的主要目標。腫瘤相關巨噬細胞(tumor-associated macrophages, TAM)的浸潤(infiltration)所誘發之發炎反應會促進腫瘤的生長和存活(proliferation and survival)並誘發腫瘤血管新生與轉移(angiogenesis and metastasis),其中巨噬細胞分泌的抗酒石酸性磷酸酶(tartrate-resistant acid phosphatase, TRAcP)可能扮演重要角色。因此本研究欲探討M2巨噬細胞是否經由調控抗酒石酸性磷酸酶的表現促進腫瘤生長速度,以及其相關機制。
方法:我們將M2巨噬細胞與JC乳腺癌細胞經由不同組合方式(單純注射JC、M2和JC一起注射,先注射M2 30分鐘後再注射JC)注射至小鼠的乳腺皮下,經過14天後測量其腫瘤生長的速度、大小和重量、腫瘤的抗酒石酸性磷酸酶表現和血清中抗酒石酸性磷酸酶5a的濃度。
結果:實驗結果發現預先注射M2巨噬細胞30分鐘後再注射JC乳腺癌細胞之組別與其他兩組(單純注射JC以及M2和JC一起注射)比較能顯著促進小鼠乳腺癌細胞之生長,而且這與腫瘤抗酒石酸性磷酸酶的表現增加有關。
結論:M2巨噬細胞可以藉由調節抗酒石酸性磷酸酶影響腫瘤相關環境,進而顯著促進小鼠乳腺癌細胞之生長。因此,抗酒石酸性磷酸酶可能可作為臨床監測乳癌癌症預後的生物指標或標靶治療的目標。
Background and purpose:
Alleviating or even eliminating tumor growth and metastasis has been the main goal of cancer treatment. Tumor-associated macrophages (TAM, also called M2 macrophage) infiltration-induced inflammatory responses not only promote tumor growth and survival, but trigger tumor angiogenesis and metastasis. The tartrate-resistant acid phosphatase (TRAcP) secreted by macrophages may play an important role. This study aimed to investigate whether M2 macrophages promoted the growth of tumor by regulating the expression of TRAcP and its mechanisms.

Method:
M2 macrophages and JC breast cancer cells were injected into the mammary glands of BALB/c mice via different combinations: 1) JC alone; 2) M2 and JC at the same time; 3) M2 was injected 30 min before the injection of JC. After 14 days, the rate and size of tumor growth, the body weight of the mice, the expression of TRAcP in tumors, and the level of TRAcP in serum were measured to compare the baseline data of above parameters.

Statistic Analysis:
All data showed by MEAN±SD, using Two-Way Repeated Measures ANOVA to compared different groups and different times for the body weight and subcutaneous tumor size of the mice. One the other hand, using One-Way ANOVA to compared different groups for tumor size of the mice. Statistical software is SPSS.

Result:
The results showed that the injection of M2 macrophages 30 min before the injection of JC significantly promoted tumor growth relative to other two groups (JC only or M2 and JC were injected at the same time), and this was associated with the expression of TRAcP in tumors and serum.

Conclusion:
M2 macrophages may regulate tumor-related environment by modulating the level of TRAcP in tumors and serum, and thereby have significant impacts on the growth of breast cancer cells in mice. Therefore, TRAcP may be a promising biomarker for clinical prognosis or a target for oncological therapy in mice breast cancer.
目錄
摘要 III
Abstract IV
關鍵字: VI
背景/簡介 1
實驗目的 4
實驗假說 4
實驗材料和方法 5
細胞培養 5
細胞轉型 5
流式細胞儀 6
蛋白質抽取 6
西方墨點轉漬法(Western blot) 7
動物實驗 9
腫瘤組織切片染色 10
酵素免疫分析法(ELISA) 11
統計分析(Statistic Analysis) 11
結果 12
討論 34
Reference 39
表目錄
表一、比較預先注射M2巨噬細胞是否會影響不同組別小鼠的體重(g)。 18
表二、比較預先注射M2巨噬細胞是否會影響小鼠乳腺癌腫瘤的惡性度。 19
表三、比較預先注射抗酒石酸性磷酸酶是否影響不同組別小鼠的體重(g)。 20
表四、比較預先注射抗酒石酸性磷酸酶是否會影響小鼠乳腺癌腫瘤的惡性度。 21
圖目錄
圖一、RAW264.7巨噬細胞(圖A)、M2巨噬細胞(圖B)與JC乳腺癌腫瘤細胞(圖C)之形態學差異。 22
圖二、利用流式細胞儀觀察RAW及M2巨噬細胞與JC腫瘤細胞之CD68、Arg-1、iNOS與TRAcP之表現差異 23
圖三、不同組別小鼠在第0、7、10、14天測得之皮下腫瘤體積(mm3)變化 24
圖四、不同組別小鼠(JC、RAW+JC、RAW 30 ´+JC、M2+JC和M2 30 ´+JC)在第14天犧牲後腫瘤體積(mm3)和腫瘤重量(g) 的變化 25
圖五、以西方墨點法觀察不同組別間(JC、RAW+JC 、RAW 30´+JC 、M2+JC、M2 30´+JC)腫瘤TRAcP蛋白質表現 26
圖六、以酵素免疫分析法觀察小鼠血清中TRAcP 5a蛋白質濃度表現 27
圖七、不同濃度抗酒石酸性磷酸酶蛋白質對小鼠腫瘤體積(mm3)(第14天)的影響 28
圖八、不同組別小鼠在第0、10、14天測得之皮下腫瘤體積(mm3)變化 29
圖九、不同組別小鼠(JC、750ng TRAcP +JC、750ng TRAcP 30 ´+JC、1.5μg TRAcP +JC和1.5μg TRAcP 30 ´+JC)在第14天犧牲後腫瘤體積(mm3)和腫瘤重量(g) 的變化 30
圖十、以西方墨點法觀察不同組別間(JC、750ng TRAcP +JC、750ng TRAcP 30 ´+JC、1.5μg TRAcP +JC和1.5μg TRAcP 30 ´+JC)腫瘤TRAcP蛋白質表現 31
圖十一、以酵素免疫分析法觀察小鼠血清中TRAcP 5a蛋白質濃度表現 32
圖十二、不同組別小鼠(JC、M2+JC、M2 30 ´+JC、750ng TRAcP +JC、750ng TRAcP 30 ´+JC、1.5μg TRAcP +JC和1.5μg TRAcP 30 ´+JC)在第14天犧牲後腫瘤體積(mm3)和腫瘤重量(g) 變化 33
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