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系統識別號 U0026-2507201115003900
論文名稱(中文) 腫瘤浸潤巨噬細胞促進壺腹癌之進行
論文名稱(英文) Tumor-associated macrophages promote progression of ampullary cancer
校院名稱 成功大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Institute of Clinical Medicine
學年度 99
學期 2
出版年 100
研究生(中文) 徐慧萍
研究生(英文) Hui-Ping Hsu
學號 s9894101
學位類別 博士
語文別 英文
論文頁數 136頁
口試委員 指導教授-林炳文
指導教授-賴明德
召集委員-林以行
口試委員-靳應臺
口試委員-呂佩融
口試委員-陳立宗
中文關鍵字 壺腹腺癌  腫瘤浸潤巨噬細胞  巨噬細胞遷移抑制因子  造骨蛋白  轉化生長因子乙型 
英文關鍵字 Ampullary cancer  Tumor-infiltrated macrophage  Macrophage migration inhibitory factor  Osteopontin  Transforming growth factor-ß 
學科別分類
中文摘要 壺腹癌是僅次於胰臟癌的第二常見壺腹周圍癌症,亦是最常見的小腸癌症;根除性手術之後,於本院過去十七年的臨床資料中,五年存活率為45.7 %,預後相關因子包括胰臟侵犯、淋巴結轉移及腫瘤分化不佳等。以免疫組織染色來檢驗手術切除的壺腹癌,發現87%的壺腹癌細胞表現巨噬細胞遷移抑制因子(MIF),也有36%的壺腹癌內浸潤之巨噬細胞表現造骨蛋白(osteopontin),兩者合併表現時與大型腫瘤病患之長期存活率有關。
目前已知腫瘤浸潤巨噬細胞在癌症細胞的影響下會轉化成第二型腫瘤浸潤巨噬細胞〈M2 TAMs〉,藉由分泌細胞激素來促進血管新生或是分解細胞間質,進而促進癌症增生並轉移。巨噬細胞遷移抑制因子是一在急慢性發炎反應中扮演重要角色的細胞激素,於多種癌症細胞中皆會表現,可以促進細胞週期進展與抑制p53之功能,亦可分泌至細胞間隙以活化巨噬細胞。我們發現巨噬細胞遷移抑制因子合併轉化生長因子乙型(TGF-beta)可調節巨噬細胞,使其分化為有造骨蛋白表現之第二型腫瘤浸潤巨噬細胞。給予來自於壺腹癌、含有巨噬細胞遷移抑制因子之條件培養液後,巨噬細胞亦會轉化為第二型腫瘤浸潤巨噬細胞;同時給予巨噬細胞遷移抑制因子之抑制劑後,第二型腫瘤浸潤巨噬細胞的特性與造骨蛋白的表現量皆大幅下降。在本研究中得知,壺腹癌細胞藉由分泌巨噬細胞遷移抑制因子,改變表現造骨蛋白之第二型腫瘤浸潤巨噬細胞,並影響壺腹癌之侵襲性。藉由抑制巨噬細胞遷移抑制因子或是造骨蛋白陽性之第二型腫瘤浸潤巨噬細胞,可能為壺腹癌之有效治療方式。
英文摘要 The carcinoma of the ampulla of Vater is the second most common periampullary cancer and also the most common neoplasm of small intestine. In our previous data from patients undergoing pancreaticoduodenectomy for adenocarcinoma of the ampulla of Vater, the 5-year survival rate was 45.7%. We used immunohistochemistry staining of tissue sections and revealed expression of macrophage migration inhibitory factor (MIF) in cancer cells in 79 of 91 patients (87%) and cytoplasmic positivity for osteopontin (OPN) in scattered infiltrating tumor-associated macrophages (ITAMs) in 36 of 100 patients (36%). Patients with bulky tumor, coexpression of MIF in ampullary cancer cells and OPN in ITAMs had poor prognosis than those with either one negative.
In literatures, the tumor-associated macrophages (TAMs) are likely differentiated into M2 macrophages, which can produce angiogenetic factors for neovascularization, protease for matrix remodeling and growth factors for tumor cells to promote metastasis. MIF is a crucial cytokine in innate and acquired immunity which is found to overexpress in several kinds of human neoplasms. MIF can inhibit p53 tumor suppressor and enhance cell cycle progression in cancer. MIF is also secreted into extracellular matrix to increase angiogenesis and activate macrophages. In present study, we found that combination of MIF and transforming growth factor-beta (TGF-beta) induced differentiation of macrophages into M2 macrophages with OPN expression. Co-culture with MIF-rich conditioned medium from ampullary cancer cells also stimulated M2 differentiation. MIF inhibitor, ISO-1, suppressed M2 cytokines and OPN expression in macrophages. The present study indicates that synergistic interactions of tumor-derived MIF and TGF-beta induce expression of OPN in M2 TAMs. M2 TAMs promote progression of ampullary cancer. Target therapy against MIF or OPN+ TAMs can improve survival of patients with ampullary cancer.
論文目次 Abstract in Chinese.......................................1
Abstract in English.......................................2
Acknowledgement...........................................4
Table of Contents.........................................6
List of Tables............................................9
List of Figures..........................................11
Abbreviations............................................13

Chapter One: Introduction................................15
I. Ampullary cancer.................................16
1. Carcinogenesis of ampullary cancer...............17
2. Inflammation in ampullary cancer.................17
II. Tumor-associated macrophages.....................19
1. M2 TAMs in cancer................................20
2. Mediators from M2 TAMs in cancer.................21
3. Macrophage-associated cytokines from cancer cells22
III. Specific aim and significance....................24
1. Hypothesis and significance of present research..24
2. Summary of research protocols....................24
3. Specific aims....................................24

Chapter Two: Materials and methods.......................27
I. Chemical drugs and reagents......................28
II. Kits.............................................29
III. Antibody.........................................30
IV. Experimental Procedures..........................30
Cell culture and temperature.............................30
Plasmid and construction of stable clone cell lines......34
Western blot analysis....................................35
Laser-assisted microdissection (LMD).....................38
Semiquantitative reverse transcription polymerase chain reaction.................................................39
Quantitative real-time polymerase chain reaction.........41
Conditioned medium.......................................42
Enzyme-linked immunosorbent assay (ELISA)................43
Immunohistochemistry.....................................44
Immunohistochemical analysis.............................45
Sorting of CD14+ macrophages.............................46
Induction of M2 macrophages..............................47
Flow cytometry...........................................49
Statistical analysis.....................................50

Chapter Three: Results...................................51
I. Crosstalk between ampullary cancer cells and tumor-associated macrophages is related to poor survival.......52
1. The patients with ampullary cancer have poor prognosis................................................52
2. Tumor-associated macrophages increase in specimens of ampullary cancer......................................53
3. Expression of M1 or M2 TAMs is correlated with clinical outcome of ampullary cancer patients............54
4. Expression of OPN in infiltrating tumor-associated macrophages is related to advanced cancer................55
5. OPN+ ITAMs in specimens is correlated with disease recurrence and long-term survival of patients with ampullary cancer.........................................56
6. Plasma OPN level in patients with ampullary cancer is higher than healthy volunteers........................57
7. Expression of macrophage-associated cytokines is detected in ampullary cancer cells.......................58
8. Overexpression of MIF is correlated with advanced tumor stage..............................................58
9. Plasma MIF level in patients with ampullary cancer is similar to healthy volunteers.........................59
10. Co-expression of MIF+ ampullary cancer cells and OPN+ ITAMs in bulky ampullary cancer predicts poor survival.................................................60
II. Expression of M2 macrophages is induced by cancer cells and partially suppressed by MIF inhibitor..........61
1. CD14+ monocytes and PMA-stimulated THP-1 cells are used as mature macrophages...............................61
2. Surface markers of mature macrophages shift to M2 markers after stimulation with TGF-beta and augment by MIF......................................................61
3. Activated macrophages express OPN and M2 markers after stimulation with TGF-beta and MIF......................................................62
4. Expression of M2 cytokines in activated macrophages is increased after stimulation with conditioned medium from MIF+ cancer cells............................62
5. MIF inhibitor (ISO-1) inhibits expression of M1/M2 cytokines and OPN in CD14+ Monocytes.....................63
6. Elucidate different response of mature macrophages after treatment with conditioned medium from MIF-high cancer cells and MIF-low cancer cells....................63

Chapter Four: Discussion.................................65
I. Crosstalk between cancer cells and tumor-associated macrophages in ampullary cancer...............66
II. In vitro induction of M2 macrophage and partial suppression by MIF inhibitor.............................71
III. Limitation and Implications......................73
IV. Future Research..................................75
V. Conclusions......................................76

References...............................................77
Tables...................................................86
Figures.................................................106
Addendum data...........................................128
Curriculum Vitae........................................133
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