與卵巢癌細胞共同培養之腫瘤相關巨噬細胞的功能可塑性和一種新的卵巢癌斑馬魚模式

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研究生：	陳婉馨
論文名稱：	與卵巢癌細胞共同培養之腫瘤相關巨噬細胞的功能可塑性和一種新的卵巢癌斑馬魚模式 The Functional Plasticity of Tumor Associated Macrophages in Ovarian Cancer cells Co-culture Assay and a Novel Ovarian Cancer Zebrafish Model
指導教授：	莊永仁
口試委員:	李宜靜鄭邑荃張幸治莊永仁
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	42
中文關鍵詞：	卵巢癌、腫瘤相關巨噬細胞、腫瘤微環境
相關次數：	點閱：3 下載：0
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　　在台灣，卵巢癌是所有婦癌科中致死率最高的癌症。其高死亡率的原因主要是由於沒有症狀或其症狀不明顯，這使得卵巢癌的早期發現成為一個十分具有挑戰性的任務。也因此，臨床上常見卵巢癌的轉移擴散且往往導致預後不佳。目前已知在晚期的卵巢癌組織中常見巨噬細胞浸潤的現象，因此，我們有必要深入探討癌細胞與巨噬細胞間的交互作用以發展新的卵巢癌治療策略。
　　巨噬細胞為發炎性細胞在卵巢癌組織中的主要組成部分。巨噬細胞大致上可區分為M1和M2兩種型態，M1型巨噬細胞為刺激免疫反應的細胞，另一方面，M2巨噬細胞則表現出促進腫瘤發展的功能，因此M2又被稱為腫瘤相關巨噬細胞。研究報告指出，腫瘤相關巨噬細胞的存在對於癌症生長，血管新生和癌細胞轉移的過程十分重要。
　　組織蛋白酶S（CTSS）為半胱氨酸蛋白酶家族的成員。據了解，CTSS與TAMs一樣都參與了腫瘤惡化的過程。我們的結果顯示，M1和M2兩種極化後的巨噬細胞在共同培養後皆對於SKOV3細胞CTSS表現量有促進的效果。根據結果推論，這些巨噬細胞可能在卵巢癌細胞存在的情況下改變其特性，進一步轉變成腫瘤促進型的巨噬細胞。因此，在本研究中，我們的目標是在巨噬細胞-卵巢癌細胞共同培養的實驗中鑑定巨噬細胞的表現型態。
　　近年來斑馬魚成為癌症研究上一個強而有力的模式生物，雖然細胞共同培養試驗對於巨噬細胞與腫瘤細胞交互作用相關研究提供了一個易於操作的模式，但它終究無法反映出活體中癌症的發展過程。為了進一步在活體中探討卵巢癌的癌化過程以及腫瘤相關巨噬細胞與癌細胞間複雜的交互作用，我們的另一個目標為發展一種新的卵巢癌斑馬魚模式。藉由斑馬魚低成本和透明成魚的優勢，我們預期這種斑馬魚癌症模式可以成為一個研究卵巢癌進展和轉移過程中病理與生物學特性的工具。

In Taiwan, ovarian cancer has the highest rate of mortality among all gynecological cancers. The high death rate is mostly due to the common absence or subtle symptom of ovarian cancer, which makes early detection a very challenging task. As a result, the metastatic spread of ovarian cancer becomes predominant and lead to poor prognosis.
Among the variety of factors known to promote tumorigenesis and metastasis, macrophages infiltration is reported link to advanced ovarian cancer. Thus, to understand the ovarian cancer cells-macrophages interactions is necessary before one can develop new diagnostic and therapeutic strategies.
Macrophages are the major components of inflammatory cells in ovarian cancer tissue. In general, macrophages can be classified into M1 and M2 phenotypes. M1 macrophages are immune-stimulating cells, while the M2 macrophages show tumor promotion properties and are also known as tumor-associated macrophages (TAMs). TAMs are shown to promote tumor growth, angiogenesis and metastasis of tumors. Many of the functions are mediated by cathepsins (CTSS). CTSS is a member of cysteine protease family. It is known that CTSS involve in tumor malignant transition, and the expression levels of CTSS are elevated in many tumor microenvironments in the presence of TAMs. In this study, we found both of M1 and M2 polarized macrophages stimulate CTSS expression in ovarian cancer cells, SKOV3 cells after co-culture. The result suggested these macrophages may change to be tumor-progression phenotype in the presence of ovarian cancer cells. Therefore, in this study, our first aim is to investigate the phenotypic switch of macrophages influenced by ovarian cancer cells via in vitro co-culture assays.
Although cell co-culture assay provide a maneuverable system to study macrophage-cancer cell interactions, it cannot reflect the complex process of cancer development in the animal model system. To further investigate the ovarian carcinogenesis and the complex interplay between TAMs and ovarian cancer cells in vivo, we also aim to develop a novel ovarian cancer zebrafish model to take advantage of this aquatic vertebrate model organism’s low-cost and transparent features. We expect this zebrafish model could become a powerful tool to study pathological and biological features during ovarian cancer progression and metastasis.

中文摘要        I
Abstract        II
致謝    IV
Abbreviations    VIII
1.    Introduction    1
1.1    Ovarian cancer and the link between immune cells and ovarian cancer malignancy    1
1.2    Immune cells in tumor microenvironment are necessary for cancer progression    1
1.3    Macrophages promote tumor malignancy in tumor microenvironment    2
1.4    The distinction between M1 and M2 macrophages    2
1.5    Cathepsin S plays an important role in tumor progression    3
1.6    Zebrafish is an alternative model organism for cancer research    4
1.7    The functions and the expression sites of ZP3 and FSHR in zebrafish    4
1.8    Objectives and significant finding of this study    5
2.    Materials and methods    7
3.    Results    12
3.1    Both of M1- and M2- polarized macrophages promote CTSS expression in SKOV3 cells after co-cultured    12
3.2    Polarized macrophages acquired M2 characteristics after serum-free starvation    13
3.3    Macrophages showed M1 phenotype after co-cultured with SKOV3 cells    14
3.4    Zebrafish zp3 and fshr are ovary-specific genes, and the zp3 and fshr promoter sequences are highly conserved across human, mouse and zebrafish    15
3.5    Generate novel ovarian cancer zebrafish models by tol2 transposon system and the promoter of zp3 and fshr are used in this model    16
3.6    F0 generation of ovarian cancer zebrafish lines were identified by using observed eGFP signal and PCR approach    18
4.    Discussion    19
4.1    Macrophages change their functional properties repeatedly to promote tumor progression in tumor microenvironment    20
4.2    Both of M1 and M2-like properties exist in tumor associated macrophages and contribute to tumor progression    21
4.3    TNF-αand other M1 macrophage-specific factors may be the regulator of CTSS to cancer cells    22
4.4    Limitations of tol2 transposon system in generating transgenic zebrafish    23
4.5    The applications of our ovarian cancer zebrafish model in ovarian cancer research    23
4.6    The structural and functional differences between human and zebrafish ovaries    24
4.7    Comparison of cell types for ovarian cancer alteration between human and zebrafish cancer model    24
Reference:    26
Table 1. The sequence of primers used for cloning    30
Table 2. The sequence of primers used for genotyping and RT-PCR    31
Table 3. The collecitons of transgenic zebrafish    32
Figure 1. The CTSS mRNA expression profile in SKOV3 cells after co-cultured with 
        M1- or M2- polarized macrophages    33
Figure 2. Polarized-macrophages lost M1 characteristics and acquired M2 
        characteristics after 24 hours starvation    35
Figure 3. Polarized macrophages acquired M1 characteristics after co-culture with 
        SKOV3    36
Figure 4. Zebrafish zp3 and fshr are specifically express in ovary    37
Figure 5. The 3 kb 5’ upstream promoter sequence of fshr is highly conserved across 
        zebrafish, mouse and human    38
Figure 6. Analysis of fshr and zp3 promoter and zebrafish zp3 promoter is highly 
        conserved    39
Figure 7. Constructs for generating ovarian cancer transgenic zebrafish model    40
Figure 8. The experimental design for Cre/lox system which applicated in inducible 
        zebrafish ovarian cancer model    41
Figure 9. Stable transgene zebrafish lines were confirmed by observing GFP signal 
        and using PCR to do genotyping    42

                                

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