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系統識別號 U0026-1409201216323900
論文名稱(中文) 活化的雌激素受體貝他促進4T1/Luc+乳癌細胞於小鼠體內擴散
論文名稱(英文) Acceleration of tumor spreading by activated ERβ in 4T1/Luc+ breast tumor bearing-mice
校院名稱 成功大學
系所名稱(中) 生理學研究所
系所名稱(英) Department of Physiology
學年度 101
學期 1
出版年 101
研究生(中文) 黃怡靜
研究生(英文) Yi-Jing Huang
學號 S36994102
學位類別 碩士
語文別 英文
論文頁數 75頁
口試委員 指導教授-蔡美玲
口試委員-蘇五洲
口試委員-張雋曦
中文關鍵字 乳癌  雌激素  雌激素受體  細胞爬行  微管蛋白  細胞擴散 
英文關鍵字 17β-estradiol  ERβ  breast tumor spreading  migration  microtubul  α-tubulin 
學科別分類
中文摘要 乳癌的發生率受到雌激素(E2)濃度的影響。表現雌激素受體(ERβ)的乳癌細胞具有較高的侵略性而且在人體內造成高度的轉移以及不良的預後。 然而, 目前並不清楚ERβ在乳癌中所扮演的角色。因此本篇研究的目的是探討雌激素如何促進乳癌的發展以及ERβ在乳癌發展中所調控的機制。首先,我們發現4T1乳癌細胞只表現ERβ。我們將乳癌細胞4T1植入小鼠的背側以建立乳癌自動轉移的動物模式。實驗結果顯示持續二到三周腹腔注射E2至小鼠體內加速原位乳癌細胞的發展。而持續三周同時注射ERβ拮抗劑PHTPP至小鼠體內抑制E2所引起的原位乳癌發展。然而, 持續五周注射E2至小鼠體內不影響原位的乳癌發展以及乳癌細胞的轉移。在細胞實驗中,E2不影響細胞數目但是透過增加細胞面積以及促進細胞突出而改變細胞型態,而PHTPP抑制由E2所促進的細胞突出。此外, PHTPP和ERβ sh-RNA 抑制由E2和DPN (an ERβ agonist) 所引發的4T1乳癌細胞爬行。另一方面,太平洋紫杉醇 (Taxol)、秋水仙素( colchicine )皆可抑制由E2所增加的4T1乳癌細胞爬行。E2處理三小時促進4T1乳癌細胞內的微管 (microtubule)呈放射線狀的散佈於細胞質且延伸至細胞突出處,而這些由E2所誘發的現象都可被PHTPP所抑制。E2處理三小時不影響4T1乳癌細胞聚合態的α-微管蛋白(polymeric α-tubulin)和非聚合態的α-微管蛋白(soluble α-tubulin)的表現。 此外, E2處理三小時不影響4T1乳癌細胞α-微管蛋白 (α-tubulin) 的表現量和乙醯化的α-微管蛋白 (acetyl α-tubulin)的表現量以及乙醯化的α-微管蛋白/α-微管蛋白的比率。因為活化態的ERβ造成α-微管蛋白的重新分布以及細胞型態的改變和增加細胞爬行還有促進乳癌的發展, 所以由這些結果顯示活化態的ERβ透過造成微管蛋白的重新分布而促進微管動態以及改變細胞型態促進細胞爬行,而這些因素最後促進乳癌細胞的擴散。
英文摘要 The higher incidence of breast cancer is positively associated with the sex hormone estrogen. Estrogen receptor β –positive breast cancer cells are more aggressive that show greater frequency of metastasis and poorer prognosis. However, the role of estrogen receptor β in the development of breast cancer remains largely unclear. The purpose of this study is to examine estrogen’s contribution to the full development of breast tumor and ERβ-mediated mechanisms in breast tumor. Here, we showed that 4T1 breast tumor cells only expressed ERβ. 4T1 cells were subcutaneous implanted into the dorsal flank of BALB/c mice to establish the spontaneous metastasis animal model. Daily intraperitoneal injection of 17β-estradiol (E2) for 2-3 wks accelerated the increase tumor mass of breast tumor at primary sites in 4T1-bearing mice. E2-induced tumor mass and area of 4T1 breast tumor at primary sites was inhibited by 3-wk co-treatment with ERβ antagonist PHTPP. Further exposure to E2 for 5 wks did not affect the development of 4T1 breast tumor at primary site and metastatic site. In 4T1 cells, E2 did not affect the cell number but caused cell morphological changes by increasing cell area and cell protrusion. PHTPP blocked the E2-induced cell protrusion. Furthermore, PHTPP or ERβ sh-RNA inhibited the E2- and DPN- (an ERβ agonist)- increased migration in 4T1 cells. Disturbance of microtubule dynamics by paclitaxel and colchicine counteracted E2-increased migration. 3-hr treatment of E2 induced the formation of long thick microtubule bundles scattered throughout the cytoplasm toward the membrane protruding regions, which was reversed by co-treatment with PHTPP. The treatment of E2 for 3 hrs did not affect the polymeric α-tubulin and solubleα-tubulin. Moreover, the treatment of E2 for 3 hrs did not affect the abundance of α-tubulin, acetylated α-tubulin, and the ratio of acetyl α-tubulin to α-tubulin. Since activated ERβ caused re-distribution of α-tubulin and morphological changes, increased cell migration, and enhancing the development of breast tumor, these results suggest that activated ERβ increases breast tumor cell migration by enhancing microtubule dynamics, specifically accelerating the redistribution of tubulin to protruding regions and morphological changes which ultimately contributes to breast tumor spreading.
論文目次 Index
中文摘要 I
Abstract III
致謝 V
List of table X
List of figures XI
List of supplementary figures XIII
Chapter 1. Literature Review 1
1.1 Current studies of breast cancer 1
1.1.1 Epidemiology of Breast Cancer 1
1.1.2 Breast cancer cell proliferation 2
1.1.3 Breast cancer cell migration and spreading 3
1.1.4 Breast cancer cell invasion and metastasis 4
1.2 Estrogen and breast development 4
1.2.1 Estrogen biosynthesis 4
1.2.2 Estrogen receptors 5
1.2.3 Estrogen in mammary gland development 6
1.3 Molecular mechanism of Estrogen on breast tumor formation 7
1.3.1 Estrogen receptors in breast cancer 7
1.3.2 Role of ERs in breast cancer cell proliferation 7
1.3.3 Role of ERs in breast cancer cell migration 8
1.3.4 The role of ERs in breast cancer cell invasion 9
Chapter 2. Characterization of breast tumor development in 4T1-bearing mice 10
2.1 Introduction 10
2.1.1 Unique features of breast tumor in Asia women 10
2.1.2 Estrogen on breast tumor development in animal model 10
2.1.3 The purpose and design of this study 11
2.2 Material and methods 12
2.2.1 Reagents 12
2.2.2 Cell line and cell culture 12
2.2.3 Animal care 12
2.2.4 In vivo tumor xenograft model 13
2.2.5 In vivo imaging 13
2.3 Results 14
2.3.1 The influence of gender on the development of breast tumors 14
2.3.2 The influence of gender on the response of tumor tissues to E2 14
2.4 Summary and conclusion 14
Chapter 3. Activation of ERβ by estrogens promotes microtubule dynamics, enhances cell migration, and enlarges the ERα-tumors in mice 16
3.1 Introduction 16
3.1.1 Clinical studies of estrogen receptors in breast cancer 16
3.1.2 The role of estrogen and its receptors in breast tumor cell migration 17
3.1.3 The role of estrogen on the development of breast tumor in animal model 17
3.1.4 The purpose and designs of this study 18
3.2 Material and methods 20
3.2.1 Reagents and antibodies 20
3.2.2 Cell line and cell culture 20
3.2.3 Animal care 21
3.2.4 In vivo tumor xenograft model 21
3.2.5 In vivo imaging 21
3.2.6 Cell counting assay 22
3.2.7 Counting of cell with morphological changes and measuring the cell area 22
3.2.8 MTT assay 23
3.2.9 Migration assay 23
3.2.10 Transfection 23
3.2.11 Immunofluorescence staining 24
3.2.12 Whole cell lysate 24
3.2.13 Isolation of insoluble polymerized microtubules and soluble tubulin dimers 25
3.2.14 Western blot analysis 25
3.2.15 Data analysis and statistical evaluation 26
3.3 Results 27
3.3.1 4T1 cells express ERβ 27
3.3.2 Estrogen accelerates the development of tumor tissue at the primary site in 4T1 breast tumor-bearing mice 27
3.3.3 Activated ERβ by E2 contributes to the development of tumor tissue at primary sites but not in secondary sites in 4T1 breast tumor-bearing mice 28
3.3.4 Activated ERβ by E2 changed cell morphology only 28
3.3.5 Activated ERβ increased 4T1 breast tumor cell migration 30
3.3.6 Polymerization and depolymerization of microtubule response for E2 -increased 4T1 cell migration 31
3.4 Discussion 34
3.4.1 Summary of this study 34
3.4.2 Effect of ERβ on cell proliferation and cell migration in breast cancer cells 35
3.4.3 Relation between tubulin and aggressiveness in different breast tumor cells 36
3.4.4 Comparison of spontaneous and experimental metastasis in 4T1-bearing mice 37
Chapter 4. Significance of this study 39
Chapter 5. References 40
Chapter 6. Tables 45
Table 1. List of primary and secondary antibodies used in this study 45
Table 2. List of drugs used in this study 47
Chapter 7. Figures 49
Chapter 8. Supplementary data 68
Curriculum vita 75
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