綠茶表沒食子唲茶素沒食子酸酯受器對於人類乳癌細胞株MCF7生長的影響

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胡毓容(Yu-Jung Hu) 查詢紙本館藏

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生命科學系

論文名稱

綠茶表沒食子唲茶素沒食子酸酯受器對於人類乳癌細胞株MCF7生長的影響
(Effect of the green tea (-)-epigallocatechin-3-gallate receptor on growth of human MCF7 cells)

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摘要(中)

LAMR1是一個295胺基酸的非粘著蛋白受器 (non-integrin receptor)，且對層粘連蛋白(laminin)有很高的親合力。此蛋白質主要被發現與癌細胞的浸潤與轉移有很大的關聯。為了研究LAMR1對癌細胞生長的影響並了解參與的LAMR1特殊胺基酸區域，我以人類乳癌細胞株-MCF7為平台，建構了數種穩定型細胞株，包含全長、1-200 a.a.、1-150 a.a.、1-100 a.a.和1-55 a.a.長的LAMR1轉染的細胞株。另外為了利於偵測，也同時建構了一組在不同片段LAMR1 之C端接有Flag序列的轉染細胞株。結果發現，無論是在有Flag或是沒有Flag的組別，全長和1-200 a.a.長的LAMR1片段轉染細胞株在八天的培養期間中，其細胞生長明顯快於控制組細胞。而在其他不同片段LAMR1轉染的細胞株，其生長情形沒有明顯快於控制組細胞。根據以上的結果，我們推測LAMR1胺基酸151-200區域在MCF7生長中扮演重要的角色。另外，因為LAMR1被認為是綠茶唲茶素-表沒食子唲茶素沒食子酸酯 ((-) - epigallocatechin-3-gallate; EGCG）的受器，並且可抑制肺癌細胞的生長。因此，我們藉由不同片段LAMR1轉染的細胞株，想要瞭解LAMR1在EGCG抑制乳癌細胞株MCF7生長中扮演的角色。首先，當我們先以LAMR1抗體處理MCF7時，可防止EGCG對MCF7細胞造成的細胞數目降低。第二、我們發現到當MCF7轉染全長LAMR1後，此轉染細胞對EGCG所抑制的細胞生長與控制組細胞相比有較敏感的現象。因此我們得知全長LAMR1對於EGCG抑制MCF7生長中扮演重要的角色，對此未來可以再利用LAMR1的訊息傳遞蛋白的表現，來幫助我們更加釐清不同片段LAMR1的作用。

摘要(英)

LAMR1 is an 295 a.a. non-integrin receptor and has a high affinity for laminin. It was found to enhance the invasion and metastasis of cancer cells. To examine whether any of the specific amino acid domain of LAMR1 protein is responsible for its regulating growth of breast cancer cells, I have stably cloned several lines of MCF7 which contain the full length or four different C-terminus-truncated forms of LAMR1(the respective insert sizes of 200, 150, 100, and 55 a.a.), which were fused with or without Flag protein. MCF7 cells transfected with full length of the LAMR1 or 1-200 a.a. of LAMR1 exhibited increase in the cell numbers more rapidly over the 8-day incubation than the cells transfected with the vehicle. Other transfected MCF7 cells did not grow quickly than did the cells transfected with the vehicle. This suggests that the region of LAMR1 at position 151-200 a.a. is important to the growth of MCF7 cells. Because the LAMR1 is also a receptor for green tea (-)-epigallocatechin-3-gallate (EGCG) and involved in the EGCG inhibition of growth of human lung cancer cells, we like to know whether any of the specific amino acid domain of LAMR1 is responsible for EGCG in regulating growth of breast cancer cells. First, pretreatment of the MCF7 cells with the LAMR1 antiserum could prevent the EGCG-reduced cell numbers. Second, MCF7 cells transfected with full length of LAMR1 were more sensitive to EGCG than were other cells transfected with truncated forms of LAMR1. Results of this study appear to find a functional region of the LAMR1 mediating the effects of EGCG on growth of human breast cancer cells. Further, we can use the signal transducer of the LAMR1 to confirm the eventually function of different C-terminus-truncated forms of LAMR1.

關鍵字(中)

★ 綠茶表沒食子唲茶素沒食子酸酯
★ 綠茶表沒食子唲茶素沒食子酸酯受器
★ 乳癌
★ MCF7

關鍵字(英)

★ EGCG
★ MCF7
★ LAMR1
★ breast cancer

論文目次

中文摘要　
英文摘要
誌謝
目錄
表目錄
圖目錄
一、序論1
　 1-1 綠茶1
　 1-2 綠茶與癌症1
　 1-3 EGCG 與癌症2
1-4 EGCG 與乳癌2
　 1-5 EGCG 抑癌機制3
　 1-6 LAMR15
　 1-7 LAMR1 與癌症7
　 1-8 LAMR1 與EGCG8
　 1-9 研究動機與目的9
二、材料與方法10
　 2-1 細胞培養10
2-2 不同片段LAMR1 的設計10
2-3 質體的建構11
　 2-4 穩定型細胞株的製備13
　 2-5 西方點墨法15
　 2-6 細胞計數17
　 2-7 偵測細胞DNA 複製17
2-8 抗體與EGCG 的處理17
三、結果19
3-1 MCF7 的LAMR1 核苷酸與胺基酸序列19
3-2 質體的建構19
3-3 穩定型細胞株的篩選19
3-4 不同片段的LAMR1 對細胞生長的影響21
　 3-5 不同片段的LAMR1 對細胞DNA 複製的影響22
　 3-6 LAMR1 隨著細胞生長的蛋白質表現情形22
　 3-7 LAMR1 參與EGCG 抑制MCF7 細胞生長的機制中23　
3-8 不同片段的LAMR1 對EGCG 作用於細胞的影響24
四、討論25
　 4-1 MCF7 的LAMR1 核苷酸與胺基酸序列25
4-2 穩定型細胞株的篩選25
4-3 LAMR1 與細胞生長26
4-4 LAMR1 與EGCG 的作用27
五、　結語28
參考文獻29
附錄一54
附錄二55
附錄三56

參考文獻

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指導教授

高永旭(Yung-Hsi Kao)

審核日期

2008-7-23

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