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研究生:周玉萍
研究生(外文):Yu-Ping Chou
論文名稱:EGCG與Theaflavin影響人類肺癌細胞(A549、H838)中貼附蛋白之表現量
論文名稱(外文):EGCG and Theaflavin affect the expression of membrane adhesion protein in the human lung cancer cells(A549, H838)
指導教授:范瑞珍范瑞珍引用關係
指導教授(外文):Ruey-Jane Fan
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:44
中文關鍵詞:兒茶素茶黃質貼附蛋白細胞外間質細胞遷移即時定量PCR細胞毒性測試
外文關鍵詞:EGCGTheaflavinadhesion proteinE-cadherinintegrin αEβ7extracellular matrixECMcell migrationReal-time PCRMTT assay
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茶葉是世界上最普遍的飲料,研究指出綠茶中的兒茶素 epigallocatechin gallate (EGCG)與紅茶中的茶黃質Theaflavin(TF)可以抑制癌細胞的增生、貼附及遷移並且可使癌細胞進行凋亡。由於綠茶中的EGCG與紅茶中的TF是否與癌細胞有直接的接觸而產生作用仍然未知,為進一步了解探討此問題,在進行細胞毒性測試(MTT assay)之前藥物以用兩種不同的方式與細胞作用:(1)細胞懸浮時(cell suspension)加藥及(2)細胞貼附(cell adhesion)後加藥。經由實驗發現,在細胞懸浮時以EGCG和TF處理,隨著時間及濃度的增加可以顯著性的抑制細胞的存活率。然而,細胞貼附後再加藥,其存活率沒有顯著性的改變。因此,判斷EGCG和TF是間接作用在細胞外間質(extracellular matrix, ECM),可能會影響某些貼附蛋白(adhesion proteins)。再經由migration assay於細胞貼附後經藥劑作用,得知EGCG及TF對於人類肺癌細胞A549及H838遷移有抑制的效果。因此推測EGCG及TF除了對細胞外間質有間接作用外,對於細胞與細胞間的貼附蛋白也有影響,且TF抑制癌細胞增生以及遷移能力比EGCG好。而針對與細胞遷移能力有關的貼附蛋白,經由Real-time PCR進一步探討癌細胞中貼附蛋白之基因表現量,經EGCG或TF處理後得到兩種結果:TF可增加肺癌細胞中E-cadherin之基因表現,而與E-cadherin相結合的integrin αEβ7基因經TF的作用下也同樣增加,而經EGCG處理後只有增加E-cadherin之基因表現。因此推論TF可經由增加E-cadherin及integrin αEβ7基因的表現量,來增進癌細胞間的貼附,降低癌細胞轉移的機會。未來,將以western blot的方式針對貼附蛋白E-cadherin及integrin αEβ7做進一步的探討,再以immunostaining方式,運用特定蛋白抗體標定,觀察蛋白在細胞內的變化。
Tea is the most popular drink in the world, previous studies show that epigallocatechin gallate (EGCG) and theaflavin(TF) inhibit cell proliferation, adhesion, migration and induce apoptosis of cancer cells. However, the mechanisms underlying the effect of EGCG or theaflavin in inhibiting the growth of cancer cells are still unknown. Therefore we investigated in view of this question. At first, we established two human lung cancer cell lines: A549 and H838. Then, we applied either EGCG or TF into the cell culture medium at either (1) cell suspension treatment with agent, or (2) after cell adhesion on the dish treatment with agent, and determine cell viability by MTT assay.
We observed the cell suspension treatment with EGCG and TF exhibited time and dose-dependent inhibitory effect. However, treatment with agent after cell adhesion, the cell viability has no significant effect. Therefore, we suggested that EGCG and TF may indirect interact with the extra cellular matrix (ECM) proteins, which possibly can reduce expression of adhesion proteins. The results of the migration assay suggest that EGCG and TF may indirect interact with the extra cellular matrix (ECM) and interact with cell-cell adhesion protein. Moreover, TF could inhibit cell growth and migration of cancer cell more than EGCG did. Finally, we investigated which gene expression of cell migration adhesion proteins were affected by the treatments of EGCG and TF. We used real-time PCR to study the gene expression at mRNA levels. Our results showed that EGCG and TF affected different ECM protein gene expression. After treating with TF, the lung cancer cells increased the expression of E-cadherin, and Integrin αEβ7, while after treating with EGCG, the cancer cells only induced the gene expression of E-cadherin. The result implies that TF could increase gene expression of E-cadherin and Integrin αEβ7, which could enhance cell-cell adhesion and subsequently reduce cancer cell metastasis. Wstern blots and immunostaining are necessary to further confirm the observations.
第一章 前言
一、茶的分類及成分-------------------------------- 1
二、茶的生理功效---------------------------------- 1
三、茶的抗癌機制---------------------------------- 1
四、茶與細胞的作用層面介紹--------------- 2
五、茶抑制癌細胞轉移(metastasis)現象---- 4
六、研究動機--------------------------------------5

第二章 材料與方法
一、細胞培養--------------------------------------------- 6
二、實驗藥物--------------------------------------------- 8
三、實驗設計--------------------------------------------- 8
四、細胞活性測試─MTT assay-------------------8
五、Wound closure assay(Migration assay)---9
六、基因表現量之分析------------------------------ 10
七、統計分析---------------------------------------------13

第三章 結果
一、綠茶中的兒茶素EGCG對於人類肺癌細胞(A549、H838)毒性之測試------14
二、紅茶中的茶黃質TF對於人類肺癌細胞(A549、H838)毒性之測試-------- 15
三、EGCG與TF對於人類肺癌細胞(A549、H838)遷移速率之影響---16
四、EGCG與TF影響細胞與細胞間貼附蛋白之基因表現量---------17
五、 EGCG與TF影響細胞與細胞間隙蛋白之基因表現量----- 19

第四章 討論------------------------------------------ 21
圖表---------------------------------------------------- 25
附錄---------------------------------------------------- 41
參考文獻--------------------------------------------- 42
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