臺灣博碩士論文加值系統

自噬是一種保護細胞免受壓力或飢餓狀況的細胞過程。該過程需要許多分子以高度有組織的方式相互作用以幫助確定細胞存活或死亡。綠茶唲茶素，尤其是EGCG具有抗氧化的作用，充當抗氧化劑和脂肪細胞活性的調節劑。然而，關於EGCG對脂肪細胞自噬的影響知之甚少。為了充分了解EGCG是否通過調節自噬調節脂肪細胞生長，研究了EGCG在自噬中的作用。發現EGCG調節特定類型的自噬標誌物，例如Beclin-1，ATG3，ATG5，ATG7，ATG12-ATG5綴合的ATG16L1蛋白，以及晚期自噬標誌物（p62和LC3B-II）。此外，6小時的3-MA傾向於通過ERK非依賴性途徑誘導自噬標誌物。此外，發現CQ在3和24小時通過不同的治療持續時間抑制自噬標記物（Beclin-1，ATG3，ATG5，ATG7，ATG12-ATG5偶聯，ATG16L1），然後積累晚期標誌物（p62和LC3B-II）。簡而言之，EGCG顯示出與CQ相似的調節自噬標誌物的作用。此外，EGCG時間和劑量依賴性地抑制3T3-L1前脂肪細胞生長。在3-MA（5mM）或CQ（20μM）存在下，它還抑制3T3-L1前脂肪細胞生長。這些發現表明EGCG可能通過調節自噬信號蛋白髮揮其抗生長作用。

Autophagy is a cellular process that protects the cell from stressful or starvation condition. This process requires a number of molecules that interact in a highly organized manner to help determine cell survival or death. Most polyphenols in green tea catechins, especially EGCG (epigallocatechin-3-gallate), act as an antioxidant and a regulator of fat cell activity. However, little information is known about the effect of EGCG on autophagy of fat cell. To fully understand whether EGCG regulates fat cell growth through regulation of autophagy, the role of EGCG in autophagy was investigated. EGCG was found to regulate a particular type of autophagy markers, such as Beclin-1, ATG3, ATG5, ATG7, ATG12-ATG5 conjugated, ATG16L1 proteins, as well as late autophagy markers (p62 and LC3B-II). In addition, 3-MA at 6 h tended to induce autophagy markers through ERK-independent pathway. Moreover, CQ was found to inhibit autophagy markers (Beclin-1, ATG3, ATG5, ATG7, ATG12-ATG5 conjugated, and ATG16L1) through different duration of treatment at 3 and 24 h, and then accumulated late phase markers (p62 and LC3B-II). Briefly, EGCG showed similar effect with CQ to modulate autophagy markers. Furthermore, EGCG time- and dose-dependently suppressed 3T3-L1 preadipocyte cell growth. Either in the presence of 3-MA (5 mM) or CQ (20 μM), it also suppressed 3T3-L1 preadipocyte cell growth. These findings indicate that EGCG might exert its anti-growth action through modulation of autophagy signaling protein.

Abstract………………………………………………………………………………… i
中文摘要……………………………………………………………………………….. ii
Acknowledgement……………………………………………………………………... iii
Table of Contents……………………………………………………………………..... iv
List of Figures…………………………………………………………………….......... vi
List of Tables……………………………………………………………………........... vii
Abbreviations………………………………………………………………….............. viii
Chapter 1 Introduction…………………………………………………………............ 1
1.1 Autophagy……………………………………………………………….............. 1
1.1.1. Origin and Discovery……………………………………………….............. 1
1.1.2. Autophagy functions………………………………………………............... 1
1.1.3. Mechanism of autophagy and its marker proteins………………................... 2
1.2 Green tea EGCG and its catechins…………………………………….............. 3
1.3 Green tea EGCG and autophagy……………………………………................. 3
1.4 Green tea EGCG regulates autophagy marker proteins in non-fat cells.............. 4
1.5 Autophagy and adipocyte and obesity…………………………….................... 5
1.6 Research motives and purposes…………………………………….................. 5
Chapter 2 Materials and Methods……………………………………………............... 7
2.1 Experimental materials………………………………………………................... 7
2.2 Cell culture…………………………………………………………..................... 7
2.3 Trypan blue exclusion method ……………………………………….................. 7
2.4 Western blot analysis……………………………………………......................... 8
2.4.1 Gel production……………………………………………………....................... 8

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