綠茶表沒食子兒茶素沒食子酸酯調節A549肺癌和HepG2肝癌細胞中ACE2和TMPRSS2蛋白的表達

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張立樺(Li-Hua Chang) 查詢紙本館藏

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論文名稱

綠茶表沒食子兒茶素沒食子酸酯調節A549肺癌和HepG2肝癌細胞中ACE2和TMPRSS2蛋白的表達
(EGCG regulates the expression of ACE2 and TMPRSS2 proteins in A549 lung and HepG2 liver cancer cells)

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至系統瀏覽論文 (2029-2-1以後開放)

摘要(中)

自從 COVID-19 爆發以來，全球已有超過 7 億多人感染，逾 600 萬人生命不幸喪
失。儘管疫苗和口服藥物已經問世，但仍然存在許多的後遺症，其持續時間因人而異，
從數週至數年不等。為尋找 COVID-19 的替代草藥或營養療法，先前的研究發現，綠茶
中的表兒茶素-3-没食子兒茶素酸酯(EGCG)在體內和體外均能有效抑制 SARS-CoV-2 感
染，並抑制 SARS-CoV-2 刺突蛋白與血管收縮素轉化酶 2(ACE-2)在無細胞系統中的結
合。在這項研究中，我們使用 A549 肺癌細胞和 HepG2 肝癌細胞作為細胞系統，探究
EGCG 是否調節 ACE2 和第 II 型跨膜絲胺酸蛋白酶(TMPRSS2)的蛋白表達和轉位。在
A549 細胞中，EGCG 在 10-80 μM 的濃度下，處理後 24 小時傾向於增加 ACE2 和
TMPRSS2 蛋白表達，但在 48 小時處理後降低了它們的蛋白表達。在 HepG2 細胞中，
EGCG 在 24 小時處理下對 ACE2 和 TMPRSS2 蛋白表達產生了類似的影響，但在 48 小
時處理下未觀察到類似情況。當在 24 小時內檢測了兩種蛋白的轉位時，發現處理或不
處理 EGCG 的 A549 細胞膜(PM)部分未檢測到 ACE2，但在 EGCG 處理 24 小時後，其
在高密度微粒體(HDM)部分增加，在低密度(LDM)部分降低。然而，EGCG 被發現劑量
依賴地增加所有部分的 TMPRSS2 蛋白表達。有趣的是，在 48 小時處理下，80 μM 的
EGCG 傾向於增加所有部分的 ACE2 蛋白表達，但未改變 TMPRSS2 蛋白表達。在 HepG2
細胞中，EGCG 在 20 和 40 μM (80 μM 除外)的濃度下，在 24 小時處理後傾向於增加 PM
和 HDM 部分的 ACE2 和 TMPRSS2 蛋白表達。在 48 小時處理後，EGCG 傾向於增加
PM 中的 ACE2 而不是 TMPRSS2 蛋白表達，並在 HDM 和 LDM 部分不改變 ACE2 但增
加 TMPRSS2 的表達。這些結果表明，EGCG 調節 ACE2 和 TMPRSS2 蛋白的表達及其
從細胞質到細胞膜的轉位在細胞類型、處理劑量和處理時間上存在差異。這些發現可能
為 EGCG 對抗 COVID-19 感染以及進入人體肺細胞和肝細胞提供了細胞基礎。

摘要(英)

Since the outbreak of COVID-19, over 700 million people worldwide have been infected,
with more than 6 million lives lost. Despite the availability of vaccines and some oral
medications, there are still many post-infection sequelae, which vary in duration from several
weeks to several years depending on the individual. To find an alternative herbal or nutritional
therapy for COVID-19, previous studies have found that green tea epigallocatechin-3-gallate
(EGCG) can effectively inhibit SARS-CoV-2 infection both in vivo and in vitro, and it
suppresses the binding of SARS-CoV-2 spike protein with angiotensin-converting enzyme
(ACE)-2 in the cell-free system. In this study using A549 lung and HepG2 liver cancer cells as
the cell-based systems, we investigated whether EGCG regulates the protein expression and
translocation of ACE2 and type II transmembrane serine protease (TMPRSS2). In A549 cells,
EGCG at 10-80 μM tended to increase ACE2 and TMPRSS2 protein levels after 24 h of
treatment, but it decreased their protein levels at 48 h. Similar effects of EGCG on ACE2 and
TMPRSS2 protein expressions in HepG2 cells were observed at 24 h but not at 48 h. When the
translocation of two proteins were examined at 24 h, the ACE2 was not detected in the plasma
membrane (PM) fraction of A549 cells treated with or without EGCG, but it was increased in
the high-density microsome (HDM) fraction and decreased in the low-density (LDM) fraction
after 24 h of EGCG treatment. However, EGCG was found to dose-dependently increase
TMPRSS2 protein levels in all the fractions. Interestingly, EGCG at 80 μM for 48 h tended to
increase ACE2 protein expression in all fractions but unaltered TMPRSS2 protein levels. In
HepG2 cells, EGCG at 20 and 40 μM but not 80 μM tended to increase ACE2 and TMPRSS2
protein levels in PM and HDM fractions after 24 h of treatment. At 48 h, EGCG tended to
increase ACE2 but not TMPRSS2 proteins in PM, while it unaltered ACE2 proteins and
increased TMPRSS2 in HDM and LDM fractions. These results suggest that EGCG
modulations of ACE2 and TMPRSS2 protein expressions and their translocation from cytosol
to plasma membrane vary with cell types, dose of treatment, and duration of treatment. These
findings may provide the cellular basis for the action of EGCG against the COVID-19 infection
and entry to human lung cells and liver cells.

關鍵字(中)

★ 綠茶兒茶素

關鍵字(英)

論文目次

摘要 I
ABSTRACT II
致謝 IV
目錄 IV
表目錄 VIII
圖目錄 VIII
英文縮寫對照表 X
壹、緒論 1
1-1 ACE2 1
1-1-1介紹 3
1-1-2調控 3
1-1-3異構體(isoform)及修飾 2
1-2 TMPRSS2 2
1-2-1 介紹 2
1-3 Furin 2
1-3-1 介紹 2
1-4 COVID-19 3
1-4-1介紹 3
1-4-2冠狀病毒 3
1-4-3傳播及感染細胞途徑 3
1-4-1病毒複製 4
1-5 茶多酚 4
1-3-1 茶與EGCG 4
1-6 研究動機 5

貳、材料與方法 6
2-1實驗材料(Experimental materials) 6
2-2細胞培養(Cell culture) 6
2-3西方墨點法(Western blot) 7
2-4 次細胞分群(Subcellular fractionation) 9
2-5 統計(Statistics) 10
參、結果 11
3-1 ACE2蛋白質在不同細胞株表達量的差異 11
3-2 EGCG對A549細胞ACE2表達的影響 11
3-3 EGCG對A549細胞TMPRSS2表達的影響 11
3-4 EGCG對HepG2細胞ACE2表達的影響 12
3-5 EGCG對HepG2細胞TMPRSS2表達的影響 12
3-6 LPS對A549以及HepG2細胞ACE2/TMPRSS2表達的影響 12
3-7 EGCG對A549細胞Furin表達的影響 13
3-8 EGCG對HepG2細胞Furin表達的影響 13
3-9 EGCG對A549細胞ACE2在細胞內分佈的影響 13
3-10 EGCG對A549細胞TMPRSS2在細胞內分佈的影響 14
3-11 EGCG對A549細胞Furin在細胞內分佈的影響 14
3-12 EGCG對HepG2細胞ACE2在細胞內分佈的影響 15
3-13 EGCG對HepG2細胞TMPRSS2在細胞內分佈的影響 15
3-14 EGCG對HepG2細胞Furin在細胞內分佈的影響 16
3-15時間對人類肺癌細胞A549中對ACE2/TMPRSS2/Furin在細胞內分布的影響 16
3-16時間對人類肺癌細胞HepG2中對ACE2/TMPRSS2/Furin在細胞內分布的影響 16

肆、討論 17
伍、結論 19
陸、參考文獻 20
柒、附錄 40

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指導教授

高永旭(Yung-Hsi Kao)

審核日期

2024-1-31

推文