博碩士論文 104821012 詳細資訊




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姓名 鍾佳芳(CHUNG,JIA-FANG)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 綠茶表兒茶素藉由microRNA-494路徑改善橫向主動脈繃紮術誘導型小鼠的心臟疾病
(Green Tea Epicatechin Improved Transverse Aortic Banding-Induced Heart Disease in Mice via the MicroRNA-494 Pathway)
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摘要(中) 心血管疾病 (Cardiovascular diseases, CVDs) 是全球第一死因。根據世界衛生組織統計,西元2016年總共有1770萬人死於CVDs。這反映了除人體健康之外,CVDs對全球死亡率的重大影響。雖然CVDs的病程發展分別被microRNA-494 (miR-494) 與表兒茶素 (Epicatechin, EC) 調控,但EC是否在心血管疾病中調控miR-494的機制還不是很清楚。本論文使用橫向主動脈繃紮術 (Transverse aortic banding, TAB) 以及H9c2大鼠心肌細胞探討EC是否透過miR-494的路徑來改善心肌肥大以及纖維化的現象。我們確認小鼠在TAB後2周和12周造成心臟功能異常,包括增加血流速、血壓、收縮與舒張時的心室中隔厚度和收縮與舒張時的左心室後壁厚度,而在12周時心臟左心室短縮分率 (Left ventricular fractional shortening, LVFS) 以及左心室射出率 (Left ventricular ejection fraction, LVEF) 顯著下降。在TAB後12周的數據中觀察到會增加心臟重量、纖維化現象,而在TAB後2周和12周時,數據指出會增加心肌肥大的生物標誌因子,如β-MHC、ANP與BNP並下降α-MHC,另外纖維化的生物標誌因子,如Col1α2和Col3α1也在TAB後2周及12周上升。此外,TAB後3、7、14和21天會增加小鼠心臟miR-494的RNA表現量,但在1天時並不會被影響,而miR-494抑制基因ATF3會在1天顯著上升,而後3~21天掉回基準點。處理0.25毫克/公斤/天的EC可以回復TAB後12周所下降的LVFS以及LVEF。另外,EC會抑制心臟中β-MHC、ANP和miR-494的RNA表現量,而且也改善TAB所導致的心臟纖維化,可能是透過抑制2周心臟中Col1α2和Col3α1表現量,而Col3α1到12周仍持續抑制,但心臟重量沒顯著差異。同時,在TAB 12周後的miR-494基因轉殖鼠,其心臟纖維化現象略高而LVFS與LVEF顯著差於野生小鼠,但心臟重量並無差異。此外,TAB 12周後的miR-494基因轉殖鼠處理EC 1毫克/隻/兩天,可以提高LVFS以及LVEF的百分比。在H9c2心肌細胞中我們預處理5 μM的EC會抑制Angiotensin-II所誘導增加的miR-494 RNA表現量。根據體內以及體外實驗數據,我們得知EC會透過抑制miR-494表現量來改善TAB誘導的心臟病,而這個論點也讓我們瞭解食用含有EC的綠茶如何保護人類避免心血管疾病的風險。
摘要(英) Cardiovascular diseases (CVDs) are the first leading cause of death globally. According to the statistics by the World Health Organization, an estimated 17.7 million people died from CVDs in 2016. This reflects the big impacts of CVDs on global mortality rate besides human health. Although development of CVDs can be respectively regulated by microRNA-494 (miR-494) and green tea (-)-epicatechin (EC), it is still unknown whether EC interacts with miR-494 on CVDs. The present thesis was designed to use transverse aortic banding (TAB)-induced mice and H9c2 rat cardiocytes and investigate whether EC improved cardiac hypertrophy and fibrosis through the miR-494 pathway. We confirmed that mice with TAB for 2 and 12 weeks caused cardiac dysfunctions, including increased blood flow velocity, blood pressure, IVSd, IVSs, LVPWd, and LVPWs, while cardiac LVFS and LVEF were decreased at 12 weeks. At 12 weeks, TAB also induced increases in heart weight, fibrosis. At 2 and 12 weeks, TAB induced increases cardiac hypertrophic marker genes, such as β-MHC, ANP, and BNP, but decrease α-MHC. Besides, fibrosis marker genes, such as Col1α2 and Col3α1, induced at 2 and 12 weeks. In addition, TAB induced an increase of miR-494 RNA expression in mice heart after 3, 7, 14, and 21 days but not 1 day of treatment, while the miR-494-downregulated gene ATF3 was increased with mRNA levels at day 1 and then significantly declined to the basal levels at day 3 until day 21. Treatment with EC (0.25 mg/kg/d) antagonized 12-week-TAB-induced decreases in cardiac LVFS and LVEF. Also, EC suppressed TAB-induced increases in levels of β-MHC, ANP and miR-494 RNA levels in the heart. Moreover, EC improved TAB-induced cardiac fibrosis, maybe via inhibited Col1α2 and Col3α1 at 2 weeks; in addition, Col3α1 held down at 12 weeks. Interestingly, EC had no effect on heart weight in TAB-treated mice. In parallel, miR-494-transgenic mice with TAB for 12 weeks exhibited slightly greater level of cardiac fibrosis and significantly less percentages of LVFS and LVEF compared with non-transgenic mice with TAB. But, no significant changes were observed in the heart weight. Moreover, treatment with 1 mg EC/mice/2d improved the percentages of LVFS and LVEF in miR-494-transgenic mice with 12-week TAB. Using H9c2 cardiocytes, we found that pretreatment with 5 μM of EC blocked angiotensin-II-induced increases of miR-494 RNA levels. According to these in vivo and in vitro findings, we conclude that EC improves TAB-induced heart disease through inhibition of miR-494 expression. Results of this thesis also led us to understand how consumption of EC-containing green tea protects humans against the risk of CVDs.
關鍵字(中) ★ 綠茶表兒茶素
★ 微小核醣核酸-494
★ 橫向主動脈繃紮術
★ 心臟疾病
關鍵字(英) ★ Green Tea Epicatechin
★ microRNA-494
★ Transverse Aortic Banding
★ Heart Disease
論文目次 中文摘要 i
Abstract ii
致謝 iv
目錄 v
縮寫對照表 viii
壹、 前言 1
1-1心肌肥大 1
1-1-1心肌肥大的風險因子 1
1-1-2心肌肥大的症狀與影響 1
1-1-3心肌肥大的診斷治療 2
1-1-4心肌肥大的細胞傳訊路徑 2
1-1-5心肌肥大的生物標誌 3
1-2心肌肥大與微小核醣核酸 3
1-2-1微小核醣核酸 4
1-2-2微小核醣核酸與心肌肥大 4
1-3茶與心血管疾病 5
1-3-1茶葉成分與表兒茶素的重要性 5
1-3-2表兒茶素的穩定性 6
1-3-3表兒茶素吸收與代謝 6
1-3-4表兒茶素與心肌肥大的訊息傳遞路徑 6
1-4研究目的與動機 7
貳、 材料方法 8
2-1實驗動物 8
2-2細胞培養 8
2-3心肌肥大的模式 9
2-3-1體內模式 (In vivo model) 9
2-3-2體外模式 (In vitro model) 9
2-4 EC給予之體內體外方式 9
2-4-1體內模式 (In vivo model) 10
2-4-2體外模式 (In vitro model) 10
2-5 miR-494基因轉殖鼠 (miR-494 transgenic mice) 10
2-6即時聚合酶鏈鎖反應 (Real-time polymerase chain reaction) 11
2-7心臟超音波分析 (Echocardiography) 11
2-8動物犧牲 (Sarcrifice) 12
2-9次世代定序 (Next Generation Sequencing) 12
2-10組織病理切片及染色 (Histology) 12
2-11 miR-494基因剔除鼠 (miR-494 knockout mice) 14
2-12統計方式 14
參、 實驗結果 15
3-1橫向主動脈繃紮術所引起的心肌肥大降低小鼠心臟功能 15
3-2橫向主動脈繃紮術所引起的心肌肥大造成小鼠心臟纖維化 16
3-3橫向主動脈繃紮術所引起的心肌肥大增加miR-494基因表現量 17
3-4 Epichatechin改善橫向主動脈繃紮術所引起的心肌肥大小鼠心臟功能 17
3-5 Epichatechin改善橫向主動脈繃紮術所引起的心肌肥大小鼠病理現象 18
3-6 Epichatechin在心肌肥大小鼠中抑制miR-494基因表現量 19
3-7用NGS分析Epichatechin在心肌肥大小鼠中可能影響的傳訊路徑 19
3-8 Epichatechin在Angiotensin II誘導心肌細胞肥大中抑制miR-494表現量 21
3-9建立miR-494基因轉殖鼠 21
3-10在橫向主動脈繃紮術後miR-494基因轉殖鼠心臟功能低於正常小鼠 22
3-11在橫向主動脈繃紮術後miR-494基因轉殖鼠病理現象 23
3-12建立miR-494基因剔除鼠 23
肆、 討論 24
伍、 結論 28
陸、 參考文獻 29
柒、 表目錄 37
捌、 圖目錄 44
附錄一 83
附錄二 84
附錄三 86
附錄四 88
附錄五 90
附錄六 92

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指導教授 高永旭 林恆(Yung-Hsi Kao Heng Lin) 審核日期 2018-3-20
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