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研究生:簡瑞涓
研究生(外文):Jui-Chuan Chien
論文名稱:北冬蟲夏草對高葡萄糖誘發人類臍帶內皮細胞氧化損傷之影響
論文名稱(外文):Effect of Cordyceps militaris on high glucose-induced oxidative damage of HUVEC
指導教授:杜平悳朱惠鈴朱惠鈴引用關係
指導教授(外文):Pin-Der DuhHeny-Ling Chu
學位類別:碩士
校院名稱:嘉南藥理科技大學
系所名稱:生物科技系暨研究所
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:2010
畢業學年度:98
語文別:中文
論文頁數:68
中文關鍵詞:北冬蟲夏草氧化壓力高血糖細胞凋亡人類臍帶內皮細胞
外文關鍵詞:Cordyceps militarisoxidative stressapoptosishyperglycemiaHUVEC
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中文摘要
高血糖誘發氧化壓力會導致內皮細胞功能不全,進而引起糖尿病血管併發症,本研究擬以北冬蟲夏草(Cordyceps militaris;CME)萃取物為材料並與冬蟲夏草(Cordyceps sinensis;CSE)作比較,探討其高濃度葡萄糖對造成內皮細胞氧化傷害之影響。於試管實驗中我們發現北冬蟲夏草對化學性誘導產生的蛋白質氧化傷害具保護性,於500 ?慊/ml時具有65% 抑制蛋白質羰基化作用。在人類臍帶內皮細胞(HUVEC)細胞模式中,北冬蟲夏草對高血糖誘發氧化壓力具保護性,其作用機制可能與降低ROS 及 NO 的產生率,減少 eNOS 、磷酸化 eNOS 及 Bax 蛋白表現量,增加抗凋亡之蛋白 Bcl-2 表現量,調整粒線體膜電位以及降低 caspase 3 活性有關,綜合以上述實驗的結果,推測北冬蟲夏草對高血糖誘發細胞傷害之保護性可能與其維持細胞內正常氧化還原系統有關。


關鍵字:人類臍帶內皮細胞、北冬蟲夏草、氧化壓力、高血糖、細胞凋亡
Abstract
  Hyperglycemia-induced oxidative stress is detrimental to endothelial cells, contributing to the vascular complications of diabetes. The present study was designed to investigate the effect of water extract of Cordyceps militaris (CME) on high glucose induced oxidative damage in human umbilical endothelial cells (HUVEC). CME at 500 ?慊/ml showed 68.5% inhibition of protein carbonylation. In HUVEC intracellular model system, the mechanism of protection against cells death was achieved mainly by decreasing ROS and NO generation, reducing eNOS and p-eNOS expression levels, up regulating Bcl-2 activation and down regulating of Bax, modulating mitochondria membrane potential as well as suppressing caspase-3 activation. On the basis of the results obtained, the protective actions of CME on hyperglycemia-induced cytotoxicity may be related to their maintenance of the normal redox status of cells.


Keywords:HUVEC; Cordyceps militaris; oxidative stress; hyperglycemia;
apoptosis
目錄
中文摘要 ……………………………………………………………………Ⅰ
Abstract ………………………………………………………………………Ⅱ
目錄 …………………………………………………………………………Ⅲ
縮寫表 ………………………………………………………………………Ⅶ
第一章 緒論 ………………………………………………………………?
1.1 糖尿病 …………………………………………………………………?
1.1.1糖尿病介紹 …………………………………………………………?
1.1.2 糖尿病診斷標準 ………………………………………………………2
1.1.3 糖尿病分類 ………………………………………………………2
1.2 高葡萄糖與氧化壓力 …………………………………………………?
1.2.1 高葡萄糖之氧化壓力對葡萄糖代謝之影響 ………………………3
1.3 糖尿病引起心血管疾病之病理機制 …………………………………8
1.3.1 高葡萄糖引起細胞傷害……………………………………………9
1.3.2 自由基(Free radical)之簡介………………………………………9
1.3.3 活性氧物種(reactive oxygen spies , ROS)………………………10
1.3.4 活性氮屬……………………………………………………………… 12
1.3.5 NO、eNOS 調控機制……………………………………………………13
1.3.6 高葡萄糖對蛋白質之氧化損傷 ………………………………………14
1.4 高葡萄糖對內皮細胞凋亡相關訊息 …………………………………15
1.4.1 人類臍帶靜脈內皮細胞(HUVEC) …………………………………15
1.4.2 高葡萄糖對內皮細胞凋亡 ……………………………………………15
1.4.3 細胞凋亡(Apoptosis)與相關訊息傳遞路徑…………………………16
1.5 冬蟲夏草(Cordyceps sinensis)及北冬蟲夏草(Cordyceps militaris)簡介??
1.5.1 冬蟲夏草(Cordyceps sinensis) ………………………………………18
1.5.2 北冬蟲夏草(Cordyceps militaris)……………………………………19
1.5.3 冬蟲夏草及北冬蟲夏草之化學成分 …………………………………19
研究動機 …………………………………………………………………21
第二章 材料方法 ………………………………………………………23
2.1 樣品 ……………………………………………………………………23
2.2 藥品 ……………………………………………………………………23
2.3 儀器設備 ………………………………………………………………24
2.4 細胞株 …………………………………………………………………24
2.5 蛋白質的羰基化(Carboxylation) ……………………………………25
2.6人類臍帶靜脈內皮細胞之細胞培養條件、繼代培養 ………………26
2.7 細胞存活率試驗 (MTT Assay) ………………………………………??
2.8 胞內活性氧 (ROS) 分析 ……………………………………………27
2.9 一氧化氮測定(NO Production test)…………………………………??
2.10 粒線體膜電位 (ΔΨ) 測定 …………………………………………29
2.11 Caspase-3活性分析 …………………………………………………31
2.12 西方墨點法 ( Western blot )  ………………………………………31
2.13 統計分析 ………………………………………………………………31
第三章 結果 ………………………………………………………………35
3.1 CSE 及 CME對於HUVEC 細胞之細胞毒性測試 ………35
3.2 CSE 及 CME對蛋白質羰基化 ( protein Carboxylation) 之保護…36
3.3 CSE 及 CME對葡萄糖下HUVEC 細胞之細胞存活能力測試 …36
3.4 CSE 及 CME對葡萄糖下HUVEC 細胞之活性氧(ROS)影響…38
3.5 CSE 及 CME 對葡萄糖下 HUVEC 細胞之清除一氧化氮能力影響39
3.6 CSE 及 CME對葡萄糖下HUVEC 細胞粒線體 eNOS 與 p- eNOS
蛋白表現之影響 ………………………………………………………39
3.7 CSE 及 CME 對葡萄糖 HUVEC 細胞之粒線體膜電位(ΔΨm)40
3.8 CSE 及 CME 對葡萄糖下HUVEC 細胞粒線體 Bcl-2 與 Bax蛋白
表現之影響……………………………………………………41
3.9 CSE 及 CME對葡萄糖下HUVEC 細胞之凋亡(Caspase-3)活性分析42
第四章 討論 ………………………………………………………………44
第五章 結論 ………………………………………………………………51
參考文獻 ……………………………………………………………………52
附表與附圖 …………………………………………………………………60
圖1. CSE及 CME 對HUVEC 細胞存活能力之影響 …………………60
圖2. CSE 及 CME對蛋白質的羰基化(Carboxylation)之保護率 ………61
圖3. CSE及CME對葡萄糖誘發內皮細胞存活率之影響 ……………62
圖4. CSE及CME對葡萄糖誘發內皮細胞之活性氧(ROS)之影響 63
圖5. CSE及CME對葡萄糖誘發內皮細胞之NO影響 ………………64
圖6. CSE及CME對葡萄糖誘發內皮細胞e-NOS與p-eNOS生成影響65
圖7. CSE 及CME 對葡萄糖誘發內皮細胞之膜電位影響 ……………66
圖8. CSE 及 CME對葡萄糖下HUVEC 細胞粒線體 Bcl-2 與 Bax蛋白
表現之影響………………………………………………………67
圖9. CSE及CME 對葡萄糖誘發內皮細胞之Caspase-3 活性評估 ……68
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