臺灣博碩士論文加值系統

糖尿病是心血管疾病風險的主要原因之一。所有糖尿病類型都以慢性高血糖為特徵。我們知道高血糖會誘導ROS的產生，導致內皮細胞功能失調或受損，這也是動脈粥樣硬化的開端。因此，到目前為止，發明或發現有效的抗氧化物來救治誘發性高血糖內皮細胞受損是個重要的議題。
近年來一種傳統的中藥草三黃瀉心湯，受到相當高度的注意，體內或體外研究被認為是有抗氧化和抗發炎的效果。因此我們希望進一步找尋三黃瀉心湯代謝物，是否可以被用來保護內皮細胞受損。內皮細胞經由高葡萄糖(33mM)刺激，所造成的細胞發炎貼附及細胞凋亡反應；因此希望進一步研究在高葡萄糖(33mM)的刺激內皮細胞後，加入三黃瀉心湯代謝物，對於抑制發炎來探討。
在本研究中我們使用以下列方法，細胞貼附(Cell adhesion)、酵素鏈結合免疫吸附法(ELISA)、明膠分解酶測定 ( Gelatin zymography )、西方墨點法(Western Blot)來驗證我們的假設。高葡萄糖取33mM (594mg/dl)和16mM (288mg/dl)來對內皮細胞刺激。三黃瀉心湯 (SHXXT) 代謝物，從低濃度到高濃度劑量0.5~100μl/ml先進行細胞存活率試驗，發現SHXXT劑量不需太高；進一步取SHXXT 5~50μl/ml進行細胞貼附性試驗，SHXXT 50μl/ml會造成細胞毒性；取低劑量0.5~2.5μl/ml進一步測試細胞貼附性試驗，發現三黃瀉心湯 (SHXXT) 代謝物，低劑量濃度足以達到抗發炎效果。
由實驗分析結果，高葡萄糖(33mM)誘發內皮細胞受損，短時間48小時內，存活率並無明顯變化；但在細胞貼附24小時，已有顯注發炎反應，再加入三黃瀉心湯代謝物低劑量後，證明三黃瀉心湯代謝物確實有很強的抗發炎和抗氧化反應；並且單獨使用三黃瀉心湯代謝物，對待內皮細胞，證實對內皮細胞無毒性。
過去已證實維他命C是很好的抗氧物。三黃瀉心湯與維他命C比較，三黃瀉心湯具有更好的抗發炎。在Gelatin zymography、western blot分析高葡萄糖(33mM)誘發動脈硬化因子，及eNOS、COX-2、ICAM-1表現，再分別加入三黃瀉心湯與維他命C比較：明顯發現在Gelatin zymography部分，評估動脈硬化，加入三黃瀉心湯後，有改善效果。eNOS部分，加入三黃瀉心湯確實具有內皮細胞保護效果。COX-2會造成內皮細胞發炎，但加入三黃瀉心湯後，確實具有改善的效果。發炎反應，會造成白血球貼附內皮細胞性增加，在ICAM-1表現上加入三黃瀉心湯比維他命C，更具有降低ICAM-1的表現，由我們的實驗證明，高葡萄糖(33mM)誘發內皮細胞受損，三黃瀉心湯代謝物確實具有很強的抗發炎和抗氧化效果。

The diabete is one of the major risk factors of cardiovascular disease. All forms of diabetes are characterized by chronic hyperglycemia. We know hyperglycemia induce reactive oxygen species (ROS) production and cause endothelial cells dysfunction or injury which initiates atherosclerotic inflammatory processing reaction. Therefore, till now, creating or discovering antioxidant items with benefits to rescue hyperglycemia-induced endothelial cell injury may be an important issue.
Arising considerable attention in recent years, San-Huang-Xie-Xin-Tang (SHXXT), one kind of traditional herbs, was identified with anti-oxidant and anti-inflammatory effects in vitro and in vivo studies. Therefore, we hoped to further investigate its metabolite to find out whether SHXXT (0.5-5μl/ml) can be used to protect endothelial cells injury, such as cell adhesion, cell apoptosis, under stimulation of high glucose (16mM, 33mM). In this study, we used the following methods：Primary cell culture HUVECs, complex drug SHXXT (contain Rhei Rhizoma、Scutellaria Radix、Coptidis Rhizoma), ELISA, Gelatin zymography, Western Blot to confirm our hypothesis.
According to data analysis, our results demonstrated the metabolite of SHXXT has strong anti-oxidant and anti-inflammatory effects on preventing high glucose-induced endothelial cells injury. In experiment of high glucose (33mM) inducing endothelial cell injury within 48 hours, the survival rate did not significantly change. However, when cell adhesion lasted for 24 hours and had a significant inflammatory response, the add of the SHXXT metabolite 0.5-5μl/ml dosage. It proved that the SHXXT metabolites cause strong anti-inflammatory and anti-oxidant reaction. In the experiment of Gelatin zymography on SHXXT and Vitamin C, the finding showed that SHXXT had better effect of reducing arteriosclerosis then Vitamin C. In the experiment of eNOS, the SHXXT metabolites resulted in endothelial cell protective effects. In the experiment of ICAM-1, adds of SHXXT caused more decrease in ICAM-1 than Vitamin C. According to data analysis, our results demonstrated the metabolite of SHXXT has strong anti-oxidant and anti-inflammatory effects on preventing high glucose-induced endothelial cells injury.

目錄 VI
圖目錄 X
表目錄 XII
表目錄 XII
第一章 緒論 1
1-1 前言 1
1-2 血糖過高症 ( Hyperglycemia ) 3
1-3 三黃瀉心湯代謝物 ( San-Huang-Xie-Xin-Tang metabolite ) 4
1-4 文獻回顧 5
1-5 研究目的 9
第二章 理論基礎 11
2-1 三黃瀉心湯San-Huang-Xie-Xin-Tang 11
2-1-1 大黃(Rhei Rhizoma；RR) 13
2-1-2 黃芩(Scutellaria Radix；SR) 13
2-1-3 黃連(Coptidis Rhizoma；CR) 14
2-2 內皮細胞 ( Endothelial cell ) 14
2-2-1 內皮細胞基本功能 14
2-2-2 內皮細胞發炎 16
2-2-3 內皮細胞與巨噬細胞 17
2-3 活性氧化自由基 ( Reactive Oxygen Species ) 20
2-4 8-hydroxy-2’-deoxyguanosine ( 8-OHdG ) 22
2-5 細胞黏附分子 ( Cell adhesion molecule ) 22
2-5-1 細胞間黏附分1 ( ICAM-1 )與高葡萄糖之關係 23
2-6 一氧化氮( NO ) 24
2-7 基質金屬蛋白酶（Matrix Metalloproteinases, MMPs） 24
2-8 COX (環氧酵素，Cyclooxygenase) 26
第三章 實驗材料與方法 27
3-1實驗儀器與培養皿 27
3-1-2藥品與試劑 27
3-1-3 實驗藥品與試劑 28
3-2 實驗流程 30
3-2-1 實驗細胞株 33
3-2-1-1人類臍帶靜脈內皮細胞(HUVECs) 33
3-2-1-2人類單核球白血病細胞(THP-1 cells) 35
3-2-1-3細胞繼代培養 35
3-2-1-4細胞(冷凍)儲存與解凍 37
3-3 細胞計數(Cells Counting) 37
3-3細胞計數方法：MTT assay 39
3-4 酵素鏈結免疫吸附法（Enzyme-linked Immunosorbent 40
Assay; ELISA） 40
3-4-1 細胞死亡分析 ( Cell Apoptosis Kit ) 40
3-4-2 8-hydroxy-2-deoxyguanosine ( 8-OHdG Kit) 42
3-5 白血球黏著之測定: THP-1 Adhesion 43
3-6 明膠分解酶測定 ( Gelatin zymography ) 44
3-7西方墨點法(Western Blot) 45
3-7-1全細胞蛋白質萃取(protein extraction) 46
3-7-2蛋白質濃度測定 46
3-7-3 電泳 ( Electrophoresis ) 47
3-7-4 轉膜( Transfer ) 48
3-7-5 封閉 ( Blocking ) 48
3-7-6 一抗孵育 ( Primary antibody incubation ) 48
3-7-7 二抗孵育 ( Secondary antibody incubation ) 48
3-7-8蛋白檢測 ( Detection of proteins ) 49
3-7-9統計分析 49
第四章 結果與討論 50
4-1 高糖對待內皮細胞(HUVECs)存活率分析：MTT assay 50
4-1-1 三黃瀉心湯代謝物(SHXXT)對待內皮細胞(HUVECs)存活率分析 51
4-1-2 在高糖對待內皮細胞(HUVECs)及加入三黃瀉心湯代謝物(SHXXT)存活率分析 52
4-2 THP-1 Adhesion(白血球黏著之測定) 53
4-2-1 高糖與三黃瀉心湯代謝物，對待在HUVECs之cell adhesion 55
4-3 細胞死亡率(cell apoptosis) 59
4-4 明膠分解酶測定 ( Gelatin zymography ) 60
4-5 西方墨點法(Western Blot) 61
4-5-1 High glucose treat COX-2 and eNOS 62
4-5-2 High glucose/SHXXT eNOS 63
4-5-3 High glucose/SHXXT COX-2、eNOS、ICAM-1 64
4-6 8-hydroxy-2’-deoxyguanosine ( 8-OHdG ) 66
第五章 結論 68
參考文獻 71

圖目錄
圖1-1 三黃瀉心湯化學結構 7
圖1-2 NO對內皮細胞的刺激反應 8
圖2-1 內皮細胞所維持的新陳代謝與各項調節機能 17
圖2-2 高葡萄糖對單核球刺激造成的發炎反應 19
圖2-3 高葡萄糖對內皮細胞刺激造成的發炎反應 20
圖2-4 活性氧化物質組成 21
圖2-5 白血球在血管內皮細胞上的作用[104] 23
圖2-6 參與巨大細胞(Mast cell)和中間介質，影響在動脈硬化上 25
圖2-7 炎症刺激COX-2活化，造成血管 26
圖3-1 三黃瀉心湯代謝物製成簡圖 27
圖3-2 萃取臍帶內皮細胞(HUVECs)簡圖 31
圖3-3 實驗流程圖 32
圖3-4 新鮮萃取出的HUVECs 34
圖3-5 HUVECs加入Trypsin-EDTA作用 36
圖3-6 HUVECs繼代培養 36
圖3-7 細胞計數盤( cell counting ) 38
圖4-1 High glucose 16 mM/33 mM對待在HUVECs上，反應不同時間之MTT 50
圖4-2 三黃瀉心湯代謝物不同濃度，對待在HUVECs上，反應48小作用之細胞存活率(n=6,*表示與控制組相比其P<0.05) 52
圖4-3 三黃瀉心湯代謝物與高葡萄糖，對待在HUVECs上，反應48小時作用之細胞存活率。 53
圖4-4 High glucose 33 mM對待在HUVECs上，反應不同時間之cell adhesion 54
圖4-5 單一加藥，對待在HUVECs上，反應24小時作用之細胞存活率(n=6,*表示與控制組相比其P<0.05) 55
圖4-6 Treat high glucose and SHXXT(0.5-50 μl/ml)，對待在HUVECs上，反應24小時作用之cell adhesion 56
圖4-7 Treat high glucose 16 mM and SHXXT(0.5-5 μl/ml)，對待在HUVECs 上，反應24小時作用之cell adhesion (n=6,*表示與控制組相比其P<0.05)。 57
圖4-8 Treat high glucose 33 mM and SHXXT(0.5-5 μl/ml)，對待在HUVECs上，反應24小時作用之cell adhesion (n=6,*表示與控制組相比其P<0.05)。 58
圖4-9 Treat high glucose 33 mM and SHXXT(2.5 μl/ml)，對待HUVECs，在顯微鏡100倍視野。 59
圖4-10 Treat high glucose 33 mM and SHXXT(2.5 μl/ml)，對待在HUVECs上，反應24小時作用之cell apoptosis (n=4,*表示與控制組相比其P<0.05)。 60
圖4-11 Treat high glucose and SHXXT(1.25 μl/ml)，對待在HUVECs上，反應48小時作用之MMP-2。 61
圖4-12 Treat high glucose and 不同時間比較COX-2與eNOS 63
圖4-13 High glucose/SHXXT Western blot:eNOS 64
圖4-14 High glucose/SHXXT Western blot:eNOS、COX-2 65
圖4-15 High glucose/SHXXT Western blot:eNOS 66
圖4-16 High glucose/SHXXT Western blot:ICAM-1 66
圖4-17 High glucose/SHXXT 8-OHdG(n=2) 67

表目錄
表1-1葡萄糖正常值 3
表1-2葡萄糖 mg/dL換算mmol/L對照表 3
表2-1三黃瀉心湯主要成份 11
表2-2三黃瀉心湯與經絡之對應[71-76] 12
表2-3內皮細胞所製造之可調節凝血作用及血管舒張的物質 16
表3-1實驗藥品與試劑 28
表3-2 Tricine Sondium Dodecyl Sulfate ( 8%SDS ) 45
表3-3 Tricine Sondium Dodecyl Sulfate ( 10%SDS ) 47

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