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研究生:戴怡芬
研究生(外文):Tai Yi-Fen
論文名稱:同半胱胺酸誘發血管平滑肌細胞增生、自由基產生與細胞鈣化之研究
論文名稱(外文):The effects of homocysteine- induced proliferation, free radicals, and calcification on vascular smooth muscle cells
指導教授:陳伯中陳伯中引用關係
學位類別:碩士
校院名稱:弘光科技大學
系所名稱:營養醫學研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
畢業學年度:100
語文別:中文
論文頁數:86
中文關鍵詞:同半胱胺酸血管平滑肌細胞氧化壓力增生血管鈣化
外文關鍵詞:HomocysteineVascular smooth muscle cellsOxidative stressCalcification and proliferationPI3K/p-Akt pathwayp-ERK
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血管鈣化 (vascular calcification) 的發生與血管疾病的死亡率有極大的相關性,尤其在腎病末期 (end-stage renal disease ; ESRD) 患者,血管鈣化之情況更是嚴重。近來文獻指出,血管鈣化的危險因子包括同半胱胺酸 (homocysteine ; Hcy)、氧化壓力 (oxidative stress) 及高血鈣 (hypercalcemia) 等。正常人血漿 Hcy 濃度約為 5~15 M,在高同半胱胺血症中 Hcy 濃度可高達 100~250 M。臨床證據亦發現,氧化壓力過高、高血鈣及血液中不正常過高的 Hcy 經常一起出現在許多病變上。然而,Hcy 在血管鈣化的作用機制上仍未完全釐清。因此,本論文旨在探討不同濃度的 Hcy 是否誘發大鼠主動脈之初代血管平滑肌細胞 (primary vascular soomth muscle cells ; VSMCs) 的氧化壓力、增生和鈣化。本將 VSMCs 分別培養在 10、50、100 和 250 M Hcy 的環境下,培養時間為 24、48、72 和 96小時。分析細胞氧化壓力、抗氧化酵素活性、細胞增生與細胞鈣化。結果發現,添加 Hcy 的濃度越高且時間越長,VSMCs 內的超氧陰離子 (superoxide anion radical)、過氧化氫 (hydrogen peroxide) 和丙二醛 (malondialdehyde ; MDA) 含量都顯著增加。進一步發現 Hcy 的濃度與培養時間的增加,明顯增加超氧化物歧化酶 (superoxide dismutase, SOD)、過氧化氫酶 (catalase) 的活性、與降低穀胱甘肽過氧化酶 (glutathione peroxidase, GPx) 活性。顯示 Hcy 改變抗氧化-氧化系統的平衡。另外,在增生方面,隨著 Hcy 濃度的增加,VSMCs 細胞數目相對增加,VSMCs 在不同濃度 Hcy 培養 96 小時後,可以活化細胞內 PI3K pathway 和下游蛋白 p-Akt 蛋白的表現。在細胞鈣化方面,添加不同濃度 Hcy 環境下,雖在第 24 和 48 小時未觀察到鈣化的證據;但在第 72 和 96 小時,可以觀察到明顯的鈣化現象。另外,在第 96 小時,隨著 Hcy濃度的增加顯著誘發 p-ERK 蛋白表現。綜合以上結果,Hcy 刺激 O2- 和 H2O2 的產生,導致 VSMCs 受到氧化傷害,進而改變抗氧化酵素 (SOD、GPx 和 catalase) 活性,誘發細胞內 PI3K/p-Akt pathway 訊息傳遞,使得 VSMCs 開始增生與增加鈣的攝入,亦明顯誘發 p-ERK 蛋白表現,加速 VSMCs 鈣化。因此,過高的 Hcy 含量,可能是加速血管鈣化的主要原因之一。
Vascular calcification is highly correlated with cardiovascular mortality, especially in patients with end-stage renal disease (ESRD). Recently evidence suggests that high blood homocysteine (Hcy), increased oxidative stress, and hypercalcemia may contribute to vascular calcification. The average ranges of plasma Hcy in healthy subjects are 5-15 uM; whereas the plasma levels of Hcy are approximately 100~250 uM in hyperhomocysteinemia. Increased plasma Hcy levels are a well-known risk of elevated oxidative damage in vitro and in vivo. Thus, the pathobiomolecular mechanisms of Hcy-induced vascular calcification need to be explored. In the present thesis, we examined the effects of oxidative stress induction by Hcy on the calcification and proliferation of rat vascular smooth muscle cells (VSMCs). VSMCs treatment with different Hcy levels (10, 50, 100 and 250 M) had showed a dose-dependent significant increase in superoxide anion radical (O2-), hydrogen peroxide (H2O2), or lipid pro-oxidant production malondialdehyde (MDA). Increased antioxidant enzymes activities of superoxide dismutase (SOD) and catalase, as well as decreased glutathione peroxidase (GPx) were also observed in Hcy-treated VSMCs followed by increased incubation periods (24, 48, 72, and 96 hours). Further, Hcy treatment significantly promoted proliferation in the VSMCs by activated PI3K/p-Akt pathway. Also, VSMCs treatment with Hcy had showed a markedly elevation in intracellular calcium and are associated with active p-ERK protein expression. In summary, Hcy significantly induced an increase in intracellular calcium concentrations and proliferation may attribute to increased oxidative damage and activated PI3K/p-Akt pathway and p-ERK protein.

目 錄
摘要 ………………………………………………………………I
目錄 ………………………………………………………………III
圖目錄 ……………………………………………………………Ⅶ
第一章 緒論………………………………………………………1
第二章 文獻回顧
一、 心血管疾病………………………………………………3
1. 血管的組成………………………………………………4
2. 動脈粥狀硬化的演進過程………………………………6
3. 血管鈣化形成之可能機制………………………………8
二、 同半胱胺酸 …………………………………………… 11
三、 氧化壓力…………………………………………………15
四、 同半胱胺酸與氧化壓力之關聯…………………………17
五、 氧化壓力與細胞增生之關聯……………………………19
六、 氧化壓力與血管鈣化之關聯……………………………20
七、 實驗假說…………………………………………………22
八、 研究目的…………………………………………………23
第三章 材料和方法
一、 實驗藥品與儀器…………………………………………24
1. 藥品………………………………………………… 24
2. 儀器………………………………………………… 27
二、 實驗流程…………………………………………………29
三、 血管平滑肌細胞之取得與培養…………………………30
四、 細胞型態螢光染色 …………………………………… 31
五、 細胞生長曲線和增生……………………………………32
六、 Lipid peroxides 含量分析………………………………33
七、 細胞內之自由基測定……………………………………34
八、 超氧化物歧化酶…………………………………………34
九、 麩胱甘肽過氧化酶………………………………………36
十、 過氧化酶之分析…………………………………………38
十一、 細胞培養液鈣含量之分析……………………………37
十二、 細胞內鈣化影響之分析………………………………39
十三、 PI3K 及 p-Akt之蛋白質表現……………………… 41
十四、 統計分析………………………………………………43
第四章 結果
一、 初代血管平滑肌細胞之取得……………………………44
二、 血管平滑肌細胞之型態觀察……………………………45
三、 血管平滑肌細胞之生長情形……………………………46
四、 同半胱胺酸對血管平滑肌細胞生長之情形……………47
五、 同半胱胺酸誘發血管平滑肌細胞內自由基的產生
1. 同半胱胺酸誘發血管平滑肌細胞內 O2- 含量…… 49
2. 同半胱胺酸誘發血管平滑肌細胞內 H2O2 含量… 51
3. 同半胱胺酸誘發血管平滑肌細胞內 MDA 含量之觀察…………………………………………………… 53
六、 同半胱胺酸對血管平滑肌細胞內抗氧化酵素活性之影響
1. 同半胱胺酸對血管平滑肌細胞內 SOD 活性之影響 ……………………………………………………55
2. 同半胱胺酸對血管平滑肌細胞內 GPx 活性之影響…………………………………………………… 57
3. 同半胱胺酸對血管平滑肌細胞內 catalase 活性之影響…………………………………………………… 59
七、 同半胱胺酸對血管平滑肌細胞增生及鈣化之情形
1. 同半胱胺酸對血管平滑肌細胞增生之影響…………61
2. 同半胱胺酸對血管平滑肌細胞外鈣含量之影響……63
3. 同半胱胺酸對血管平滑肌細胞鈣化之分析…………65
4. 同半胱胺酸對血管平滑肌細胞鈣化之影像觀察……67

八、 同半胱胺酸對血管平滑肌細胞內 PI3K/p-Akt pathway 及
p-ERK 蛋白質表現之影響……………………………… 69
第五章 討論
一、 同半胱胺酸對血管平滑肌細胞生長之影響……………71
二、 同半胱胺酸誘發血管平滑肌細胞氧化壓力上升………72
三、 同半胱胺酸對血管平滑肌細胞內抗氧化酵素之影響…73
四、 同半胱胺酸對血管平滑肌細胞增生……………………74
五、 同半胱胺酸對血管平滑肌細胞的鈣化…………………76
第六章 結論 ……………………………………………………78
第七章 參考文獻 ………………………………………………80

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