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研究生:陳信宇
研究生(外文):Sin-Yu Chen
論文名稱:ApoE基因缺乏母鼠高膽固醇血症促進子代動脈粥狀硬化進程之機制研究
論文名稱(外文):Mechanistic study of maternal hypercholesterolemia modulating the progression of atherosclerosis in adult ApoE deficient offspring
指導教授:林甫容
指導教授(外文):Fu-Jung Lin
口試委員:黃青真呂紹俊蘇慧敏張美玲
口試委員(外文):Ching-Jang HuangShao-Chun LuHui-Min Su
口試日期:2018-07-03
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科技學系
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:2018
畢業學年度:106
語文別:中文
論文頁數:137
中文關鍵詞:動脈粥狀硬化發展規劃高膽固醇血症發炎
相關次數:
  • 被引用被引用:1
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心血管疾病如動脈粥狀硬化在近年來是廣泛盛行且死亡率高的慢性疾病之一,主要是由長期高脂飲食攝取與組織慢性發炎所導致。現今研究中提到子代在親代母親子宮內或是嬰兒時期受到的某些因素而對於其後來發育與健康程度有長久影響,此為著名「發展規劃」學說。流行病學研究中發現親代母親在懷孕期間的營養過剩或健康情況較差的狀況將會使得子代在成年期罹患代謝性症候群如第二型糖尿病、脂質代謝與心血管疾病的機率大幅增加。目前認為表觀遺傳的調控模式在發展規劃中可能扮演關鍵的角色,但是如何影響子代罹患動脈粥狀硬化病症代謝疾病的分子機制尚未有詳盡的研究。
我們假設親代母體在懷孕及泌乳期間暴露於西方飲食,將透過表觀遺傳的方式促進子代發炎反應,使得罹患動脈粥狀硬化的機會提高。本研究使用脂蛋白缺乏 (ApoE-/-) 母鼠,在懷孕前後透過西方飲食的誘導使之產生高膽固醇血症,再觀察其子代小鼠罹患疾病的嚴重度。我們發現到子代小鼠受到親代高膽固醇血症的影響,增加成年後罹患代謝性症候群及動脈粥狀硬化的機會。親代餵食西式飲食的子代雌鼠血液中促發炎反應的Ly6Chigh單核球數量顯著提升,主動脈中大量表現發炎介質及巨噬細胞相關基因,進而引起血管壁的發炎反應,此外,我們也發現母鼠高膽固醇血脂症也會促進M1巨噬細胞的極化分化,導致血管壁中巨噬細胞/M1促發炎反應巨噬細胞的數量上升進而促進動脈粥狀硬化斑塊的形成。
總結,我們發現親代母鼠在懷孕及泌乳期間持續食用西式飲食,使得子代小鼠透過促進組織慢性發炎的方式來提高動脈粥狀硬化病症的發生率。
Atherosclerosis is one of the chronic inflammatory vascular diseases with high prevalence and mortality worldwide. It is mainly resulted from accumulative uptake of lipid and prolonged chronic inflammation in tissue. Exposure to adverse maternal environment in utero or at infancy stage may permanently influence the development and health later in life. This phenomenon is so called “developmental programming” or “fetal programming”. Epidemiological studies showed that adverse condition such as overnutrition or ill state of mother may increase the risk of cardiovascular diseases and metabolic disorders in adult offspring. However, the mechanisms underlying the increased susceptibility of offspring to atherosclerosis and metabolic diseases are still unclear.
We hypothesized that diet-induced maternal hypercholesterolemia increased the susceptibility to atherosclerosis in adult offspring by inflammation. Apolipoprotein E deficient (ApoE-/-) female mice were fed with control diet (CD) or western diet (WD) prior to and throughout pregnancy and lactation. All mice were fed with WD after weaning. We found that offspring from WD-fed dams were more prone to develop metabolic syndrome and atherosclerosis later in life. Offspring from WD-fed dams exhibited an increased percentage of pro-inflammatory Ly6Chigh monocyte in the blood compared with offspring from CD-fed dams. The expression of inflammatory mediators and macrophages were significantly up-regulated in aorta of female offspring from WD-fed dams, suggesting acceleration of vascular inflammation. Moreover, we showed that maternal hypercholesterolemia promoted M1 macrophage polarization. Together, our study showed that maternal hypercholesterolemia promoted the development of atherosclerosis in female offspring through augmenting inflammation.
致謝 I
中文摘要 II
Abstract III
代號與縮寫對照表 IV
總目錄 VIII
圖目錄 XIII
表目錄 XV
第一章 緒論 1
第一節 前言 1
第二節 文獻回顧 3
一、代謝症候群與心血管疾病 3
1. 代謝症候群 (Metabolic syndrome, MetS) 3
2. 心血管疾病與動脈粥狀硬化 4
3. 脂蛋白運輸代謝與動脈粥狀硬化形成的關係 7
二、發炎反應與動脈粥狀硬化之關係 9
1. 急性與慢性發炎反應 9
2. 促發炎反應細胞激素 10
3. 慢性發炎反應與動脈粥狀硬化病症形成 12
4. 巨噬細胞與動脈粥狀硬化病症形成 13
5. 巨噬細胞的極化分化與動脈粥狀硬化病症形成 14
6. 周圍血液單核細胞 (PBMCs) 與動脈粥狀硬化病症形成 20
三、母體效應與表觀遺傳學 23
1. DNA甲基化 (DNA methylation) 24
2. 組蛋白修飾 (Histone modification) 26
3. RNA干擾 (RNA interference) 27
第三節 研究假說 28
第二章 實驗設計與材料方法 29
第一節 前言與實驗設計 29
一、前言 29
二、實驗設計 30
第二節 實驗材料與方法 31
一、實驗動物與飼育環境 31
二、子代實驗鼠繁殖 (Mating) 31
三、基因型分型 (Genetyping) 31
四、飼料配製 32
1. 控制組飲食 (Control diet, CD) 32
2. 西方飲食 (Western diet, WD) 33
五、血液、血清樣本收集 34
六、血液生化指標分析 34
1. 三酸甘油酯 (Triglyceride, TG) 檢測: 34
2. 總膽固醇 (Total cholesterol, TC) 檢測: 35
3. 非酯化脂肪酸 (Nonesterified fatty acid, NEFA) 檢測: 35
4. 胰島素 (Insulin) 檢測: 36
七、腹腔注射葡萄糖耐受性測試 (IPGTT) 37
1. 藥品配製: 37
2. 實驗流程: 37
八、腹腔注射胰島素耐受性測試 (IPITT) 38
1. 藥品配製: 38
2. 實驗流程: 38
九、子代小鼠動物犧牲及樣本收集 38
1. 器具準備與溶液配製: 38
2. 犧牲流程: 39
十、子代小鼠肝臟脂質分析 39
1. 肝臟脂質萃取 39
2. 肝臟中三酸甘油酯 (Triglyceride, TG) 檢測 40
2. 肝臟中總膽固醇 (Total cholesterol, TC) 檢測 40
十一、子代小鼠陰電性低密度脂蛋白萃取與分離 41
1. 低密度脂蛋白 (LDL) 分離 (等密度超高速離心法) 41
2. 陰電性低密度脂蛋白 (LDL(-),L5) 分離 41
十二、血液中發炎指標之細胞激素分析 42
十三、主動脈mRNA基因表現分析 43
1. 主動脈總RNA抽取 43
2. 去除DNA步驟 44
3. Total RNA反轉錄為cDNA 44
4. Quantitative Real-time PCR 45
十四、子代小鼠脾臟細胞發炎反應分析 45
1. 脾臟細胞之取得與分離 45
2. 脾臟內免疫細胞之刺激誘導與細胞激素檢測 46
3. 脾臟內免疫細胞之刺激誘導與mRNA表現測定 46
4. 脾臟內免疫細胞計數與比例測定 47
十五、骨髓衍生巨噬細胞 (BMDMs) 之分化潛勢分析 49
1. 骨髓衍生巨嗜細胞 (BMDM) 之分離、培養與誘導分化 49
2. M1/M2巨噬細胞計數與表面協同刺激分子比例測定 53
3. M1/M2巨噬細胞細胞激素含量檢測 53
4. M0/M1/M2巨噬細胞基因mRNA表現測定 54
十六、血液內單核細胞 (PBMCs) 分析 55
1. 血液內免疫單核細胞 (PBMCs) 之分離 55
2. 血液內免疫單核細胞 (PBMCs) 計數與比例分析 55
十七、主動脈竇 (Aortic sinus) 組織切片染色分析 57
1. 主動脈竇石蠟切片與冷凍切片製作 57
2. 主動脈竇油紅O (Oil red O, ORO) 組織染色 57
3. 主動脈竇免疫螢光組織染色 58
4. 主動脈竇橫切面組織染色面積定量 59
十八、腹腔內常駐型巨噬細胞 (PEMs) 之分析 59
1. 腹腔內常駐型巨噬細胞 (PEMs) 之分離 59
2. M1/M2巨噬細胞基因mRNA表現測定 60
3. M1/M2巨噬細胞細胞激素含量檢測 61
十九、骨髓造血幹細胞 (HSCs) 與骨髓前驅細胞 (CMPs) 之分析 61
1. 骨髓造血幹細胞 (HSCs) 與骨髓前驅細胞 (CMPs) 之分離 61
二十、主動脈中巨噬細胞與內皮細胞之分離 63
1. 酵素分解主動脈 63
2. 主動脈中巨噬細胞與內皮細胞之分離 64
二十、統計分析 65
第三章 實驗結果 69
第一節 親代母鼠高膽固醇血脂症對於子代小鼠代謝性疾病的影響 69
一、親代孕母鼠體重與血液生化指標分析 69
二、子代小鼠3週離乳至13週齡期間之體重變化分析 70
三、子代小鼠3週離乳至13週齡期間之血脂濃度變化分析 70
四、子代小鼠葡萄糖耐受性與胰島素阻抗性之試驗分析 70
五、子代小鼠16週齡犧牲時組織重量分析 71
六、子代小鼠16週齡犧牲時血液生化指標分析 72
七、子代小鼠的肝臟脂肪含量分析 72
第二節 親代母鼠高膽固醇血脂症對於子代小鼠動脈粥狀硬化的影響 73
一、子代小鼠主動脈竇橫切面油紅染色與免疫螢光染色分析 73
二、子代小鼠血液中負電性低密度膽固醇分析 73
三、子代小鼠主動脈mRNA表現量分析 74
1. 促發炎反應相關細胞激素之基因表現量分析 74
2. 發炎反應相關趨化因子/受器之基因表現量分析 74
3. 血管壁細胞吸附因子之基因表現量分析 74
4. 巨噬細胞極化分化之基因表現量分析 75
四、脾臟發炎反應與免疫細胞之組成分析 75
五、血液中促發炎反應細胞激素分析 75
六、血液中周邊單核細胞 (PBMCs) 之組成分析 76
七、骨髓衍生巨噬細胞 (BMDMs) 的極化分化潛勢分析 76
八、骨髓衍生巨噬細胞 (BMDMs) 之PPAR-γ與LXR-α基因表現量分析 78
第四章 實驗討論與結論 103
第一節 實驗討論 103
一、親代母鼠高膽固醇血症對於子代出生體重的影響 103
二、子代小鼠體重變化與組織重量差異 103
三、親代母鼠高膽固醇血症惡化子代血糖調節的風險 104
四、親代母鼠高膽固醇血症促進子代雌鼠血管壁發炎反應 105
五、母鼠高膽固醇血症增加子代雌鼠主動脈吸附單核球細胞的能力 106
六、親代母鼠的高膽固醇血症影響子代雌鼠巨噬細胞的極化分化 107
七、親代母鼠高膽固醇血症增加子代雌鼠血液中促發炎單核球細胞之數量 108
八、親代母鼠高膽固醇血症加速子代雌鼠動脈粥狀硬化形成之性別差異 110
第二節 實驗結論 112
第五章 參考文獻 113
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