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研究生:陳昱璇
研究生(外文):Yu-Hsuan Chen
論文名稱:利用自發性高血壓大鼠模式評估克弗爾預防高血壓併發症之功效
論文名稱(外文):Functional Assessment of Kefir on Preventing Complications of Hypertension in Spontaneously Hypertensive Rats.
指導教授:Chuan-Mu Chen 陳全木
口試委員:Chi-Mei Hsueh 葛其梅Fu-Chou Cheng 陳甫州Kowit Yu Chong 張國友
口試日期:2014-07-29
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:119
中文關鍵詞:克弗爾高血壓中風CatWalk磁振造影自發性易中風型高血壓大鼠
外文關鍵詞:sodium chloridekefir producthypertensionstrokeCatwalkMRIstroke prone spontaneously hypertensive rats
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不論在開發中或發展中國家,腦血管、心血管疾病、心臟衰竭與慢性腎臟疾病都是造成高血壓危險因素之一,當血壓下降則能夠使得中風與心血管疾病的發生率降低;然而高血壓除了藥物的控制之外,遺傳因素、生活方式及營養攝取都佔著極大的影響因素。因此本試驗將以實驗室自行由酵母菌與乳酸菌共同發酵產製之克弗爾發酵胜?餵食,在過去研究中克弗爾被認為具有抗氧化、降血壓、抗微生物、調節免疫功能及促進鈣質吸收等益生功能,在實驗室先前研究中指出發酵乳製品對於自發性高血壓動物模式具有降低血壓的作用,本研究試驗使用動物模式為自發性易中風型高血壓大鼠 (Stroke prone spontaneously hypertensive rat;SHRSP),其本身帶有高血壓相關併發症且容易因小血管問題而發生腦中風之情況,造成全身性疾病問題,因此將探討不同發酵胜?於自發性易中風型高血壓大鼠之高血壓相關併發症預防功效。首先我們利用CatWalk系統進行步態評估及磁振造影系統 (MRI) 進行腦部與腎臟相關影像監測。本試驗以50-60週齡WKY當作外對照組 (Normal/Control);另外使用50-60週齡完全不餵食鹽水及發酵胜?之SHRSP作為未處理對照組 (Untreated/Control);另外三組分別以鈉含量當作基準並於飲水添加入1%鹽水同時進行誘導分為:(1) 與克弗爾和可爾必思相當鈉含量 (NaCl/Mock, 0.178mg/kg/day);(2) 克弗爾 (NaCl/Kefir, 140mg/kg/day);(3) 可爾必思 (NaCl/Trade? , 140mg/kg/day),雄鼠進行四週試驗;另外中風之大鼠則以不同年齡進行磁振造影系統 (MRI) 進行腦部、血管及腎臟之早期評估。試驗結果發現克弗爾醱酵胜?於1%鹽水攝入下,為期4週之預防試驗具有顯著降低高血壓、血液生化肌酸激?、並維持體重、步態行為,並防止心臟主動脈弓厚度,保護腎臟所造成的慢性腎臟病變及纖維化且降低中風的發生率,效果優於商業化可爾必思胜?組;反之於1%鹽水攝入額外管餵與醱酵胜?之相同鈉含量之Mock組別其體重嚴重下降甚至死亡,步態行為嚴重受影響,且其腎臟嚴重萎縮並造成腎小球體纖維化,主動脈弓變厚,出現癲癇及腦水腫。因此可以知道克弗爾發酵胜?具有保護腎臟及預防腦中風之發生率,本試驗經由選育成功培育出穩定具有高血壓及腦中風之自發性易中風型高血壓大鼠,以及利用磁振造影系統建立能夠即時早期預知腎臟損害程度之評估參數與方法,並可由CatWalk步態系統評估其中風側邊及神經損傷程度。

Cerebrovascular, cardiovascular disease, heart failure and chronic kidney diseases are caused by one of the risk factors for hypertension in development or developing countries. When lowering blood pressure is able to make reduced of stroke incidence and cardiovascular disease;however, addition to control high blood pressure with drugs, genetic component, lifestyle and nutritional have occupied a great factor. Kefir product is a kind of fermented dairy products made by kefir grains. In the current studies, it is known that fermentation of milk causes degradation of milk proteins into various peptides with health-promoting effects including antioxidant, immunomodulating, antihypertensive, antithromobotic calcium-absorption enhancing, and antimicrobial bioactivities. In our previous study, the effects of these fermented milk products exhibited antihypertensive effect at the spontaneously hypertensive rat. In this study, we use a stroke prone spontaneously hypertensive (SHRSP) rat as an animal model, itself with hypertension-related complications and problems of the small blood vessels with causing systemic disease to case of stroke. The aim of this study is to functional assessments of kefir on preventing complications of hypertension. To assess locomotor function recovery, the CatWalk footprint analysis system and magnetic resonance imaging system (MRI) of the brain and kidney-related conduct monitoring were used. In this study, 55-60 week-old male wistar rats (WKY) and stroke prone spontaneously hypertensive rats (SHRSP) were assigned into five groups for treatment: (1) WKY normal control group, (2) SHRSP Untreated control, (3) SHRSP 1% NaCl/Mock, (4) SHRSP 1% NaCl/Kefir 140 mg/kg/day and (5) SHRSP 1% NaCl/Trade? 140 mg/kg/day. Rats were sacrificed at 4 weeks after different treatment. Results showed that rats in kefir treated group significantly reduced high blood pressure and blood biochemistry of creatine kinase, maintained body weight and footprint behavior, prevented heart arch thickness, protected kidney injury by chronic kidney disease with fibrosis and reduced the incidence of stroke. The contrary of 1% saline Mock group that body weight were serious decline with gait behavior and even death, caused a serious decline in glomerular fibrosis in kidney, thicker of the aortic arch, epilepsy and cerebral edema. Data suggested that the kefir supplementation exhibits a anti-hypertensive activity and prevents the incidence of stroke.

中文摘要…………………………………………………………………………………i
英文摘要………………………………………………………………………………...ii
目次……………………………………………………………………………………..iii
表次……………………………………………………………………………………viii
圖次……………………………………………………………………………………..ix
壹、 緒言………………………………………………………………………………...1
貳、 文獻探討…………………………………………………………………………...2
一、 高血壓…………………………………………………………………………..2
(一)、高血壓的簡介………………………..……………………………………...2
1. 高血壓分類..............................................................................................3
2. 人口分布及年齡層..................................................................................6
3. 高血壓所衍生疾病..................................................................................8
(二)、體內血壓的調控機制……………………...………………………………10
1. 腎素血管收縮素調節系統(RAAS)..................................10
2. 市售控制血壓及中風相關藥物...............................................................10
(三)、抑制RAAS系統之活性胜?……………………………………………...12
1. 抑制RAAS系統活性胜?之種類........................................................12
2. 活性胜?之胺基酸組成與功能......................................14
3. 克弗爾醱酵乳之保健功效及應用........................................................14
二、 高血壓所引起之中風…………………………………………………………17
(一)、中風………….....................................................................………………..17
(二)、腦中風的種類及特性……………………………………………………..17
(三)、高鹽飲食對高血壓及中風之影響………...........…………………………20
(四)、高鹽引發腦中風的機制……………….…………………………………..20
三、 高血壓引起的腎臟病……………………………........………………………21
(一)、腎臟病變種類…………………….......................................................……21
(二)、高鹽飲食對高血壓與腎臟病變之影響…………...............………………21
(三)、高血壓引發腎臟病變之機制…………………....………………………...23
四、 中風型高血壓鼠動物模式介紹………………………............………………27
(一)、動物的由來……………......................................................................…….27
(二)、中風型高血壓大鼠之類型與其不同之病症………….…………………..27
1. 腦部.........................................................................................................27
2. 腎臟..........................................................................................................29
五、 生物醫學影像…………………....……………………………………………30
(一)、生物活體影像系統 (In vivo biolumineescence) 發展…....................…….30
(二)、磁共振影像術 (MRI)之應用........................................................................30
(三)、磁共振影像術之特性及優點.........................................................................31
六、 動物模式之行為測試……............……………………………………………31
(一)、動物行為種類及其特性....................................................................................31
(二)、步態測試在腦中風模式鼠之應用....................................................................32
七、研究動機與策略...................................................................................................33
參、 材料與方法……………………………………………………………………….35
一、 試驗動物之品系…………………..........................................................…......35
(一)、試驗動物品系來源與飼養………………………………………………...35
(二)、自發性易中風型高血壓大鼠之紀錄判定………..…..…………………...35
(三)、動物神經行為評估方式...............................................................................36
二、 試驗流程、餵食模式、動物犧牲及樣品取得…………...........……………….36
(一)、試驗前置流程……………………..…..…………………………………...36
(二)、醱酵胜?試驗設計與分組………………..............……………………….36
(三)、犧牲前準備………………………………………………………………...37
(四)、血壓之測定…………………………....…………………………………...37
(五)、癲癇狀況之觀察...........................................................................................37
(六)、尿液生化之測定...........................................................................................37
(七)、動物犧牲.......................................................................................................41
(八)、臟器重、肉眼觀察病理變化.........................................................................41
(九)、2.3.5-triphenyltertrazolium hydrochloride 染色..........................................42
三、 CatWalk步態分析系統…………………………..............................……...….42
四、 磁振造影影像 (MRI)分析…………….....................................……….……..42
五、 腎臟組織蛋白質表現分析………………….....................………………...…47
(一)、總蛋白質 (total protein)之萃取………………...………………………..47
(二)、蛋白質濃度測…………………………………….………………………..47
(三)、蛋白質膠體電泳 (SDS-PAGE)…………………….……………………..47
六、 SHRSP大鼠體重測定…………………...............................................………48
七、 組織切片染色分析 (H&E staining)......................................................….......49
八、 特殊組織化學染色............................................................................................49
(一)、Masson’s Trichrome staining………………….....…………………………49
(二)、Sirius Red staining………………………………..………………………...49
(三)、Cresyl violet(Nissl staining)…………………......………………………...49
九、 免疫組織化學染色分析 (IHC staining)….......................................................50
十、 統計分析…………....................................................................................….50
肆、 結果……………………………………………………………………………….52
一、 自發性易中風型高血壓大鼠之相關生理特徵………………………………52
二、 餵食不同醱酵胜?於自發性易中風型高血壓大鼠之試驗評估....................53
(一)、自發性易中風型高血壓雄鼠體重變化……........................................…...53
(二)、自發性易中風型高血壓雄鼠血壓變化分析…...........................................53
(三)、自發性易中風型高血壓雄鼠代謝變化分析………...................................56
(四)、自發性易中風型高血壓雄鼠血液生化變化………............……………...56
(五)、自發性易中風型高血壓雄鼠臟器重量.......................................................61
(六)、自發性易中風型高血壓雄鼠臟器巨觀形態病變.......................................61
(七)、體內臟器組織病理分析之評分...................................................................66
(八)、自發性易中風型高血壓雄鼠死亡率評估...................................................66
三、 步態分析……………………................................................................………69
(一)、不同醱酵胜?處理對於大鼠行為上CatWalk系統腳印呈像之評估.........69
(二)、不同醱酵胜?處理對於大鼠行為上CatWalk系統之數據分析.................71
四、 腎臟分析………………………………………………………………………77
(一)、不同醱酵胜?處理於大鼠腎臟之病理切片分析…………................…...77
(二)、不同醱酵胜?處理於大鼠腎臟之腎上腺病理變化……….............……..83
(三)、不同醱酵胜?處理於大鼠腎臟之動脈病理變化…………................…...83
五、 心血管分析…………………………………………………………..………...83
六、 腦部分析…………………………………………………………..……….......87
(一)、不同醱酵胜?處理於大鼠腦部病理切片分析..........................................87
七、自發性易中風型高血壓大鼠於中風後之變化...................................................89
(一)、中風之自發性易中風型高血壓大鼠行為變化..........................................89
(二)、比較中風高血壓雄鼠與其他組別臟器之變化..........................................89
八、中風之高血壓大鼠之磁振造影與組織病理之相關性.......................................96
(一)、腦部磁振造影與組織病理分析..................................................................96
(二)、腎臟磁振造影與組織病理分析................................................................103
伍、 討論.......................................................................................................................106
一、 不同發酵胜?於自發性易中風型高血壓大鼠模式生理數質改善之效果...106
二、 不同發酵胜?於自發性易中風型高血壓大鼠模式不同臟器之保護效果...107
三、 不同發酵胜?於自發性易中風型高血壓大鼠行為與中風之相關性……...108
四、 不同發酵胜?降低自發性易中風型高血壓大鼠之死亡率…………….......108
五、 自發性易中風型高血壓大鼠模式於中風狀態之腦部磁振造影與病理
相關...................................................................................................................109
六、 自發性易中風型高血壓大鼠模式於中風狀態之腎臟磁振造影與病理
相關...................................................................................................................109
陸、 結論……………………………………………………………………………...111
柒、 參考文獻………………………………………………………………………...112


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