第一部分 探討具有遺傳性肥胖症的Zucker Rats其心肌擴張及細胞程式性凋亡現象之研究 第二部分 探討間歇性缺氧對Zucker Rats的心肌細胞肥大及凋亡之影響

Part I The Effect of Dilated Cardiomyopathy and Apoptosis in Hereditary Obese Zucker Rats Part II The Effect of Intermittent Hypoxia on Hypertrophy and Apoptotic Signalings in Zucker Rats Heart Cells

吳芳俐(Fang-Li Wu)

指導教授 ： 李信達 ； 黃志揚

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2005年)

摘要

第一部份:肥胖與心血管疾病、高血壓、糖尿病等慢性疾病的罹病率與死亡率有極大的正相關，除了生活型態的差異導致肥胖外，遺傳的影響也是一個重要的因素。肥胖症所產生的冠狀動脈阻塞的病人（Ischemic heart disease），因自由基大量侵襲低密度脂蛋白（LDL），使得LDL形成oxLDL複合體，並引起血小板的吸附聚集產生血塊，血塊增大阻塞血管則產生心肌細胞缺氧的現象，進而促使血紅素喪失與氧結合的能力，導致缺氧情形更嚴重而使Ischemic heart disease病人之心肌細胞產生嚴重的擴張性肥大及凋亡的現象，但究竟如何使心肌細胞走向擴張性肥大及凋亡，其機制卻尚未完全得知。本實驗乃藉由具有遺傳性肥胖症的Zucker male rats，來加以模擬並探討因肥胖而產生Ischemic heart disease的病人缺氧後所受的傷害。本研究採用組織切片、Western blotting、RT-PCR等實驗方法來加以分析其影響機制。本篇研究發現因遺傳性肥胖而缺氧會引發心肌擴張的現象，而此現象可能是透過活化IL6、MEK5、ERK5的途徑。另外，遺傳性肥胖症會透過活化粒腺體上的Pro-apoptotic protein（BNIP3、Bad），以及減少Anti- apoptotic protein (Bcl2)的活性，促使粒腺體上產生能量的電子傳遞鏈遭到損害，造成細胞色素c ( Cytochrome c) 釋出到細胞質中，進而活化Caspase 9以及Caspase 3，使得心肌細胞走向凋亡；並且也會造成Death receptor dependent pathway中Fas、FADD、活化態的Caspase 8增加，進而活化Caspase 3，使得心肌細胞走向凋亡。

第二部份: 間歇性缺氧在調節身體功能方面，扮演著ㄧ個重要性的角色，然而對於肥胖者之心臟的影響，目前仍尚未十分清楚。本論文的主要目的是在探討長期間歇性缺氧對於遺傳性肥胖症的Zucker Rat，其心肌細胞肥大及產生細胞凋亡的現象有何影響。實驗大鼠分為四組，依序為常氧且瘦的體型對照組、常氧且胖的體型組、間歇性缺氧且瘦的體型組、間歇性缺氧且胖的體型組。間歇性缺氧組則置於12% O2 及88% N2 之低氧箱每天八小時，連續七天。之後我們利用Western blotting及RT-PCR 技術分析心肌細胞肥大及細胞凋亡的相關途徑。實驗結果顯示，與常氧組比較下，我們發現長期間歇性缺氧的obese Zucker rats 會更加活化IL6、MEK5、ERK5的這條途徑。並且其Death receptor pathway，如Fas、FADD、Bid、activated-Caspase8 和activated-Caspase 3 也有明顯的增加。另外，在粒腺體上的pro-apoptosis factor如：BNIP3及Bad皆有明顯的上升，而 anti-apoptosis factor（Bcl2）則有明顯的下降，並且粒腺體中決定有氧呼吸鏈功能之細胞色素c 大量釋放到細胞質中，這顯示長期間歇性缺氧對粒腺體有明顯的損害，並有apoptosis的趨勢。本研究結果顯示長期間歇性低氧促進心肌細胞肥大的機制，會透過活化IL6、MEK5、ERK5這條途徑；另外造成心肌細胞凋亡的途徑機制包含：(1)活化Death receptor dependent pathway (2) 以及活化 mitochondrial dependent apoptotic pathway。

關鍵字

肥胖 ； 缺氧 ； 心肌細胞

並列摘要

PartI: The obese Zucker rat, a genetic model of morbid obesity, presents many of the same cardiopulmonary deficits as noted in obese humans. Severe obesity in human has long been recognized as causing a form of cardiomyopathy characterized by increased rates of hypertension, chronic volume overload, left ventricular hypertrophy and the development of heart failure. However, the precise mechanisms of cardiac apoptosis and hypertrophy in sever obesity remain uncertain. The purpose of this study was to evaluate the key components of apoptotic and hypertrophic pathways in excised heart from obesity.
Twelve obese Zucker rats were studied at 4~5 months of age and twelve age-matched lean Zucker rats served as control. The myocardial architecture and key components of mitochondrial-dependent apoptotic pathway, Fas recetopr-dependent apoptotic pathway, IL-6-MEK5-ERK-5 hypertrophic pathway in the excised left ventricle from rats were measured by histological analysis, Western blotting and reverse transcription polymerase chain reaction (RT-PCR).In the mitochondria dependent apoptotic pathway, we detect the pro-apoptotic protein（BNIP3、Bad）increased and anti-apoptotic protein (Bcl2) decreased. Besides, Cytochrome c released to the cytoplasm from mitochondria highly then Caspase 9 and Caspase 3 would be activated to make cardiac muscle apoptosis. In the Fas-death receptor dependent pathway,we detect Fas、FADD、acitvated-Caspase 8 and acitvated-Caspase 3 increased to make obese Zucker rats cardiac muscle apoptosis. In IL-6-MEK5-ERK-5 hypertrophic pathway, we found that dilated cardiomyopathy is may be mediated through IL-6 and MEK5-ERK5 pathway activation identified by Western blotting experiment.The cardiac apoptotic and hypertrophic pathways were more active in obese Zucker rat, which may provide one of possible apoptotic and hypertrophic mechanism for developing heart failure in obesity.

Part II:The obese Zucker rat, a genetic model of morbid obesity, presents many of the same cardiopulmonary deficits as noted in obese humans. Nocturnal hypoxemia during sleeping hours or intermittent hypoxia implicated in the pathogenesis of cardiac complications were often reported in severe obesity. The main purpose of this study was to explore the cardiac hypertrophy and apoptosis in obese Zucker Rat under long-term intermittent hypoxia.
Twelve obese Zucker rats and twelve age-matched lean Zucker rats were divided into four groups including normoxia in lean group, normoxia in obese group，hypoxia in lean group, and hypoxia in obese group. The hypoxia group were housed in a hypoxic chamber (12% O2, 88% N2 ), 8 hour per day for 7 days. The myocardial architecture and key components of mitochondrial-dependent apoptotic pathway, Fas recetopr-dependent apoptotic pathway, IL-6-MEK5-ERK-5 hypertrophic pathway in the excised left ventricle from rats were measured by histological analysis, Western blotting and reverse transcription polymerase chain reaction (RT-PCR).
The activity levels of IL-6-MEK5-ERK5 hypertrophic pathway, death-receptor pathway (Fas、FADD、Bid、activated-Caspase 8 and activated-Caspase 3) and mitochondrial dependent apoptotic pathway (BNIP3, Bad and cytosolic cytochrome c, activated caspase 9) were increased in lean rat heart after long-term intermittent hypoxia. The activity levels of IL-6-MEK5-ERK5 hypertrophic pathway, death-receptor pathway and mitochondrial dependent apoptotic pathway were increased in obese rat hearts, compared with lean rat heart and further increased after long-term intermittent hypoxia in obese rat hearts, compared with either lean in hypoxia group or obese in normoxia group. Long term intermittent hypoxia and obesity itself would make additively deleterious effects on cardiac tissues in obese Zucker Rat. Our findings imply that intermittent hypoxia and obesity itself will additively increases cardiac hypertrophy through IL6-MEK5-ERK5 pathway and additively increases cardiac apoptosis in obese rat heart through mitochondrial dependent apoptotic pathway and death receptor dependent pathway.

並列關鍵字

Zucker Rat ； Obese ； Intermittent Hypoxia

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