| 摘要: | 瘦體素（leptin）是一種由169 個胺基酸所組成的蛋白質，由脂肪組織合成，主要作用在下視 丘以抑制食慾，並可增加能量消耗，藉此維持身體脂肪含量。除了能量的攝取與消耗的調節外， 瘦體素亦參與心血管系統之調控，細胞培養的實驗結果進一步發現瘦體素有誘發鼠心臟細胞肥 大的作用，然而其作用的相關細胞分子機轉，目前尚不是很清楚。內皮素,為目前已知具有強力 促進血管收縮作用的內生性物質,它除了與高血壓及冠狀動脈硬化的形成有關之外,還會造成心 臟細胞的肥大作用,然而有關瘦體素與內皮素兩者之間,在心臟細胞作用的交互關聯性,就目前 已知尚不是很清楚。經由報告基因轉染入細胞的研究方法,已知內皮素基因啟動區上存在著活化 蛋白-1 (activator protein-1; AP-1)轉錄因子的結合部位,而瘦體素經由活化細胞內訊息傳遞 的機轉(如胞外訊號調節激脢路徑),具有影響細胞內AP-1 的生成及結合親和力的作用,是以瘦體 素經由增加細胞內AP-1 的生成,再輾轉於基因轉錄的層面上,可能可以進一步活化內皮素的基因 表現。最近的研究報告: 於培養的心臟細胞的研究觀察,發現瘦體素確實會誘發內皮素的生合成; 此外瘦體素造成心臟細胞肥大的作用,亦與內生性的內皮素有關。由以上的研究報告可推論:於 心臟細胞,瘦體素經由增加細胞內AP-1 的生成,再輾轉於基因轉錄的層面上,可能可以進一步活 化內皮素基因表現的假設是可以成立的,然而目前在心臟細胞,有關瘦體素對內皮素基因表現的 直接作用,及其作用的細胞內訊息傳遞的機轉,亦或導致心臟的重塑,目前都不是很清楚。在本 計劃中,我們欲瞭解在心臟細胞及心臟纖維細胞,內皮素接受器的拮抗劑,是否可對抗瘦體素的 造成心臟細胞肥大及心臟纖維細胞增生,另瘦體素是否會直接活化內皮素基因的表現,及其作用 的細胞內訊息傳遞的機轉。應用培養之鼠心臟細胞及心臟纖維細胞在瘦體素作用下探討: (1) 經 由報告基因轉染入細胞的研究方法,確定內皮素接受器的拮抗劑,是否可對抗瘦體素的活化心臟 細胞肥大相關基因─蛋白質重鍊基因的表現以及細胞內蛋白質的合成; (2) 經由酵素連結免疫吸 附分析法、北方氏點墨法及報告基因轉染入細胞的研究方法,確定瘦體素是否會直接活化內皮素 基因的表現; (3) 探索瘦體素活化內皮素基因表現其細胞內的訊息傳遞的機轉; (4) 決定瘦體素 誘發內皮素基因表現,於基因啟動區上的正向活化之序列。並使用老鼠動物模式，以慢性（2 禮 拜）皮下注射瘦體素，觀察是否引發活體心臟肥大或心室血管周圍纖維化，並評估施打白蘆黎 醇等抗氧化物是否可以抑制瘦體素所引發心臟的重塑。
Leptin, a 16-kDa peptide encoded by the obese gene, is produced by adipocytes and released into the bloodstream. It has been suggested that leptin may contribute to cardiovascular disease, independently of obesity such as in hypertension. Recent clinical evidence has implicated leptin as a potential independent risk factor for coronary heart disease, and increased plasma leptin levels have been found in patients with congestive heart failure. Heart failure is generally preceded by myocardial remodeling, involving cardiomyocyte hypertrophy and other maladaptive responses (such as cardiac fibroblast proliferation and extracellular matrix protein accumulation), although whether leptin contributes to these events and the detailed intracellular mechanisms has not been well defined. Elevated serum concentrations of endothelin-1 (ET-1), a potent vasoconstrictor and mitogen, were also observed in cardiovascular disease subjects. Recent studies indicate that stimulation with leptin of cultured cardiomyocytes induced synthesis of ET-1. Endogenously produced ET-1 was found to contribute to the hypertrophic response of cardiomyocytes to leptin and thereby to cardiac hypertrophy. Therefore, it is tempting to hypothesize that endogenous ET-1 does play a role in the effects of leptin. Thus, ET-1 antagonism may offer an additional weapon for therapeutic interventions aimed at preventing cardiovascular damage. However, the direct effect of leptin on ET-1 gene expression and its intracellular signal transduction mechanism and the pathogenesis of cardiac remodeling have not been well examined in vitro and in vivo. In this 3-year project, the applicant will observe ET-1 receptor antagonist on leptin-induced cardiac remodeling, and investigate whether leptin affects ET-1 gene expression and explore its molecular mechanism in culture system. In cultured rat cardiac myocytes (or fibroblasts) in response to leptin, we will (1) inspect the effect of ET-1 receptor antagonist on leptin-nduced protein synthesis, DNA synthesis and myosin heavy chain gene expression by chloramphenicol acetyltransferase (CAT) assay, (2) analyze effect of leptin on ET-1 gene expression by enzyme-linked immunosorbent assay (ELISA), Northern blot analysis and CAT assay, (3) explore the detailed intracellular signal transduction pathway of leptin-induced ET-1 gene expression, (4) determine which cis-acting sequences in the ET-1 promoter region are important for leptin-induced transcription of the gene, (5) determine the development of cardiac remodeling in a rat model with chronically subcutaneously infused leptin, and (6) determine the effect of resveratrol on leptin-induced cardiac remodeling (cardiac hypertrophy and left ventricular fibrosis) in vivo. |
