探討Genistein對Leptin誘導大鼠血管平滑肌細胞增生之抑制作用

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题名:	探討Genistein對Leptin誘導大鼠血管平滑肌細胞增生之抑制作用 The Effect of Genistein on the Leptin-induced Cell Proliferaion in Rat Aortic Smooth Muscle Cell
作者:	呂思穎
贡献者:	中國藥學暨中藥資源學系碩士班
关键词:	細胞增生;瘦體素 cell proliferation;leptin
日期:	2011-07-26
上传时间:	2011-10-17 16:01:03 (UTC+8)
出版者:	中國醫藥大學
摘要:	動脈粥狀硬化(atherosclerosis)是心血管疾病中的其中一種，它的調控因子包含了內皮細胞的損傷、發炎、血管平滑肌細胞的增生以及細胞間質的改變。其中血管平滑肌細胞的增生在動脈粥狀硬化的發展上扮演著關鍵的角色。瘦體素(leptin)是一種由ob基因所分泌的胜肽荷爾蒙(peptide hormone)，在控制體重上佔有重要的地位，leptin會造成潛在的動脈粥樣硬化的效果，例如誘導內皮細胞的損傷、刺激發炎反應、血管平滑肌細胞遷移、增生以及增厚。Genistein是一種由植物所分離出的化合物，目前已知具有預防乳癌、前列腺癌、心血管疾病的作用和停經後所產生的問題。因此，本論文的目標在於探討genistein是否能抑制由leptin所誘導而形成的主動脈平滑肌細胞增生，以及了解其相關機制。大鼠主動脈平滑肌細胞(A10)經過低濃度血清飢餓並接著給予leptin 0.06, 0.6, 6 nM (1, 10, 100 ng/mL)72小時。對A10細胞來說，leptin有濃度依存性的誘導細胞增生。在Transwell遷移實驗中，會將A10細胞種至insert中，細胞會爬行穿越insert底層的微膜，也具有濃度依存性。總結來說，leptin顯著誘發主動脈平滑肌細胞的增生與遷移。除此之外，給A10細胞投以genistein (5 μM)後能顯著的降低由leptin所誘發而產生的細胞增生現象。至於genistein是經由何種機制去抑制leptin誘導血管平滑肌細胞增生作用，實驗發現，經過leptin處理後細胞p44/42 MAPK磷酸化程度會隨leptin iv 劑量隨之增加，而此作用會受genistein (5 μM)抑制。為了更進一步研究leptin對血管平滑肌增生作用的影響性，cyclin D1對細胞成長是重要的，且在細胞週期過程中cyclin D1是一個關鍵性的訊息傳導蛋白。本實驗也發現Leptin (1 ng/mL、10 ng/mL、100 ng/mL)濃度上升會誘發血管平滑肌細胞中cyclin D1蛋白表現量上升，並且具有濃度依存性。p21是一個強效的cyclin-dependent kinase inhibitor (CDK1)，在本實驗中，細胞經leptin處理後，隨著leptin濃度上升會降低細胞中p21蛋白質表現量，而genistein會抑制leptin對p21蛋白質表現的影響。總結，genistein可明顯降低leptin誘發血管平滑肌細胞增生作用，血管平滑肌細胞增生可以經由許多不同的機制，包含了促進細胞周期的進行，調控cyclin D1和p21的表現，刺激p44/42 MAPK的磷酸化。Genistein的作用機轉可能與抑制cyclin D1蛋白質表現，p44/42 MAPK磷酸化以及增加p21蛋白質表現有關。 Atherosclerosis is one of the cardiovascular diseases. The control events of atherosclerosis include endothelial dysfunction, inflammation, vascular smooth muscle cells proliferation and matrix alteration. The proliferation of vascular smooth muscle cells (VSMCs) plays a key role in the development of atherosclerosis. Leptin is a peptide hormone that is encoded by the ob gene and plays a central role in the regulation of body weight. Leptin exerts many potentially atherogenic effects such as induction of endothelial dysfunction, stimulation of inflammatory reaction, migration, hypertrophy and proliferation of VSMCs. Genistein is a plant-derived compound possessing well-known preventive activity in breast and prostate cancer, cardiovascular diseases and post-menopausal problem. Therefore, the aim of this study was to investigate whether genistein can inhibit the VSMC proliferation induced by leptin and the possible molecular mechanism of its action. Rat aortic smooth muscle cells (A10 cells) were serum-starved and subsequently treated with leptin at increasing concentrations 0.06, 0.6, or 6 nM (1, 10, and 100 ng/mL) for 72h. As observed for A10 cells, leptin enhanced the proliferation of cell in a dose-dependent manner. In transwell migration assay, the A10 cells were seeded into the insert and move through the pores of the membrane at the bottom of the insert in a dose-dependent. To concludes, leptin significantly induced VSMCs proliferation and migration. In addition, pretreatment with genistein(5 μM) significantly decreased the cell proliferation induced by leptin in VSMCs. We are needed to clarify the anti-proliferatory mechanism of genistein. It is well know that p44/42 MAPK activation plays an important role in cell proliferation. So, we investigated the contribution of p44/42 MAPK activation to VSMCs proliferation induced by vi leptin. After cells were treated with expression of phosphorylated p44/42 MAPK was enhanced in a leptin concentration-dependent manner. The phosphorylation of p44/42 MAPK induced by leptin was inhibited by pre-treatment with genistein (5 μM). To further investigate the effect of leptin on VSMC proliferation, cyclin D1 is important for cell growth and is a key cell-cycle signaling protein in cell-cycle progression. Leptin induced a dose-dependent increase in the cyclin D1 protein level at 1 ng/ml、10 ng/ml. P21 is a potent cyclin-dependent kinase inhibitor (CK1). In our study, leptin induced a dose-dependent decrease in the p21 protein level and genistein increased the p21 protein level induced by leptin. In conclusion, leptin stimulated VSMC proliferation by several mechanisms, namely enhancing cell cycle progression, up-regulating cyclin D1 and p21 expression, motivating ERK1/2 phosphorylation. Genistein significantly decreased the cell proliferation induced by leptin in A10 cells. The beneficial effect of genistein on leptin-induced cell proliferation may result from its inhibition of cyclin D1 expression and p44/42 MAPK phosphorylation and the increased of p21cip1 expression. Genistein may thus prove a potential agent against atherosclerosis in obesity.
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