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研究生:劉俊馳
研究生(外文):Chun-Chih Liu
論文名稱:RhoKinase參與小鼠長期砷暴露其耳朵皮膚經芥子油刺激引起的血管高滲漏
論文名稱(外文):Rho Kinase-mediated Vascular Hyperpermeability of Ear Skin Challenged with Mustard Oil in Mice Treated with Chronic Exposure of Arsenic
指導教授:陳世杰陳世杰引用關係
指導教授(外文):Shih-Chieh Chen
學位類別:碩士
校院名稱:高雄醫學大學
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:96
中文關鍵詞:血管通透性芥子油Y-27632RhoARho kinase
外文關鍵詞:arsenicvascular permeabilitymustard oilY-27632RhoARho kinase
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流行病學研究指出慢性砷暴露與烏腳病及動脈硬化等血管相關疾病的相關性。小型血管如小靜脈受到刺激時通透性會增加導致滲漏,主要原因為血管內皮細胞間出現缺口,而內皮細胞間隙缺口的大小取決於內皮細胞之細胞骨架的收縮狀態,許多研究報告已指出內皮細胞內微細絲重組、收縮時,RhoA/Rho kinase是其訊號傳遞鏈。RhoA可活化下游的受體Rho kinase 1(ROCK1)和ROCK2,進而磷酸化MYPT1 (Myosin phosphatase targeting subunit 1),此機制在細胞骨架的收縮中扮演了關鍵的角色。除此之外也有研究指出血管內皮生長因子VEGF-A(Vascular endothelial growth factor-A)是調控血管通透性的重要因素。動物模式已證明在餵食亞砷酸鈉的小鼠皮膚可藉由芥子油誘發血管大量滲漏,已知芥子油可刺激神經纖維釋放P物質啟動內皮細胞NK1R (Neurokinin-1 receptor)而誘發血漿滲出。本論文的假設是慢性砷暴露的小鼠,其耳朵皮膚受芥子油誘發而產生的血管高滲漏是經由RhoA/Rho kinase訊號傳遞鏈所調控。研究實驗項目包含以慢性砷暴露的動物模式測試Rho kinase的活性抑制劑Y-27632對芥子油所誘發的血管滲漏有無抑制效果,並使用西方墨點法及聚合?○s鎖反應的技術來測定與血管通透性相關基因的表現。首先以胃管方式依分組投予小鼠純水(對照組)或亞砷酸鈉(NaAsO2)溶液(實驗組),經二、四或八週餵食後,利用芥子油誘發伊凡氏藍染劑或印地安墨汁膠體碳粒的滲漏來檢測血管通透性。實驗結果顯示餵食亞砷酸鈉二、四或八週的鼠耳皮膚在芥子油誘發下皆有大量血管滲漏,而且由砷暴露增強的高度滲漏反應可以被Y-27632明顯抑制,顯示Rho kinase參與由砷暴露再受芥子油誘發血管內皮細胞骨架收縮。小鼠耳朵組織中與血管通透性相關的基因(NK1R、RhoA、ROCK1、ROCK2與VEGF-A)表現,在慢性砷暴露後皆沒有顯著的變化。實驗結果顯示Rho kinase參與了慢性砷暴露小鼠受到芥子油誘發所產生的血管高滲漏現象,而Rho kinase調控的血管高滲漏與NK1R、RhoA、ROCK1、ROCK2的基因表現無關。
Vascular-related diseases including Blackfoot disease and atherosclerosis are prominent clinical findings with chronic arsenic exposure. Increased endothelial permeability is a vascular reaction to stimuli, resulting in vascular leakage. The intercellular gaps are regulated by the cytoskeletal contraction of endothelial cells. Recent studies indicate that the rho/rho kinase signaling transduction pathway plays a role in cytoskeleton reorganization and endothelial contraction. RhoA activates its downstream effector rho kinase 1 (ROCK1) and ROCK2. ROCK subsequently phosphorylates myosin phosphatase targeting subunit 1 (MYPT1). This mechanism plays a critical role in the cytoskeletal contraction. Besides, vascular endothelial growth factor - A (VEGF-A) is a key factor to regulate the vascular permeability. Previous studies developed an animal model to demonstrate that the vascular hyperpermeability was induced by mustard oil in mice fed with sodium arsenite. It is known that application of mustard oil can induce release of substance P from sensory nerves and substance P activates neurokinin-1 receptor (NK1R). My hypothesis is rho/rho kinase signaling transduction pathway mediating vascular hyperpermeability of ear skin challenged with mustard oil in mice treated with chronic exposure to arsenic. One of the objectives is to test if the rho kinase inhibitor Y-27632 could reduce the vascular hyperpermeability induced by mustard oil in mice received chronic exposure of arsenic. The expression levels of permeability related genes were measured by techniques of Western and PCR. Mice were orally fed with water (control group) and sodium arsenite (experimental group) for 2, 4 or 8 weeks. Vascular permeability changes were evaluated by the Evans blue assay and the India ink tracer techniques. The results showed that vascular leakage induced by mustard oil was enhanced in mice fed with sodium arsenite for 2, 4 or 8 weeks. Furthermore, the arsenic-induced vascular leakage was significantly reduced when the rho kinase activity was inhibited by Y-27632. The expression of NK1R, RhoA, ROCK1, ROCK2 and VEGF-A appeared to be no difference in gene and protein levels between the two groups. The present studies indicate that rho kinase is involved in vascular hyperpermeability induced by mustard oil in arsenic-fed mice. The effects of rho kinase-mediated vascular hyperpermeability are not related with expression of NK1R, RhoA, ROCK1, and ROCK2.
Abstract 1
中文摘要 3
緒論 5
縮寫表 16
藥品與儀器 17
試劑配製 23
材料與方法 29
結果 41
討論 46
參考文獻 57
圖 63
表 84
附件一 核糖核酸(Total RNA) 萃取 87
附件二 SDS-PAGE膠體電泳及西方墨點法 89
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