臺灣博碩士論文加值系統

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

Amano, M., Ito, M., Kimura, K., et al., 1996. Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J Biol Chem. 271, 20246-9.
B?鬣khed, F., Crawford, P. A., O''Donnell, D., et al., 2007. Postnatal lymphatic partitioning from the blood vasculature in the small intestine requires fasting-induced adipose factor. Proc Natl Acad Sci. 104, 606-611.
Barbara, E. R., Nathalie, F. W., Cameron, J. W., et al., 2005. Rho and vascular disease. Atherosclerosis. 183, 1-16.
Barchowsky, A., Dudek, E. J., Treadwell, M. D., et al., 1996. Arsenic induces oxidant stress and NF-kappa B activation in cultured aortic endothelial cells. Free Radic Biol Med. 21, 783-90.
Benbrahim-Tallaa, L., Waalkes, M. P., 2008. Inorganic arsenic and human prostate cancer. Environ Health Perspect. 116, 158-64.
Breslin, J. W., Yuan, S. Y., 2004. Involvement of RhoA and Rho kinase in neutrophil-stimulated endothelial hyperpermeability. Am J Physiol Heart Circ Physiol. 286, H1057-62.
Brouwer, O. F., Onkenhout, W., Edelbroek, P. M., et al., 1992. Increased neurotoxicity of arsenic in methylenetetrahydrofolate reductase deficiency. Clin Neurol Neurosurg. 94, 307-10.
Calo, L. A., Pessina, A. C., 2007. RhoA/Rho-kinase pathway: much more than just a modulation of vascular tone. Evidence from studies in humans. J Hypertens. 25, 259-64.
Campbell, J. P., Alvarez, J. A., 1989. Acute arsenic intoxication. Am Fam Physician. 40, 93-7.
Carter, D. E., Aposhian, H. V., Gandolfi, A. J., 2003. The metabolism of inorganic arsenic oxides, gallium arsenide, and arsine: a toxicochemical review. Toxicol Appl Pharmacol. 193, 309-34.
Celik, I., Gallicchio, L., Boyd, K., et al., 2008. Arsenic in drinking water and lung cancer: A systematic review. Environmental Research. 108, 48-55.
Chadzinska, M., Kolaczkowska, E., Seljelid, R., et al., 1999. Morphine modulation of peritoneal inflammation in Atlantic salmon and CB6 mice. J Leukoc Biol. 65, 590-6.
Chang, W. C., Chen, S. H., Wu, H. L., et al., 1991. Cytoprotective effect of reduced glutathione in arsenical-induced endothelial cell injury. Toxicology. 69, 101-10.
Chen, A. L., Lewis, S. P., Su, Z., et al., 1992. Superconductivity in arsenic at high pressures. Phys Rev B Condens Matter. 46, 5523-5527.
Chen, K., Chiout, P., 2001. Oral treatment of mule ducks with arsenicals for inducing fatty liver. Poult Sci. 80, 295-301.
Chen, S. C., Chen, W. C., 2008. Vascular leakage induced by exposure to arsenic via increased production of NO, hydroxyl radical and peroxynitrite. Microvasc Res. 75, 373-80.
Chen, S. C., Tsai, M. H., Wang, H. J., et al., 2004. Vascular permeability alterations induced by arsenic. Hum Exp Toxicol. 23, 1-7.
Chen, S. C., Tsai, M. H., Wang, H. J., et al., 2007. Involvement of substance P and neurogenic inflammation in arsenic-induced early vascular dysfunction. Toxicol Sci. 95, 82-8.
Chiou, H. Y., Huang, W. I., Su, C. L., et al., 1997. Dose-response relationship between prevalence of cerebrovascular disease and ingested inorganic arsenic. Stroke. 28, 1717-23.
D?朦z-Villase?瓨r, A., Burns, A. L., Hiriart, M., et al., 2007. Arsenic-induced alteration in the expression of genes related to type 2 diabetes mellitus. Toxicol Appl Pharmacol. 225, 123-133.
D?朦z-Villase?瓨r, A., Burns, A. L., Salazar, A. M., et al., 2008. Arsenite reduces insulin secretion in rat pancreatic [beta]-cells by decreasing the calcium-dependent calpain-10 proteolysis of SNAP-25. Toxicol Appl Pharmacol. 231, 291-299.
Dvorak, H. F., 2006. Discovery of vascular permeability factor (VPF). Exp Cell Res. 312, 522-526.
Engel, R. R., Hopenhayn-Rich, C., Receveur, O., et al., 1994. Vascular effects of chronic arsenic exposure: a review. Epidemiol Rev. 16, 184-209.
Esper, R. J., Vilarino, J. O., Machado, R. A., et al., 2008. Endothelial dysfunction in normal and abnormal glucose metabolism. Adv Cardiol. 45, 17-43.
Gazitt, Y., Akay, C., 2005. Arsenic trioxide: an anti cancer missile with multiple warheads. Hematology. 10, 205-13.
Guo, H. R., 2007. Cancer risks associated with arsenic in drinking water. Environ Health Perspect. 115, A339-40; author reply A340-1.
Hall, A. H., 2002. Chronic arsenic poisoning. Toxicol Lett. 128, 69-72.
Hayakawa, T., Kobayashi, Y., Cui, X., et al., 2005. A new metabolic pathway of arsenite: arsenic–glutathione complexes are substrates for human arsenic methyltransferase Cyt19. Archives of Toxicology. 79, 183-191.
Hu, J., Fang, J., Dong, Y., et al., 2005. Arsenic in cancer therapy. Anticancer Drugs. 16, 119-27.
Inoue, H., Asaka, T., Nagata, N., et al., 1997. Mechanism of mustard oil-induced skin inflammation in mice. Eur J Pharmacol. 333, 231-40.
Isabella Ruocco, A. C. C., Ryuichi Shigemoto, Alfredo Ribeiro-da-Silva,, 2001. Light and electron microscopic study of the distribution of substance P-immunoreactive fibers and neurokinin-1 receptors in the skin of the rat lower lip. The Journal of Comparative Neurology. 432, 466-480.
Ishizaki, T., Uehata, M., Tamechika, I., et al., 2000. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Mol Pharmacol. 57, 976-83.
Izquierdo-Vega, J. A., Soto, C. A., Sanchez-Pena, L. C., et al., 2006. Diabetogenic effects and pancreatic oxidative damage in rats subchronically exposed to arsenite. Toxicol Lett. 160, 135-42.
Jancso, N., Jancso-Gabor, A., Szolcsanyi, J., 1967. Direct evidence for neurogenic inflammation and its prevention by denervation and by pretreatment with capsaicin. Br J Pharmacol Chemother. 31, 138-51.
Jin, L., Burnett, A. L., 2006. RhoA/Rho-kinase in erectile tissue: mechanisms of disease and therapeutic insights. Clin Sci (Lond). 110, 153-65.
Kao, Y. H., Yu, C. L., Chang, L. W., et al., 2003. Low concentrations of arsenic induce vascular endothelial growth factor and nitric oxide release and stimulate angiogenesis in vitro. Chem Res Toxicol. 16, 460-8.
Kirk, S. L., Karlik, S. J., 2003. VEGF and vascular changes in chronic neuroinflammation. J Autoimmun. 21, 353-363.
Kumagai, Y., Sumi, D., 2007. Arsenic: Signal Transduction, Transcription Factor, and Biotransformation Involved in Cellular Response and Toxicity. Annual Review of Pharmacology and Toxicology. 47, 243-262.
Kusama, K., Nozu, F., Awai, T., et al., 2003. Deactivation of ROCK-II by Y-27632 enhances basolateral pancreatic enzyme secretion and acute pancreatitis induced by CCK analogues. Biochem Biophys Res Commun. 305, 339-44.
Larsen, G. L., Henson, P. M., 1983. Mediators of inflammation. Annu Rev Immunol. 1, 335-59.
Lee, M. Y., Jung, B. I., Chung, S. M., et al., 2003. Arsenic-induced dysfunction in relaxation of blood vessels. Environ Health Perspect. 111, 513-7.
Lee, M. Y., Lee, Y. H., Lim, K. M., et al., 2005. Inorganic arsenite potentiates vasoconstriction through calcium sensitization in vascular smooth muscle. Environ Health Perspect. 113, 1330-5.
Leung, T., Chen, X. Q., Manser, E., et al., 1996. The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol Cell Biol. 16, 5313-27.
Liu, B., Pan, S., Dong, X., et al., 2006. Opposing effects of arsenic trioxide on hepatocellular carcinomas in mice. Cancer Sci. 97, 675-81.
Louis, S. M., Jamieson, A., Russell, N. J., et al., 1989. The role of substance P and calcitonin gene-related peptide in neurogenic plasma extravasation and vasodilatation in the rat. Neuroscience. 32, 581-6.
MCDONALD, D. M., 2001. Angiogenesis and Remodeling of Airway Vasculature in Chronic Inflammation. Am. J. Respir. Crit. Care Med. 164, 39S-45.
McDonald, D. M., Bowden, J. J., Baluk, P., et al., 1996. Neurogenic inflammation. A model for studying efferent actions of sensory nerves. Adv Exp Med Biol. 410, 453-62.
Nagy, J., Benjamin, L., Zeng, H., et al., 2008. Vascular permeability, vascular hyperpermeability and angiogenesis. Angiogenesis. 11, 109-119.
Navas-Acien, A., Silbergeld, E. K., Streeter, R. A., et al., 2006. Arsenic exposure and type 2 diabetes: a systematic review of the experimental and epidemiological evidence. Environ Health Perspect. 114, 641-8.
Nordstrom, D. K., 2002. PUBLIC HEALTH: Enhanced: Worldwide Occurrences of Arsenic in Ground Water. Science. 296, 2143-2145.
Pysher, M. D., Chen, Q. M., Vaillancourt, R. R., 2008. Arsenic alters vascular smooth muscle cell focal adhesion complexes leading to activation of FAK-src mediated pathways. Toxicol Appl Pharmacol. 231, 135-41.
Ren, X. D., Schwartz, M. A., 2000. Determination of GTP loading on Rho. Methods Enzymol. 325, 264-72.
Rossman, T. G., 2003. Mechanism of arsenic carcinogenesis: an integrated approach. Mutat Res. 533, 37-65.
Saria, A., Hua, X., Skofitsch, G., et al., 1984. Inhibition of compound 48/80--induced vascular protein leakage by pretreatment with capsaicin and a substance P antagonist. Naunyn Schmiedebergs Arch Pharmacol. 328, 9-15.
Simeonova, P. P., Luster, M. I., 2004. Arsenic and atherosclerosis. Toxicol Appl Pharmacol. 198, 444-9.
Smith, A. H., Hopenhayn-Rich, C., Bates, M. N., et al., 1992. Cancer risks from arsenic in drinking water. Environ Health Perspect. 97, 259-67.
Soucy, N. V., Ihnat, M. A., Kamat, C. D., et al., 2003. Arsenic stimulates angiogenesis and tumorigenesis in vivo. Toxicol Sci. 76, 271-9.
Straub, A. C., Stolz, D. B., Vin, H., et al., 2006. Low level arsenic promotes progressive inflammatory angiogenesis and liver blood vessel remodeling in mice. Toxicol Appl Pharmacol.
Styblo, M., Del Razo, L. M., LeCluyse, E. L., et al., 1999. Metabolism of arsenic in primary cultures of human and rat hepatocytes. Chem Res Toxicol. 12, 560-5.
Sun, H., Breslin, J. W., Zhu, J., et al., 2006. Rho and ROCK signaling in VEGF-induced microvascular endothelial hyperpermeability. Microcirculation. 13, 237-47.
Takahashi, H., Shibuya, M., 2005. The vascular endothelial growth factor (VEGF)/VEGF receptor system and its role under physiological and pathological conditions. Clin Sci (Lond). 109, 227-41.
Tapio, S., Grosche, B., 2006. Arsenic in the aetiology of cancer. Mutat Res. 612, 215-46.
Tesfamariam, B., DeFelice, A. F., 2007. Endothelial injury in the initiation and progression of vascular disorders. Vascul Pharmacol. 46, 229-37.
Touyz, R. M., Schiffrin, E. L., 2004. Reactive oxygen species in vascular biology: implications in hypertension. Histochem Cell Biol. 122, 339-52.
Tsai, M.-H., Chen, S.-C., Wang, H.-J., et al., 2005. A Mouse Model for the Study of Vascular Permeability Changes Induced by Arsenic. Toxicology Mechanisms and Methods. 15, 433 - 437.
Tseng, C. H., 2002. An overview on peripheral vascular disease in blackfoot disease-hyperendemic villages in Taiwan. Angiology. 53, 529-37.
Tseng, C. H., 2005. Blackfoot disease and arsenic: a never-ending story. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 23, 55-74.
Tseng, C. H., Chong, C. K., Chen, C. J., et al., 1996. Dose-response relationship between peripheral vascular disease and ingested inorganic arsenic among residents in blackfoot disease endemic villages in Taiwan. Atherosclerosis. 120, 125-33.
Tseng, W. P., 1961. Experimental studies on the influences of the antineoplastic chemotherapeutic agents on the leukocyte picture of the peripheral blood. J Formos Med Assoc. 60, 898-925.
Tseng, W. P., 1977. Effects and dose--response relationships of skin cancer and blackfoot disease with arsenic. Environ Health Perspect. 19, 109-19.
Tseng, W. P., 1989. Blackfoot disease in Taiwan: a 30-year follow-up study. Angiology. 40, 547-58.
Utsunomiya, I., Yoshihashi, E., Tanabe, S., et al., 2008. Expression and localization of Kv1 potassium channels in rat dorsal and ventral spinal roots. Experimental Neurology. 210, 51-58.
van Nieuw Amerongen, G. P., Koolwijk, P., Versteilen, A., et al., 2003. Involvement of RhoA/Rho kinase signaling in VEGF-induced endothelial cell migration and angiogenesis in vitro. Arterioscler Thromb Vasc Biol. 23, 211-7.
Wang, C. H., Jeng, J. S., Yip, P. K., et al., 2002. Biological gradient between long-term arsenic exposure and carotid atherosclerosis. Circulation. 105, 1804-9.
Wang, S. L., Chiou, J. M., Chen, C. J., et al., 2003. Prevalence of non-insulin-dependent diabetes mellitus and related vascular diseases in southwestern arseniasis-endemic and nonendemic areas in Taiwan. Environ Health Perspect. 111, 155-59.
Weis, S. M., 2008. Vascular permeability in cardiovascular disease and cancer. Curr Opin Hematol. 15, 243-9.
Yoneda, A., Multhaupt, H. A., Couchman, J. R., 2005. The Rho kinases I and II regulate different aspects of myosin II activity. J Cell Biol. 170, 443-53.
Zhao, D., Kuhnt-Moore, S., Zeng, H., et al., 2002. Substance P-stimulated interleukin-8 expression in human colonic epithelial cells involves Rho family small GTPases. Biochem J. 368, 665-72.
蔡明憲, 神經性發炎在砷引起的血管傷害中所扮演的角色. 高雄醫學大學醫學研究所碩士論文, 2006.