臺灣博碩士論文加值系統

懷孕時母體上的所有系統尤其以內分泌以及新陳代谢系統都會因胎兒的到來，而產生不同程度的變化。已知基質金屬蛋白水解酵素（matrix metalloproteinases；MMPs）參與胚胎及成體多種生長修護反應，而gelatine更是ECM 中最主要的組成物質，因此基質金屬蛋白水解酵素中之gelatinases（MMP–2、MMP–9） 也被視為胚胎及成體成長修護的重要酵素。然而雖金屬蛋白水解酵素的活性和分泌在許多過程中都有研究，唯獨在懷孕過程中的表現則仍甚少討論。因此為了探討懷孕過程中生理的改變所造成的影響，首先我們收集40位孕婦血清依類別及時間，將實驗組檢體分成孕期2個月孕婦、孕期7個月孕婦血清檢體，探討母體在正常懷孕過程中血清內臨床生化數值相關性及MMP -2和MMP-9活性。發現臨床生化項目的檢查中， Cholesterol（總膽固醇）、LDL-C（低密度脂蛋白）及LDH（血清乳酸脫氫酶）之檢驗數值在孕婦後期顯著升高。雖MMP-2活性表現量沒有太大的差異，但發現孕婦MMP-9活性表現量比非孕婦高，說明懷孕的變化的過程中MMP-9活性表現量受到影響。且懷孕二個月孕婦MMP-9活性表現量是高於孕期七個月孕婦MMP-9活性表現量，顯示懷孕末期MMP-9活性表現量日漸趨緩，說明母體對懷孕產生的變化漸趨適應。同時已知血清內MMP-2與MMP-9的變化與B型肝炎患者轉變為肝硬化甚至肝癌具有絕對相關，因此我們想進一步探討懷孕過程中，是否對於B型肝炎疾病所誘發之MMPs有所影響。我們收集B型肝炎患者孕婦15人與B型肝炎患者非孕婦26人血清，以Gelatin zymography檢測血清中MMP-2、MMP-9活性，發現B型肝炎患者其血清中MMP-2活性表現量有明顯的增加，但B型肝炎患者孕婦血清中之MMP-2活性表現量則與正常人相近；同時B型肝炎患者與B肝孕婦血清中MMP-9活性表現量皆有明顯的增加，顯示出B型肝炎會誘導或增加MMP-9活性表現。但B型肝炎患者血清中MMP-9活性表現量較懷孕的B型肝炎患者血清中MMP-9活性表現量高，顯示懷孕之過程影響B型肝炎所誘導之MMP-9活性表現。結果顯示B型肝炎感染促進了血清中MMP-9活性，且懷孕之過程具有保護及減緩B型肝炎對宿主的傷害作用。

In order to accommodate the arrival of newborns, pregnancy leads to various degrees of changes to mother’s body. The main changes include changes in endocrine system and body metabolism. Numerous published literatures indicated that matrix metalloproteinases MMPs is involved in significant number of physiological reactions. Such as embryo development and adult system repair. Since gelatin is the most critical substance in ECM, and gelatinases (MMP-2, MMP-9) is considered as the most important MMPs. The activity and secretion of matrix metalloproteinases in diverse metabolic pathways have been investigated; however, the expression of MMP during pregnancy was rarely discussed. In order to examine the influence of physiological changes and MMP activity during pregnancy, serum samples from 40 pregnant women were collected. Based on category and time frame, the samples were classified into 2-month and 7-month pregnant serum sample to investigate the changes in MMP-2 and MMP-9 activities in mothers, and possible trends with respect to clinical biochemistry data. Clinical biochemistry data indicated that cholesterol, LDL-C(low density lipoprotein-cholesterol) and LDH (lactate dehydrogenase) elevated to significant level during the latter stage of pregnancy. No significant difference was observed in the MMP-2 activity level of pregnant women in different trimesters and health individuals. However,the MMP-9 activity in pregnant women was higher than those who were not pregnant, suggesting changes during pregnancy would influence the activity level of MMP-9. Besides, MMP-9 activity level in 2-month pregnant women is higher than those 7-month pregnant, suggesting that certain adaptive mechanism during the progress of pregnancy caused the gradual decline of MMP-9 activity level. We already known that changes of MMP-2 and MMP-9 in serum of hepatitis B infected patients can used as an indicator for hepatitis progressing into cirrhosis or even liver cancer. Therefore, the effect of pregnancy on hepatitis B infected liver disease induced MMPs was investigated. Serum samples were collected from hepatitis B patients who were pregnant and not pregnant. Activity level of MMP-2 in hepatitis B patients showed significant increase; yet, when comparing the MMP-2 activity level in pregnant hepatitis B patients and health individuals, it was observed that MMP-2 activity levels in serum of pregnant hepatitis B patients was comparable to the health individuals.In addition, MMP-9 activity level in serum demonstrated significant increase in both pregnant hepatitis B patients and hepatitis B patients, indicating that hepatitis B would induce or increase the activity level of MMP-9. Nevertheless, MMP-9 activity level in hepatitis B patients was higher than those who were pregnant, implying that pregnancy would modulate the activity level of hepatitis B-induced MMP-9.Taken together,the activity level of MMP-9 in serum would increase due to hepatitis B infection; moreover, pregnancy status would modulate the effect of hepatitis B on the host.

中文摘要 1
英文摘要 3
縮寫表 5
前言 6
背景介紹 10
實驗動機 25
實驗材料與實驗方法 26
統計分析 34
實驗結果 35
討論 38
參考文獻 44
圖表與說明 55
附圖 60

1. Bernhard H Heidemann and John H McClure, Changes in maternal physiology during pregnancy CEACCP, Jun 2003; 3: 65 - 68.
2.W.A.H.J.Van Stiphout,A.Hofman , Bruijnserum,Lipids in young women before during and after pregnancy. Am.J.Epidemiol..
1987; 126: 922-92
3.RossR.The pathogenesis of atherosclerosis：a perspective for the 1990s.Nature.362(6423)：801-9.Review(1993)
4.LibbyP.Current concepts of the pathogenesis of the acute coronary syndrome.Circulation.104:365-372(2001)
5.Paul H. Black*, Lisa D. Garbutt(2002) ,Stress inflammation and cardiovascular disease Journal of Psychosomatic; Research 52,1–23
6.Davies, M.J., Richardson, P.D., and Woolf, N. (1993). Risk of
thrombosis in human atherosclerotic plaques: role of extracellular ipid, macrophage, and smooth muscle cell content. Br. Heart J. 69,377–381.
7.Cuchel M, Rader DJ.（2002）The role of high density lipoproteins in thrombosis. ScientificWorldJournal. Jan 12;2(1):89-95.
8.Diaz MN, Frei B and Vita JA (1997) Antioxidants and atherosclerotic heart disease. N Engl J Med 337:408-416.
9.Galis, Z.S., Sukhova, G.K., Lark, M.W., and Libby, P. (1994).Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J. Clin. Invest. 94, 2493–2503.
10.Carmeliet, P. (2000). Proteins in cardiovascular aneurysms and rupture:targets for therapy? J. Clin. Invest. 105, 1519–1520.
11.M ason D P , K enagy R D , H asenstab D , et al. Matrix
metalloproteinase-9 over expression enhances vascular smooth muscle cell migration and altersrem odeling in the injured rat carotid artery [J]. C irc R es, 1999, 85(12):1179-1185.
12.Lau JYN, Wright TL. Molecular virology and pathogenesis of hepatitis B. Lancet 1993; 342: 1335–40.
13.Chang, M. H., Chen, C. J., Lai, M. S., Hsu, H. M., Wu, T. C., Kong, M. S., et al. (1997).Universal hepatitis B vaccination in Taiwan and the incidence of hepatocellularcarcinoma in children. The New England Journal of Medicine, 336(26), 1855-1859.
14.Friedman SL. Cytokines and fibrogenesis. Semin. Liver Dis. 1999; 19: 129–40.
15.Murawaki Y, Yamada S, Ikuta Y, Kawasaki H. Clinical usefulness of serum matrix metalloproteinase-2 concentration in patients with chronic viral liver disease. J. Hepatol. 1999; 30: 1090–8.
16.Takahara T, Furui K, Yata Y et al. Dual expression of matrix metalloproteinase-2 and membrane-type 1-matrix metalloproteinase in fibrotic human livers. Hepatology 1997; 26: 1521–9.
17.Arii S, Mise M, Harada T et al. Overexpression of matrix metalloproteinase 9 gene in hepatocellular carcinoma with invasive potential. Hepatology 1996; 24: 316–22.
18.Hayasaka A, Suzuki N, Fujimoto N et al. Elevated plasma levels of matrix metalloproteinase-9 (92-kd type IV collagenase/gelatinase B) in hepatocellular carcinoma. Hepatology 1996; 24: 1058–62.
19.Xu, G.F., et al., Dynamic changes in the expression of matrix metalloproteinases and their inhibitors, TIMPs, during hepatic fibrosis induced by alcohol in rats. World J Gastroenterol, 2004. 10(24): p. 3621-7.
20.Benyon, R.C. and M.J. Arthur, Extracellular matrix degradation and the role of hepatic stellate cells. Semin Liver Dis, 2001. 21(3): p. 373-84.
21.Pijuan Domenech A, Gatzoulis MA. Pregnancy and heart disease Rev Esp Cardiol. 2006 Sep;59(9):971-84. Review. Spanish
22.Yamada, Y., Doi, T., Hamakubo, T., and Kodama, T. (1998). Scavenger receptor family proteins: roles for atherosclerosis, host defence and disorders of the central nervous system. Cell. Mol. Life .54,628–640.
23.Chan AW, Bhatt DL, Chew DP, Reginelli J, Schneider JP, Topol EJ, Ellis SG（2003）Relation of inflammation and benefit of statins after percutaneous coronary interventions. Circulation ;107:1750-1756.
24.Tambunting, F., et al., Increased lung matrix metalloproteinase-9 levels in extremely premature baboons with bronchopulmonary dysplasia. Pediatr Pulmonol, 2005. 39(1): p. 5-14.
25.Iglesias, D., et al., Metalloproteinases and tissue inhibitors of metalloproteinases in exudative pleural effusions. Eur Respir J, 2005. 25(1): p. 104-9.
26.May, A.E., et al., Plasminogen and matrix metalloproteinase activation by enzymatically modified low density lipoproteins in monocytes and smooth muscle cells. Thromb Haemost, 2005. 93(4): p. 710-5.
27.Haruta I, Tokushige K, Komatsu T, Ikeda I, Yamauchi K, Hayashi N. Clinical implication of vascular cell adhesion molecule-1 and very late activation antigen−4 interaction, and matrix metalloproteinase-2 production in patients with liver disease. Can. J. Gastroenterol. 1999; 13: 721–7.
28.Chung TW, Kim JR, Suh JI, Lee YC, Chang YC, Chung TH, Kim CH. Correlation between plasma levels of matrix metalloproteinase (MMP)-9 /MMP-2 ratio and alpha-fetoproteins in chronic hepatitis carrying hepatitis B virus.J Gastroenterol Hepatol. 2004 May;19(5):565-71.
29.Jiang, Z.Q., et al., [Relationship between expression of matrix metalloproteinase (MMP-9) and tumor angiogenesis, cancer cell proliferation, invasion, and metastasis in invasive carcinoma of cervix]. Ai Zheng, 2003. 22(2): p. 178-84.
30.Wang, L., et al., [Expression of MMP-9 and MMP-9 mRNA in gastric carcinoma and its correlation with angiogenesis]. Zhonghua Yi Xue Za Zhi, 2003. 83(9): p. 782-6.
31.Zhang, S., et al., Imbalance between expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in invasiveness and metastasis of human gastric carcinoma. World J Gastroenterol, 2003. 9(5): p. 899-904.
32.Chen, J.J., et al., Tumor-associated macrophages: the double-edged sword in cancer progression. J Clin Oncol, 2005. 23(5): p. 953-64.
33.Gu, Z.D., et al., Clinical significance of matrix metalloproteinase-9 expression in esophageal squamous cell carcinoma. World J Gastroenterol, 2005. 11(6): p. 871-4.
34.De Vicente, J.C., et al., Expression and clinical significance of matrix metalloproteinase-2 and matrix metalloproteinase-9 in oral squamous cell carcinoma. Oral Oncol, 2005. 41(3): p. 283-93.
35.Yamada, Y., Doi, T., Hamakubo, T., and Kodama, T. (1998). Scavenger receptor family proteins: roles for atherosclerosis, host defence and disorders of the central nervous system. Cell. Mol. Life .54,628–640.
36.Chan AW, Bhatt DL, Chew DP, Reginelli J, Schneider JP, Topol EJ, Ellis SG（2003）Relation of inflammation and benefit of statins after percutaneous coronary interventions. Circulation ;107:1750-1756.
37.Nagase H, Woessner JF Jr. (1999) Matrix metalloproteinases. J Biol Chem 274:21491–21494.
38.Sternlicht MD, Werb Z. (2001) How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol 17:463–516.
39.Dollery CM, McEwwan JR, Henney AM. (1995) Matrix metalloproteinases and cardiovascular disease. Circ Res 77:863-868.
40.S. L. Parsons, S. A. Watson, P. D. Brown, H. M. Collins,
and R. J. C. Steele：Matrix metalloproteinases. British
ournal of Surgery, Vol. 84, 160–166, 1997
41.Jukka Westermarck and Veli–Matti Khri：Regulation of
matrix metalloproteinase expression in tumor invasion.
FASEB J., Vol. 13, 781–792, 1999
42.Gum R, Lengyel E, Juarez J, Chen JH, Sato H, Seiki M, Boyd D. (1996) Stimulation of 92-kDa gelatinase B promoter activity by ras is mitogen-activated protein kinase kinase 1-independent and requires multiple transcription factor binding sites including closely spaced PEA3/ets and AP-1 sequences. J Biol Chem 271:10672-10680.
43.Creemers EE, Cleutjens JP, Smits JF, Daemen MJ. (2001) Matrix metalloproteinase inhibition after myocardial infarction: a new approach to prevent heart failure? Circ Res 89:201–210.
44.Spinale FG. (2002) Matrix metalloproteinases: regulation and dysregulation in the failing heart. Circ Res 90:520–530.
45.Galis ZS, Khatri JJ. (2002) Matrix metalloproteinases in vascular remodeling and atherogenesis: the good, the bad, and the ugly. Circ Res 90:251–262.
46.Rohde LE, Ducharme A, Arroyo LH, Aikawa M, Sukhova GH, Lopez-Anaya A, McClure KF, Mitchell PG, Libby P, Lee RT. (1999) Matrix metalloproteinase inhibition attenuates early left ventricular enlargement after experimental myocardial infarction in mice. Circulation 99:3063-3070.
47.Heymans S, Luttun A, Nuyens D, Theilmeier G, Creemers E, Moons L, Dyspersin GD, Cleutjens JPM, Shipley M, Angellilo A, Levi M, Nube O, Baker A, Keshet E, Lupu F, Herbert JM, Smits JF, Shapiro SD, Baes M, Borgers M, Collen D, Daemen MJ, Carmeliet P. (1999) Inhibition of plasminogen activators or matrix metalloproteinases prevent cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure. Nat Med 10:1135–1142.
48.Ducharme A, Frantz S, Aikawa M, Rabkin E, Lindsey M, Rohde LE, Schoen FJ, Kelly RA, Werb Z, Libby P, Lee RT. (2000) Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction. J Clin Invest 106:55-62.
49.Barker, L. F., & Murray, R. (1972). Relationship of virus dose to incubation time of clinical hepatitis and time of appearance of hepatitis-associated antigen. American Journal of the Medical Sciences, 263(1), 27-33.
50.Dienstag, J. L., & Isselvacher, K. J. (1994). Harrison''s Principles of Internal Medicine (13 ed.). New York: McGRAW-Hill.
51.De Clercq, E. Perspectives for the treatment of hepatitis B virus
infections. J. Antimicrob. Agents 12: 81-95, 1999
52.Ganem, D., and Varmus, H. E. The molecular biology of the hepatitis Bviruses. Ann. Rev. Biochem. 56: 651-93, 1987.
53.Monjardino, J., 1998. Molecular biology of human hepatitis viruses.Imperial College Press pp21-61.
54.Murphy, F., et al., N-Cadherin cleavage during activated hepatic stellate cell apoptosis is inhibited by tissue inhibitor of metalloproteinase-1. Comp Hepatol, 2004. 3 Suppl 1: p. S8.
55.Gardi, C., et al., Effect of free iron on collagen synthesis, cell proliferation and MMP-2 expression in rat hepatic stellate cells. Biochem Pharmacol, 2002. 64(7): p. 1139-45.
56. Iredale, J.P., et al., Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors. J Clin Invest, 1998. 102(3): p. 538-49.
57.Arthur MJP. Degradation of matrix proteins in liver fibrosis. Pathol. Res. Pract. 1994; 190: 825–33.
58.Makkonen M, Puhakainen E, Hänninen O, Castrén O，Lactate dehydrogenase isoenzymes in human myometrium during pregnancy and labor. Acta Obstet Gynecol Scand. 1982;61(1):35-7.
59.R. Visse and H. Nagase, Matrix metalloproteinases and tissue inhibitors of metalloproteinases. Structure, function, and biochemistry, Circ Res 92 (2003), pp. 827–839.
60.M.P. Jacob, Extracellular matrix remodeling and matrix metalloproteinases in the vascular wall during aging and in pathological conditions, Biomed Pharmacother 57 (2003), pp. 195–202.
61.Brown, D. L., Hibbs, M. S., Kearney, M., Loushin, C. and Isner, J. M. (1995) Identification of 92-kD gelatinase in human coronary atherosclerotic lesions. Association of active enzyme synthesis with unstable angina. Circulation 91 , pp. 2125-2131.
62.Nagase H, Woessner Jr JF 1999 Matrix metalloproteinases. J Biol Chem 274:21491–21494
63.Woessner Jr JF, Nagase H 2000 Matrix metalloproteinases and TIMPs. New York: Oxford University Press
64.Shimonovitz S, Hurwitz A, Dushnik M, Anteby E, Geva-Eldar T, Yagel S 1994 Developmental regulation of 72 kD and 92 kD type IV collagenases in human trophoblasts: a possible mechanism for control of trophoblast invasion. Am J Obstet Gynecol 171:832–838
65.Lippi G,Albiero A, et al., Lipid and lipoprotein profile in physiological pregnancy. Clin Lab. 2007;53(3-4):173-7.
66.Steinberg, D., and Witztum, J.L. (1999). Lipoproteins, Lipoprotein,Oxidation, and Atherogenesis, K.R. Chien, ed. (Philadelphia: W.B.Saunders Co.).
67.Navab, M., Berliner, J.A., Watson, A.D., Hama, S.Y., Territo, M.C.,Lusis, A.J., Shih, D.M., Van Lenten, B.J., Frank, J.S., Demer, L.L.,et al. (1996). The yin and yang of oxidation in the development ofthe fatty streak. Arterioscler. Thromb. Vasc. Biol. 16, 831–842.
68.Ross, R. (1999). Atherosclerosis— an inflammatory disease. N. Eng.J. Med 340, 115–126.
69.Montano, L., Miescher, G. C., Goodall, A. H., Wledimann, K. H., Janossy, G., & Thomas, H.C. (1982). Hepatitis B virus and HLA antigen display in the liver during chronic hepatitis B virus infection. Hepatology, 2(5), 557-561
70.Haruta I, Tokushige K, Komatsu T, Ikeda I, Yamauchi K, Hayashi N. Clinical implication of vascular cell adhesion molecule-1 and very late activation antigen−4 interaction, and matrix metalloproteinase-2 production in patients with liver disease. Can. J. Gastroenterol. 1999; 13: 721–7.
71.Chung TW, Kim JR, Suh JI, Lee YC, Chang YC, Chung TH, Kim CH. Correlation between plasma levels of matrix metalloproteinase (MMP)-9 /MMP-2 ratio and alpha-fetoproteins in chronic hepatitis carrying hepatitis B virus. J Gastroenterol Hepatol. 2004 May;19(5):565-71.
72.Xu, L., et al., Human hepatic stellate cell lines, LX-1 and LX-2:
new tools for analysis of hepatic fibrosis. Gut, 2005. 54(1): p. 142-51.
73.Reif, S., et al., Matrix Metalloproteinases 2 and 9 Are Markers of Inflammation but Not of the Degree of Fibrosis in Chronic Hepatitis C. Digestion, 2005. 71(2): p. 124-130.
74.Jong GP, Ma T., et al., Serum MMP-9 activity as a diagnosing marker for the developing heart failure of post MI patients. Chin J Physiol. 2006 Apr 30;49(2):104-9
75.Ichiro Shimizu, Susumu Ito, Protection of estrogens against the progression of chronic liver disease. Hepatology Research Vol. 37 Issue 4 Page 239 April 2007