肝纖維化代表對慢性肝損傷後反覆癒合、結疤反應所造成的結果。Interleukin-10（IL-10）是一個強力抑制發炎反應的細胞激素、它具有調節肝硬化的作用。本論文的目的在於探討IL-10基因治療是否具有抗小鼠肝纖維化的作用。於飲水中長期添加thioacetamide以誘發小鼠肝纖維化產生，在肝硬化產生後，經由電穿孔基因傳送法將人類IL-10表現載體導入。結果顯示，IL-10基因治療可反轉已形成的肝纖維化、以及抑制肝硬化所引起的細胞凋亡。反轉錄聚合酶鏈反應顯示IL-10基因治療可減緩TGF-β1、TNF-α、細胞粘連分子、和TIMPs mRNA的增表現。同時IL-10基因治療，也顯著抑制α-SMA和COX-2的活化。簡言之，電穿孔IL-10基因治療可提供肝硬化一個新穎的治療途徑，並具有臨床應用的潛力。

Hepatic fibrosis represents a process of healing and scarring in response to chronic liver injury. Interleukin-10 (IL-10) is a cytokine that downregulates the proinflammatory response and has a modulatory effect on hepatic fibrogenesis. The aim of this study was to investigate whether IL-10 gene therapy possesses anti-hepatic fibrogenesis in mice. Liver fibrosis was induced by long-term thioacetamide administration in mice. Human IL-10 expression plasmid was delivered via electroporation after liver fibrosis established. IL-10 gene therapy reversed hepatic fibrosis and prevented cell apoptosis in a thioacetamide-treated liver. RT-PCR revealed IL-10 gene therapy could reduce liver transforming growth factor-β1（TGF-β1）, tumor necrosis factor-α（TNF-α）, collagen α1, cell adhesion molecule, and tissue inhibitors of metalloproteinase（TIMPs）mRNA upregulation. Following gene transfer, the activation of α-smooth muscle actin（α-SMA）and cyclooxygenase-2（COX-2）were significantly attenuated. In brief, electroporative IL-10 gene therapy might be an effective therapeutic reagent for liver fibrosis with potential future clinical applications.

背 景 介 紹 ••••••••••••••••••••• 1
肝纖維化簡介 ••••••••••••••••••••• 2
肝纖維化病理機轉 ••••••••••••••••••• 4
肝臟纖維化細胞內訊號調控 ••••••••••••••• 6
肝纖維化是可逆? • • •••••••••••••••• 6
肝纖化相關的細胞激素 ••••••••••••••••• 7
肝纖維化的治療 •••••••••••••••••••• 8
細胞激素Interleukin-10 •••••••••••••••• 9
材 料 與 方 法 •••••••••••••••••• 11
動物 • ••••••••••••••••••••••• 12
Interleukin-10表現載體 •••••••••••••••• 12
肝纖維化誘發和基因治療 •••••••••••••••• 14
組織病理和免疫化學染色 •••••••••••••••• 15
可溶性膠原纖維的測量 ••••••••••••••••• 18
反轉錄聚合酶鏈 •••••••••••••••••••• 18
統計分析••••••••••••••••••••••• 20
結 果 •••••••••••••••••••••••• 21
肝臟病理變化 ••••••••••••••••••••• 22
IL-10和COX-2的免疫化學染色•••••••••••••• 22
TUNEL染色 •••••••••••••••••••••• 23
反轉錄聚合酶鏈反應 •••••••••••••••••• 23
討 論 ••••••••••••••••••••••• 32
參 考 文 獻 ••••••••••••••••••••• 38
圖
圖一、• ••••••••••••••••••••••• 25
圖二、•••••••••••••••••••••••• 26
圖三、 •••••••••••••••••••••••• 27
圖四、 •••••••••••••••••••••••• 28
圖五、 •••••••••••••••••••••••• 29
圖六、 •••••••••••••••••••••••• 30
表一、 •••••••••••••••••••••••• 31
附錄
附錄一、•••••••••••••••••••••• 58
附錄二、••••••••••••••••••••••• 59
附錄三、••••••••••••••••••••••• 60
附錄四、••••••••••••••••••••••• 61
附錄五、••••••••••••••••••••••• 62
附錄六、•••••••••••••••••••••••• 63

1. Bataller R, Brenner DA. Liver fibrosis. J Clin Invest 2005;115:209-18.
2. Gines P, Cardenas A, Arroyo V, Rodes J. Management of cirrhosis and ascites. N Engl J Med 2004;350:1646-54.
3. Popper H, Uenfriend S. Hepatic fibrosis. Correlation of biochemical and morphologic investigations. Am J Med 1970;49:707-21.
4. Soyer MT, Ceballos R, Aldrete JS. Reversibility of severe hepatic damage caused by jejunoileal bypass after re-establishment of normal intestinal continuity. Surgery 1976;79:601-4.
5. Friedman SL, Roll FJ, Boyles J, Bissell DM. Hepatic lipocytes: the principal collagen-producing cells of normal rat liver. Proc Natl Acad Sci USA 1985;82:8681-5.
6. Rockey DC, Boyles JK, Gabbiani G, Friedman SL. Rat hepatic lipocytes express smooth muscle actin upon activation in vivo and in culture. J Submicrosc Cytol Pathol 1992;24:193-203.
7. Poynard T, Bedossa P, Opolon P. Natural history of liver fibrosis progression in patients with chronic hepatitis C. The OBSVIRC, METAVIR, CLINIVIR, and DOSVIRC groups. Lancet 1997;349:825-32.
8. Everson GT. Treatment of hepatitis C in patients who have decompensated cirrhosis. Clin Liver Dis 2005;9:473-86.
9. Bataller R, North KE, Brenner DA. Genetic polymorphisms and the progression of liver fibrosis: a critical appraisal. Hepatology 2003;37:493-503.
10. Afdhal NH, Nunes D. Evaluation of liver fibrosis: a concise review. Am J Gastroenterol 2004;99:1160-74.
11. Hui AY, Liew CT, Go MY, Chim AM, Chan HL, Leung NW, Sung JJ. Quantitative assessment of fibrosis in liver biopsies from patients with chronic hepatitis B. Liver Int 2004;24:611-8.
12. Imbert-Bismut F, Ratziu V, Pieroni L, Charlotte F, Benhamou Y, Poynard T; MULTIVIRC Group. Biochemical markers of liver fibrosis in patients with hepatitis C virus infection: a prospective study. Lancet 2001;357:1069-75.
13. Lichtinghagen R, Bahr MJ. Noninvasive diagnosis of fibrosis in chronic liver disease. Expert Rev Mol Diagn 2004;4:715-26.
14. Callewaert N, Van Vlierberghe H, Van Hecke A, Laroy W, Delanghe J, Contreras R. Noninvasive diagnosis of liver cirrhosis using DNA sequencer-based total serum protein glycomics. Nat Med 2004;10:429-34.
15. Desmet VJ, Roskams T. Cirrhosis reversal: a duel between dogma and myth. J Hepatol 2004;40:860-7.
16. Pinzani M, Rombouts K, Colagrande S. Fibrosis in chronic liver diseases: diagnosis and management. J Hepatol 2005;42(Suppl):S22-36.
17. Arthur MJ. Fibrogenesis II. Metalloproteinases and their inhibitors in liver fibrosis. Am J Physiol Gastrointest Liver Physiol 2000;279:G245-9.
18. Gabele E, Brenner DA, Rippe RA. Liver fibrosis: signals leading to the amplification of the fibrogenic hepatic stellate cell. Front Biosci 2003;8:D69-77.
19. Pinzani M. PDGF and signal transduction in hepatic stellate cells. Front Biosci 2002 ;7:D1720-6.
20. Wandzioch E, Kolterud A, Jacobsson M, Friedman SL, Carlsson L. Lhx2-/- mice develop liver fibrosis. Proc Natl Acad Sci USA 2004;101:16549-54.
21. Tsukada S, Westwick JK, Ikejima K, Sato N, Rippe RA. SMAD and p38 MAPK signaling pathways independently regulate α1(I) collagen gene expression in unstimulated and transforming growth factor-β-stimulated hepatic stellate cells. J Biol Chem 2005;280:10055-64.
22. Marra F, Arrighi MC, Fazi M, Caligiuri A, Pinzani M, Romanelli RG, Efsen E, Laffi G, Gentilini P. Extracellular signal-regulated kinase activation differentially regulates platelet-derived growth factor's actions in hepatic stellate cells, and is induced by in vivo liver injury in the rat. Hepatology 1999;30:951-8.
23. Schwabe RF, Uchinami H, Qian T, Bennett BL, Lemasters JJ, Brenner DA. Differential requirement for c-Jun NH2-terminal kinase in TNFα- and Fas-mediated apoptosis in hepatocytes. FASEB J 2004;18:720-2.
24. Schnabl B, Kweon YO, Frederick JP, Wang XF, Rippe RA, Brenner DA. The role of Smad3 in mediating mouse hepatic stellate cell activation. Hepatology 2001;34:89-100.
25. Dooley S, Hamzavi J, Breitkopf K, Wiercinska E, Said HM, Lorenzen J, Ten Dijke P, Gressner AM. Smad7 prevents activation of hepatic stellate cells and liver fibrosis in rats. Gastroenterology 2003;125:178-91.
26. Boya P, Larrea E, Sola I, Majano PL, Jimenez C, Civeira MP, Prieto J. Nuclear factor-kappa B in the liver of patients with chronic hepatitis C: decreased RelA expression is associated with enhanced fibrosis progression. Hepatology 2001;34:1041-8.
27. Toyama T, Nakamura H, Harano Y, Yamauchi N, Morita A, Kirishima T, Minami M, Itoh Y, Okanoue T. PPARα ligands activate antioxidant enzymes and suppress hepatic fibrosis in rats. Biochem Biophys Res Commun 2004;324:697-704.
28. Benyon RC, Iredale JP. Is liver fibrosis reversible? Gut 2000;46:443-6.
29. Arthur MJ. Reversibility of liver fibrosis and cirrhosis following treatment for hepatitis C. Gastroenterology 2002;122:1525-8.
30. Issa R, Zhou X, Constandinou CM, Fallowfield J, Millward-Sadler H, Gaca MD, Sands E, Suliman I, Trim N, Knorr A, Arthur MJ, Benyon RC, Iredale JP. Spontaneous recovery from micronodular cirrhosis: evidence for incomplete resolution associated with matrix cross-linking. Gastroenterology 2004;126:1795-808.
31. Shi Z, Wakil AE, Rockey DC. Strain-specific differences in mouse hepatic wound healing are mediated by divergent T helper cytokine responses. Proc Natl Acad Sci USA 1997;94:10663-8.
32. Schwabe RF, Bataller R, Brenner DA. Human hepatic stellate cells express CCR5 and RANTES to induce proliferation and migration. Am J Physiol Gastrointest Liver Physiol 2003;285:G949-58.
33. Shek FW, Benyon RC. How can transforming growth factor-β be targeted usefully to combat liver fibrosis? Eur J Gastroenterol Hepatol 2004;16:123-6.
34. Sudo K, Yamada Y, Moriwaki H, Saito K, Seishima M. Lack of tumor necrosis factor receptor type 1 inhibits liver fibrosis induced by carbon tetrachloride in mice. Cytokine 2005;29:236-44.
35. Campbell JS, Hughes SD, Gilbertson DG, Palmer TE, Holdren MS, Haran AC, Odell MM, Bauer RL, Ren HP, Haugen HS, Yeh MM, Fausto N. Platelet-derived growth factor C induces liver fibrosis, steatosis, and hepatocellular carcinoma. Proc Natl Acad Sci USA 2005;102:3389-94.
36. Bataller R, Sancho-Bru P, Gines P, Brenner DA. Liver fibrogenesis: a new role for the renin-angiotensin system. Antioxid Redox Signal 2005;7:1346-55.
37. Planaguma A, Claria J, Miquel R, Lopez-Parra M, Titos E, Masferrer JL, Arroyo V, Rodes J. The selective cyclooxygenase-2 inhibitor SC-236 reduces liver fibrosis by mechanisms involving non-parenchymal cell apoptosis and PPARgamma activation. FASEB J 2005;19:1120-2.
38. Shen XH, Cheng WF, Li XH, Sun JQ, Li F, Ma L, Xie LM. Effects of dietary supplementation with vitamin E and selenium on rat hepatic stellate cell apoptosis. World J Gastroenterol 2005;11:4957-61.
39. Lieber CS, Leo MA, Cao Q, Ren C, DeCarli LM. Silymarin retards the progression of alcohol-induced hepatic fibrosis in baboons. J Clin Gastroenterol 2003;37:336-9.
40. Oe S, Fukunaka Y, Hirose T, Yamaoka Y, Tabata Y. A trial on regeneration therapy of rat liver cirrhosis by controlled release of hepatocyte growth factor. J Control Release 2003;88:193-200.
41. Ueki T, Kaneda Y, Tsutsui H, Nakanishi K, Sawa Y, Morishita R, Matsumoto K, Nakamura T, Takahashi H, Okamoto E, Fujimoto J. Hepatocyte growth factor gene therapy of liver cirrhosis in rats. Nat Med 1999;5:226-30.
42. Asadullah K, Sterry W, Trefzer U. Cytokines: interleukin and interferon therapy in dermatology. Clin Exp Dermatol 2002;27:578-84.
43. Mosmann TR, Coffman RL. Heterogeneity of cytokine secretion patterns and functions of helper T cells. Adv Immunol 1989;46:111-47.
44. Asadullah K, Sterry W, Volk HD. Interleukin-10 therapy--review of a new approach. Pharmacol Rev 2003;55:241-69.
45. Zdanov A. Structural features of the interleukin-10 family of cytokines. Curr Pharm Des 2004;10:3873-84.
46. Williams LM, Ricchetti G, Sarma U, Smallie T, Foxwell BM. Interleukin-10 suppression of myeloid cell activation--a continuing puzzle. Immunology 2004;113:281-92.
47. Moore KW, de Waal Malefyt R, Coffman RL, O'Garra A. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 2001;19:683-765.
48. Rennick DM, Fort MM. Lessons from genetically engineered animal models. XII. IL-10-deficient IL-10(-/-) mice and intestinal inflammation. Am J Physiol Gastrointest Liver Physiol 2000;278:G829-33.
49. Ikizceli I, Yurumez Y, Avsarogullari L, Kucuk C, Sozuer EM, Soyuer I, Yavuz Y, Muhtaroglu S. Effect of interleukin-10 on pancreatic damage caused by organophosphate poisoning. Regul Toxicol Pharmacol 2005;42:260-4.
50. Mekala DJ, Alli RS, Geiger TL. IL-10-dependent suppression of experimental allergic encephalomyelitis by Th2-differentiated, anti-TCR redirected T lymphocytes. J Immunol 2005;174:3789-97.
51. Goudy KS, Burkhardt BR, Wasserfall C, Song S, Campbell-Thompson ML, Brusko T, Powers MA, Clare-Salzler MJ, Sobel ES, Ellis TM, Flotte TR, Atkinson MA. Systemic overexpression of IL-10 induces CD4+CD25+ cell populations in vivo and ameliorates type 1 diabetes in nonobese diabetic mice in a dose-dependent fashion. J Immunol 2003;171:2270-8.
52. Saidenberg-Kermanac'h N, Bessis N, Deleuze V, Bloquel C, Bureau M, Scherman D, Boissier MC. Efficacy of interleukin-10 gene electrotransfer into skeletal muscle in mice with collagen-induced arthritis. J Gene Med 2003;5:164-71.
53. Oberholzer C, Oberholzer A, Bahjat FR, Minter RM, Tannahill CL, Abouhamze A, LaFace D, Hutchins B, Clare-Salzler MJ, Moldawer LL. Targeted adenovirus-induced expression of IL-10 decreases thymic apoptosis and improves survival in murine sepsis. Proc Natl Acad Sci USA 2001;98:11503-8.
54. Hong IC, Mullen PM, Precht AF, Khanna A, Li M, Behling C, Lopez VF, Chiou HC, Moss RB, Hart ME. Non-viral human IL-10 gene expression reduces acute rejection in heterotopic auxiliary liver transplantation in rats. Microsurgery 2003;23:432-6.
55. Martins S, de Perrot M, Imai Y, Yamane M, Quadri SM, Segall L, Dutly A, Sakiyama S, Chaparro A, Davidson BL, Waddell TK, Liu M, Keshavjee S. Transbronchial administration of adenoviral-mediated interleukin-10 gene to the donor improves function in a pig lung transplant model. Gene Ther 2004;11:1786-96.
56. van Roon J, Wijngaarden S, Lafeber FP, Damen C, van de Winkel J, Bijlsma JW. Interleukin 10 treatment of patients with rheumatoid arthritis enhances Fc gamma receptor expression on monocytes and responsiveness to immune complex stimulation. J Rheumatol 2003;30:648-51.
57. Weiss E, Mamelak AJ, La Morgia S, Wang B, Feliciani C, Tulli A, Sauder DN. The role of interleukin 10 in the pathogenesis and potential treatment of skin diseases. J Am Acad Dermatol 2004;50:657-75.
58. Nelson DR, Lauwers GY, Lau JY, Davis GL. Interleukin 10 treatment reduces fibrosis in patients with chronic hepatitis C: a pilot trial of interferon nonresponders. Gastroenterology 2000;118:655-60.
59. Andersen SR, Lambrecht LJ, Swan SK, Cutler DL, Radwanski E, Affrime MB, Garaud JJ. Disposition of recombinant human interleukin-10 in subjects with various degrees of renal function. J Clin Pharmacol 1999;39:1015-20.
60. Meng X, Sawamura D, Tamai K, Hanada K, Ishida H, Hashimoto I. Keratinocyte gene therapy for systemic diseases. Circulating interleukin 10 released from gene-transferred keratinocytes inhibits contact hypersensitivity at distant areas of the skin. J Clin Invest 1998;101:1462-7.
61. Fontana L, Moreira E, Torres MI, Fernandez I, Rios A, Sanchez de Medina F, Gil A. Dietary nucleotides correct plasma and liver microsomal fatty acid alterations in rats with liver cirrhosis induced by oral intake of thioacetamide. J Hepatol 1998;28:662-9.
62. Schnur J, Olah J, Szepesi A, Nagy P, Thorgeirsson SS. Thioacetamide-induced hepatic fibrosis in transforming growth factor β-1 transgenic mice. Eur J Gastroenterol Hepatol 2004;16:127-33.
63. Molnar MJ, Gilbert R, Lu Y, Liu AB, Guo A, Larochelle N, Orlopp K, Lochmuller H, Petrof BJ, Nalbantoglu J, Karpati G. Factors influencing the efficacy, longevity, and safety of electroporation-assisted plasmid-based gene transfer into mouse muscles. Mol Ther 2004;10:447-55.
64. Lehr HA, van der Loos CM, Teeling P, Gown AM. Complete chromogen separation and analysis in double immunohistochemical stains using Photoshop-based image analysis. J Histochem Cytochem 1999;47:119-26.
65. Vergnes L, Phan J, Strauss M, Tafuri S, Reue K. Cholesterol and cholate components of an atherogenic diet induce distinct stages of hepatic inflammatory gene expression. J Biol Chem 2003;278:42774-84.
66. Wang CH, Chen YJ, Lee TH, Chen YS, Jawan B, Hung KS, Lu CN, Liu JK. Protective effect of MDL28170 against thioacetamide-induced acute liver failure in mice. J Biomed Sci 2004;11:571-8.
67. Wang CH, Jawan B, Lee TH, Hung KS, Chou WY, Lu CN, Liu JK, Chen YJ. Single injection of naked plasmid encoding alpha-melanocyte-stimulating hormone protects against thioacetamide-induced acute liver failure in mice. Biochem Biophys Res Commun 2004;322:153-61.
68. Lotersztajn S, Julien B, Teixeira-Clerc F, Grenard P, Mallat A. Hepatic fibrosis: molecular mechanisms and drug targets. Annu Rev Pharmacol Toxicol 2005;45:605-28.
69. Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res 2003;92:827-39.
70. Xu GF, Li PT, Wang XY, Jia X, Tian DL, Jiang LD, Yang JX. Dynamic changes in the expression of matrix metalloproteinases and their inhibitors, TIMPs, during hepatic fibrosis induced by alcohol in rats. World J Gastroenterol 2004;10:3621-7.
71. Parsons CJ, Bradford BU, Pan CQ, Cheung E, Schauer M, Knorr A, Krebs B, Kraft S, Zahn S, Brocks B, Feirt N, Mei B, Cho MS, Ramamoorthi R, Roldan G, Ng P, Lum P, Hirth-Dietrich C, Tomkinson A, Brenner DA. Antifibrotic effects of a tissue inhibitor of metalloproteinase-1 antibody on established liver fibrosis in rats. Hepatology 2004;40:1106-15.
72. Nie QH, Cheng YQ, Xie YM, Zhou YX, Cao YZ. Inhibiting effect of antisense oligonucleotides phosphorthioate on gene expression of TIMP-1 in rat liver fibrosis. World J Gastroenterol 2001;7:363-9.
73. Safadi R, Ohta M, Alvarez CE, Fiel MI, Bansal M, Mehal WZ, Friedman SL. Immune stimulation of hepatic fibrogenesis by CD8 cells and attenuation by transgenic interleukin-10 from hepatocytes. Gastroenterology 2004;127:870-82.
74. Reitamo S, Remitz A, Tamai K, Uitto J. Interleukin-10 modulates type I collagen and matrix metalloprotease gene expression in cultured human skin fibroblasts. J Clin Invest 1994;94:2489-92.
75. Verma IM, Weitzman MD. Gene therapy: twenty-first century medicine. Annu Rev Biochem 2005;74:711-38.
76. Dean DA. Nonviral gene transfer to skeletal, smooth, and cardiac muscle in living animals. Am J Physiol Cell Physiol 2005;289:C233-45.
77. Claria J. Cyclooxygenase-2 biology. Curr Pharm Des 2003;9:2177-90.
78. Yamamoto H, Kondo M, Nakamori S, Nagano H, Wakasa K, Sugita Y, Chang-De J, Kobayashi S, Damdinsuren B, Dono K, Umeshita K, Sekimoto M, Sakon M, Matsuura N, Monden M. JTE-522, a cyclooxygenase-2 inhibitor, is an effective chemopreventive agent against rat experimental liver fibrosis. Gastroenterology 2003;125:556-71.
79. Mohammed NA, Abd El-Aleem SA, El-Hafiz HA, McMahon RF. Distribution of constitutive (COX-1) and inducible (COX-2) cyclooxygenase in postviral human liver cirrhosis: a possible role for COX-2 in the pathogenesis of liver cirrhosis. J Clin Pathol 2004;57:350-4.
80. Berg DJ, Zhang J, Lauricella DM, Moore SA. IL-10 is a central regulator of cyclooxygenase-2 expression and prostaglandin production. J Immunol 2001;166:2674-80.
81. Giron-Gonzalez JA, Martinez-Sierra C, Rodriguez-Ramos C, Rendon P, Macias MA, Fernandez-Gutierrez C, Diaz F, Martin-Herrera L. Adhesion molecules as a prognostic marker of liver cirrhosis. Scand J Gastroenterol 2005;40:217-24.
82. Burra P, Hubscher SG, Shaw J, Elias E, Adams DH. Is the intercellular adhesion molecule-1/leukocyte function associated antigen 1 pathway of leukocyte adhesion involved in the tissue damage of alcoholic hepatitis? Gut 1992;33:268-71.
83. Simeonova PP, Gallucci RM, Hulderman T, Wilson R, Kommineni C, Rao M, Luster MI. The role of tumor necrosis factor-α in liver toxicity, inflammation, and fibrosis induced by carbon tetrachloride. Toxicol Appl Pharmacol 2001;177:112-20.
84. Ip E, Farrell G, Hall P, Robertson G, Leclercq I. Administration of the potent PPARα agonist, Wy-14,643, reverses nutritional fibrosis and steatohepatitis in mice. Hepatology 2004;39:1286-96.
85. Kawachi S, Jennings S, Panes J, Cockrell A, Laroux FS, Gray L, Perry M, van der Heyde H, Balish E, Granger DN, Specian RA, Grisham MB. Cytokine and endothelial cell adhesion molecule expression in interleukin-10-deficient mice. Am J Physiol Gastrointest Liver Physiol 2000;278:G734-43.
86. Deng J, Kohda Y, Chiao H, Wang Y, Hu X, Hewitt SM, Miyaji T, McLeroy P, Nibhanupudy B, Li S, Star RA. Interleukin-10 inhibits ischemic and cisplatin-induced acute renal injury. Kidney Int 2001;60:2118-28.