肝癌衍生生長因子（Hepatoma-derived growth factor; HDGF）是一種已被證實可在胎肝細胞和肝癌組織中高度表達的核癌蛋白。先前的研究指出其可藉由上調肝內TGF-β1蛋白表現而在肝纖維化過程中扮演促纖維化的作用。本研究旨在探討HDGF在非酒精性脂肪性肝病（Non-alcoholic fatty liver disease; NAFLD）發展過程中的病理生理性角色。
本研究選用C57BL/6成年雄性小鼠作為誘發非酒精性脂肪性肝病實驗動物模式。在餵飼以高油飼料(High fat diet; HFD)4週後，轉換餵飼膽鹼缺乏飼料(Choline-deficient diet; CDD) 4~12週。對其血清和肝組織中的生化、組織學和脂肪肝相關分子進行檢測。之後藉由腺病毒介導的HDGF過表達或敲除被用來評估其可能的預防效果。
在血清的檢測數值中，總膽固醇（Total cholesterol; TC）、高密度脂蛋白膽固醇（HDL-C）和低密度脂蛋白膽固醇（LDL-C）的明顯上升都證實了非酒精性脂肪肝病的誘導成功。組織學油紅染色結果也顯示肝臟內脂肪有明顯的異常累積與肝細胞變性現象。定量PCR和Western blot檢測結果明確顯示：動物在餵養6周CDD後，肝臟組織中的HDGF，TGF-β1，COL1A1和PPAR-γ表現量均呈現上調。
腺病毒介導的HDGF基因修飾在NAFLD小鼠並無影響血清中AST、ALT和TG水平。HDGF siRNA傳遞卻可顯著抑制血清TC含量。油紅染色和組織形態學分析表明，這兩種HDGF過表達和基因沉默實驗組均呈現可抑制NAFLD肝臟中脂肪微滴分布區域及微滴顆粒的平均大小，這暗示HDGF可能參與肝內脂肪酸合成或運輸的機制，藉此改善肝臟脂肪性病變的發生。
這些結果表明，HDGF修飾影響NAFLD形成過程中脂肪酸的沉積，代表著HDGF功能是參與肝臟代謝的脂肪生成。因此HDGF基因修飾可被視為一種治療NAFLD的治療方式。

Hepatoma-derived growth factor (HDGF), a nuclear oncoprotein highly expressed in both fetal hepatocytes and hepatoma tissues, has been previously demonstrated to play a profibrogenic role in hepatic fibrogenesis through aggravating TGF-1 signaling. This study examined the role of HDGF in the development of non-alcoholic fatty liver disease (NAFLD).
In this study, adult male C57BL/6 mice were fed with high fat diet for 4 wk and subsequent choline-deficient diet (CDD) for 8 wks to induce NAFLD. Sera and liver tissues were subjected to biochemical, histopathological, and molecular examinations. Adenoviral-mediated HDGF over-expression or knockdown was used to evaluate its possible prophylactic effect.
Elevation of total cholesterol (TC), high density lipoprotein-cholesterol (HDL-C), and low density lipoprotein-cholesterol (LDL-C) in mouse sera confirmed the successfulness of NAFLD induction. Histologically, oil red O staining data showed a typical pattern of fatty droplet deposition in liver tissues. Quantitative PCR and Western blotting detection indicated that HDGF, TGF-1, COL1A1, and PPAR- were up-regulated in steatotic livers after 6 weeks of CDD feeding. Adenovirus-mediated HDGF gene overexpression did not affect AST, ALT, and TG serum levels in NAFLD mice. Conversely, HDGF siRNA delivery significantly suppressed TC level. Oil red staining and morphometric analysis revealed that both HDGF over-expression and gene silencing suppressed the spreading area of and the averaged size of the fatty droplets deposited in NAFLD livers, suggesting that HDGF might play a role in fatty acid synthesis and/or transportation, thereby ameliorating fatty liver retention in livers.
These findings suggest that HDGF is functionally involved in lipogenesis of liver metabolism and that HDGF modifications ameliorate fatty acid deposition during NAFLD development. Accordingly, HDGF gene modifications may be regarded a therapeutic modality for NAFLD treatment.

目 錄
書頁名………………………………………………………………………… i
論文審定書…………………………………………………………… ii
中文摘要………………………………………………………….….. iii
英文摘要………………………………………..……………………. V
目錄…………….………….……………………..……………………. Vi
表目錄….………………………………………..……………………. Viii

第 一 章 緒論…………………………………………………….. 1
第一節 非酒精性脂肪肝疾病…………………………. 1
第二節 肝癌衍生生長因子概述…………………….. 4
第三節 研究目的……………………..…………………….. 9
第 二 章 材料方法………………………………………………… 10
第一節 材料…….…………………………………………….... 10
第二節 實驗方法…….…………………………………….... 13
第 三 章 實驗結果………………………………………………… 21
第一節 餵養特殊飼料誘發脂肪肝病變並探討不同時間點的肝內脂肪變化…………………….……....21
第二節 利用腺病毒攜帶基因探討脂肪肝病變小鼠的肝內脂肪變化 …………….………..…..…….... 24
第 四 章 討論…….………………………………………………… 27
第 五 章 結論…….………………………………………………… 30

參考文獻 …….………………………………………….……………… 31
表目錄 …….………………………………………….………….……… 39

1. Brunt EM, Janney CG, Di Bisceglie AM, et al.: Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions. Am J Gastroenterol 1999; 94:2467-2474.
2. Schaffner F, Thaler H: Nonalcoholic fatty liver disease. Prog Liver Dis 1986; 8:283-298.
3. Mouralidarane A, Oben J A, Soeda J:Pathophysiology and clinical management of nonalcoholic fatty liver disease. Medicine 2011;39:592-6.
4. Clark JM, Diehl AM: Nonalcoholic fatty liver disease: an underrecognized cause of cryptogenic cirrhosis. JAMA 2003; 289: 300-304.
5. Chalasani N, Youmossi Z, Lavine JE et al: The diagnosis and management of non-alcoholic fatty liver disease: practice guideline by the American Gastroenterological Association, American Association for the Study of Liver Diseases, and American College of Gastroenterology. Gastroenterol. 2012;142:1592-609.
6. Machado MV, Cortez-Pinto H: Non-invasive diagnosis of non-alcoholic fatty liver disease. A critical appraisal. J Hepatol 2013;58:1007-19.
7. Farrell GC, Chitturi S, Lau GKK, Sollano JD: Guidelines for the assessment and management of non-alcoholic fatty liver disease in the Asia-Pacific region: executive summary. J Gastroenterol Hepatol 2007;22:775-7.
8. Day CP, James OFW: Steatohepatitis: a tale of two ‘hits’? Gastroenterology 1998; 114: 842-845.
9. Sanyal AJ, Campbell-Sargent C, Mirshahi F, et al.: Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities. Gastroenterology 2001; 120: 1183-1192.
10. Fan JG. Steatohepatitis studies in China. Shijie Huaren Xiaohua Zazhi 2001; 9:6-10
11. Haque M, Sanyal AJ.The metabolic abnormalities associated with non-alcoholic fatty liver disease. Best Pract Res Clin Gastroenterol 2002; 16:709-731
12. Angelico F,Del Ben M, Conti R, Francioso S, Feole K, Maccioni D, Antonini TM,Alessandri C. Non-alcoholic fatty liver syndrome: a hepatic consequence of common metabolic diseases. J Gastroenterol Hepatol 2003; 18:588-594
13. Falck-Ytter Y, Younossi ZM, Marchesini G, McCullough AJ. Clinical features and natural history of nonalcoholic steatosis syndromes. Semin Liver Dis 2001; 21:17-26
14. Clark JM, Brancati FL, Diehl AM. The prevalence and etiology of elevated aminotransferase levels in the United States. Am J Gastroenterol 2003; 98:960-967
15. Brunt EM: Nonalcoholic steatohepatitis: pathologic features and differential diagnosis. Semin Diagn Pathol 2005; 22: 330-338.
16. 譚健民、吳昭新： 脂肪肝之超音波影像診斷. 台灣醫誌1986; 85; 45-53.
17. Yang PM, Huang GT, Sheu JC. Mass screening of hepatobiliary disease by real-time ultraonography. J Formos Med Assoc 1986:85 1144-50.
18. Araujo LM, De Oliveira DA, Nunes DS. Liver and biliary ultrasonography in diabetic and non-diabetic obese women. Diabetes Metab 1998; 24:458-462
19. Hultcrantz R, Gabrielsson N. Patients with persistent elevation of aminotransferases: Investigation with ultrasonography, radionuclide imaging and liver biopsy. J Intern Med 1993; 233:7-12
20. Farrell GC.Non-alcoholic steatohepatitis: what is it,and why is it important in the Asia-Pacific region? J Gastroenterol Hepatol 2003; 18:124-138
21. Nakamura H, Kambe H, Egawa T et al. Partial purification and characterization of hepatoma-derived growth factor. Clin. Chem. Acta 1989; 183: 273–84.
22. Izumoto Y, Kuroda T, Harada H, Kishimoto T, Nakamura H. Hepatoma-derived growth factor belongs to a gene family in mice showing significant homology in the amino terminus. Biochem. Biophys. Res. Commun. 1997, 238, 26–32.
23. Ikegame, K.; Yamamoto, M.; Kishima, Y.; Enomoto, H.; Yoshida, K.; Suemura, M.; Kishimoto, T.; Nakamura, H. A new member of a hepatoma-derived growth factor gene family can translocate to the nucleus. Biochem. Biophys. Res. Commun. 1999, 266, 81–87.
24. Dietz, F.; Franken, S.; Yoshida, K.; Nakamura, H.; Kappler, J.; Gieselmann, V. The family of hepatoma-derived growth factor proteins: Characterization of a new member HRP-4 and classification of its subfamilies. Biochem. J. 2002, 366, 491–500.
25. Gregory SG, Barlow KF, Mclay KE, et al. The DNA sequence and biological annotation of human chromosome l. Nature, 2006,441(7091):315- 321.
26. Nakamura H, Izumoto Y, Kambe H, et al. Molecular cloning of complementary DNA for a novel hurnan hepatoma-derived growth factor: its homology with high mobility group-1 protine. J Biol Chem, 1994,269(40):25 143-25 149.
27. Nameki N, Tochio N, Koshiba S, et al. Solution structure of the PWWP domain of the hepatoma-derived growth factor family. Protein Sci, 2005, 14(3):756-764.
28. Lukasik SM, Cierpicki T, Borloz M, et al. High resolution structure of the HDGF PWWP domain: a potential DNA binding domain. Protein Sci, 2006,15(2):314-323.
29. Everett AD, Stoops T, Mcnamara CA. Nuclear targeting is required for hepatoma-derived growth factor stimulated mitogenesis in vascular smoothmusclecells.JBiolchem,2001,276(40):37564-37568.
30. Chen, S.C., et al. Hepatoma-derived growth factor regulates breast cancer cell invasion by modulating epithelial--mesenchymal transition. J Pathol 228, 158-169 (2012).
31. Enomoto H, Yoshida K, Kishima Y, et al. Hepatoma-derived growth factor is highly expressed indeveloping liver and promotes fetal hepatocyte proliferation. Hepatology, 2002,36(6):1519-1527.
32. Kishima Y, Yamamoto H, Izumoto Y et al. Hepatomaderived growth factor stimulates cell growth after translocation to the nucleus by nuclear localization signals. J. Biol. Chem. 2002; 277: 10315–22.
33. Everett AD, Lobe DR, Matsumura ME, Nakamura H, McNamara CA. Hepatoma-derived growth factor stimulates smooth muscle cell growth and is expressed in vascular development. J. Clin. Invest. 2000; 105: 567– 75.
34. Oliver JA, Al-Awqati Q. An endothelial growth factor involved in rat renal development. J. Clin. Invest. 1998; 102: 1208–19.
35. Zhou, Y., Zhou, N., Fang, W. & Huo, J. Overexpressed HDGF as an independent prognostic factor is involved in poor prognosis in Chinese patients with liver cancer. Diagn Pathol 5, 58 (2010).
36. Cilley RE, Zgleszewski SE, Chinoy MR. Fetal lung development: airway pressure enhances the expression of developmental genes. J Pediatr Surg 2000; 35: 113-118; discussion 119
37. Everett A.D.. Identification, cloning, and developmental expression of hepatoma-derived growth factor in the developing rat heart. Dev Dyn, 2001; 222:450-458.
38. Enomoto H., Yoshida K., Kishima Y., Kinoshita T., Yamamoto M., Everett A.D., Miyajima A., Nakamura H. Hepatoma-derived growth factor is highly expressed in developing liver and promotes fetal hepatocyte proliferation. Hepatology, 2002; 36:1519-1527.
39. Everett A.D., Bushweller J. Hepatoma derived growth factor is a nuclear targeted mitogen. Curr Drug Targets, 2003; 4:367-371.
40. Sedlmaier, A., et al. Overexpression of hepatoma-derived growth factor in melanocytes does not lead to oncogenic transformation. BMC Cancer 11, 457 (2011).
41. Okuda Y,Nakamura H,Yoshida K, et al. Hepatoma-derived growth factor induces tumorigenesis in vivo through both direct angiogenic activity and induction of vascular endothelial growth factor. CancerSci, 2003, 94 (12): 1034-1041.
42. Narron, J.V., Stoops, T.D., Barringhaus, K., Matsumura, M. & Everett, A.D. Hepatoma-derived growth factor is expressed after vascular injury in the rat and stimulates smooth muscle cell migration. Pediatr Res 59, 778-783 (2006).
43. Abouzied MM,EI-TahirHM,PrennerL,etal.Hepatoma-derived growth factor. Significance of amino acid residues 81-100 in cell surface interaction and proliferative activity. J Biol Chem, 2005, 280 (12):10945-10954.
44. Yoshida K, Tomita Y, Okuda Y, et al. Hepatoma-derived growth factor is a novel prognostic factor for hepatocellular carcinoma. Ann Surg Oncol, 2006,13(2):159-167.
45. Hu, T.H., et al. Expression of hepatoma-derived growth factor in hepatocellular carcinoma. Cancer 98, 1444-1456 (2003).
46. Yoshida, K.; Nakamura, H.; Okuda, Y.; Enomoto, H.; Kishima, Y.; Uyama, H.; Ito, H.; Hirasawa, T.; Inagaki, S.; Kawase, I. Expression of hepatoma-derived growth factor in hepatocarcinogenesis. J. Gastroenterol. Hepatol. 2003, 18, 1293–1301.
47. MaoJ, XuZ, FangY, et al. Hepatoma–derived growth factor involved in the carcinogenesis of gastric epithelial cells through promotionof cell pro-liferation by Erk1/2 activation. Cancer Sci,2008,99(11)：2120-2127.
48. Chang, K.C., et al. Hepatoma-derived growth factor is a novel prognostic factor for gastrointestinal stromal tumors. Int J Cancer 121, 1059-1065 (2007).
49. Wang S, Fang W. Increased expression of hepatoma-derived growth factor correlates with poor prognosis in human nasopharyngeal carcinoma. Histopathology,2011,58(2):217-224.
50. Ren, H., et al. Expression of hepatoma-derived growth factor is a strong prognostic predictor for patients with early-stage non-small-cell lung cancer. J Clin Oncol 22, 3230-3237 (2004).
51. Uyama, H., et al. Hepatoma-derived growth factor is a novel prognostic factor for patients with pancreatic cancer. Clin Cancer Res 12, 6043-6048 (2006).
52. Guo, S.; Liu, H.D.; Liu, Y.F.; Liu, L.; Sun, Q.; Cui, X.J. Hepatoma-derived growth factor: A novel prognostic biomarker in intrahepatic cholangiocarcinoma. Tumour Biol. 2015, 36, 353–364.
53. Bao, C.; Wang, J.; Ma, W.; Wang, X.; Cheng, Y. HDGF: A novel jack-of-all-trades in cancer. Future Oncol. 2014, 10, 2675–2685.
54. Bao, C.H.; Liu, K.; Wang, X.T.; Ma, W.; Wang, J.B.; Wang, C.; Jia, Y.B.; Wang, N.N.; Tan, B.X.; Song, Q.X.; et al. Prognostic role of hepatoma-derived growth factor in solid tumors of Eastern Asia: A systematic review and meta-analysis. Asian Pac. J. Cancer Prev. 2015, 16, 1803–1811.
55. Tsai, C.C.; Huang, S.C.; Tai, M.H.; Chien, C.C.; Huang, C.C.; Hsu, Y.C. Hepatoma-derived growth factor upregulation is correlated with prognostic factors of early-stage cervical adenocarcinoma. Int. J. Mol. Sci. 2014, 15, 21492–21504.
56. Tao, F.; Ye, M.F.; Sun, A.J.; Lv, J.Q.; Xu, G.G.; Jing, Y.M.; Wang, W. Prognostic significance of nuclear hepatoma-derived growth factor expression in gallbladder cancer. World J. Gastroenterol. 2014, 20, 9564–9569.
57. Reeves, H.L. & Friedman, S.L. Activation of hepatic stellate cells--a key issue in liver fibrosis. Front Biosci 7, d808-826 (2002).
58. Angulo P, Keach JC, Batts KP, Lindor KD. Independent predictors of liver fibrosis in patients with nonalcoholic steatohepatitis.Hepatology 1999; 30:1356-1362
59. Mormone, E., George, J. & Nieto, N. Molecular pathogenesis of hepatic fibrosis and current therapeutic approaches. Chem Biol Interact 193, 225-231 (2011).
60. Ahmad, A. & Ahmad, R. Understanding the mechanism of hepatic fibrosis and potential therapeutic approaches. Saudi J Gastroenterol 18, 155-167 (2012).
61. Kao YH, Chen CL, Jawan B, Chung YH, Sun CK, et al: Upregulation of hepatoma-derived growth factor is involved in murine hepatic fibrogenesis. 2010 Jan;52(1):96-105. doi: 10.1016/j.jhep.2009.10.002. Epub 2009 Oct 24.
62. Morán-Salvador E, López-Parra M, García-Alonso V. et al. Role for PPARγ in obesity-induced hepatic steatosis as determined by hepatocyte- and macrophage-specific conditional knockouts. FASEB J. 2011; 25: 2538-2550
63. Elbashir S, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001,411 (6836): 494–8
64. Fedorov,Y.,et al. "Off-targeting By siRNA Can Induce Toxic Phenotype." RNA Accepted (2006).
65. Sledz, C.A., Holko, M., de Veer, M.J., Silverman, R.H. & Williams, B.R. Activation of the interferon system by short-interfering RNAs. Nat. Cell Biol. 5, 834–839 (2003).
66. Sledz, C.A. & Williams, B.R. RNA interference in biology and disease. Blood 106, 787–794 (2005).
67. Hornung, V. et al. Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat. Med. 11, 263–270 (2005).
68. 53Morrissey, D.V. et al. Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Nat. Biotechnol. 23, 1002–1007 (2005).
69. Joao T Marques & Bryan R G Williams. Activation of the mammalian immune system by siRNAs.Nature Biotechnology 23, 1399–1405 (2005)
70. Sioud M. Single-stranded small interfering RNA are more immunostimulatory than their double-stranded counterparts: a central role for 2’-hydroxyl uridines in immune responses. Eur J Immunol (2006) 36:1222–30.
71. Sioud M. RNA interference and innate immunity. Adv Drug Deliv Rev (2007) 59:153–63. doi:10.1016/j.addr.2007.03.006
72. Kishima Y, Yoshida K, Enomoto H et al. Antisense oligonucleotides of hepatoma-derived growth factor (HDGF) suppress the proliferation of hepatoma cells. Hepatogastroenterology 2002; 49: 1639–44.