臺灣博碩士論文加值系統

肝癌之治療方式因人而異，除了手術切除，其它尚有內科療法、化療等，其中化療的成敗與否，部分取決於藥物影響腫瘤細胞內之訊息傳導調控而決定。Nuclear Factor-kappa B (NF-κB)是一個控制細胞免疫相關因子表現之轉錄因子，在腫瘤生長的組織有大量表現的情形，而異奎寧生物鹼—berberine具有抑制肝腫瘤細胞中NF-κB之活性，因此本實驗之目的將評估berberine在活體之治療效果。首先，以BALB/c小鼠進行藥物濃度實驗，取得合適之藥物治療濃度，並透過臨床檢查、血液學檢查、病理學檢查監測藥物對實驗動物之影響；之後經過劑量換算，選擇一適用於Fischer 344(F 344)大鼠之治療藥物濃度(20 mg/kg)。接著，建立一穩定且易於觀察F 344大鼠皮下轉殖GP7TB腫瘤細胞之動物模式，分別以腹腔或皮下注射方式給藥，藉由腫瘤體積變化、體重變化、血液學與血清生化學檢查、病理學檢查等項目評估berberine在生物體治療效果。結果顯示腹腔注射berberine對於皮下轉植肝腫瘤之生長速度有減緩之趨勢，僅出現攝食量減少、體重下降等臨床症狀，而其他檢驗項目結果則與對照組差異不大，皮下注射則無腫瘤生長抑制之效果，且實驗動物生理狀況良好，最後則對F 344大鼠之GP7TB細胞之肝腫瘤動物模式進行療效評估。在實驗終點測量，發現腹腔注射berberine之組別(實驗組)之肝腫瘤體積小於對照組，但無顯著差異，其它檢驗項目結果與對照組相去不遠。實驗結果顯示，腹腔注射berberine除了對於皮下轉殖肝腫瘤生長具抑制效果，對於原位肝腫瘤亦有縮小體積之傾向，且除了減低食慾而使體重下降之副作用外，對於血液相與實質臟器幾無影響，因此認為berberine有發展修飾成為化療藥物之潛力。

The treatments of hepatoma differ according to patients’ conditions, and the success of chemotherapy is partially decided by the effects of the drug used for affecting the signal transductions of tumor cells. In this study, we estimated the effects of the treatment of hepatoma by berberine, an isoquinoline alkaloid. Nuclear Factor-kappa B（NF-κB）, one of the transcription factors controls cell-mediated immune response, sometimes overexpresses in tumor cells or around tissues. According to previous researches, it’s known that berberine is capable of restraining the activity of NF-κB in liver tumor cells, therefore we designed the object in vivo. In order to get the proper injection concentration of berberine, we tested in BALB/c mice first and monitored the reactions. Then we transformed the dosage from BALB/c mice to Fischer 344 rats so we got the concentration of berberine in rats－20 mg/kg. Furthermore, we established a stable model to transplant GP7TB hepatoma cells into subcutaneous space of F344 rats and evaluated the treatment effect via monitoring the tumor volume, changes in hematology, serum biochemistry, and pathology. The results show that intraperitoneal injection of berberine can slow down the growing rate of the subcutaneous tumor with clinical symptoms of reducing food intake and loosing body weight, whereas it showed no suppression effect on the growth of tumor in subcutaneous injection group. At last, we estimated the animal model of hepatoma with GP7TB cell in F344 rats, and found that tumor size was smaller in the treated group. Apart from the side effects of reducing appetite and loosing weight, berberine has few influences on blood and organs, so we considered that berberine has the potential of being applied in chemical therapy of hepatoma after re-constructing.

第一章 前言.................................................1
第二章 文獻探討
第一節 肝臟腫瘤治療模式之探討............................2
第二節 誘導肝臟腫瘤之實驗動物模式........................3
第三節 Berberine之背景資料................................5
第三章 材料與方法
第一節 藥物濃度實驗......................................9
1.1 實驗動物...........................................9
1.2 藥物投予日程表與生理資料之收集…...................9
1.3 病理學檢查........................................10
第二節 藥物劑量換算方法.................................11
第三節 GP7TB細胞培養...................................11
第四節 皮下腫瘤與肝腫瘤實驗.............................12
4.1 實驗動物..........................................12
4.2 皮下腫瘤誘導......................................12
4.3 肝腫瘤誘導........................................13
4.4藥物投予日程表與生理資料之收集....................14
4.5 病理學檢查........................................15
第四章 結果
第一節 藥物濃度試驗結果.................................20
第二節 Berberine對GP7TB皮下腫瘤效應.....................21
第三節 Berberine對GP7TB肝腫瘤效應.......................22
第五章 討論
第一節 藥物濃度試驗結果..................................37
第二節 皮下腫瘤實驗與肝腫瘤實驗實驗結果..................38
第六章 結論................................................42
參考文獻....................................................43

梁鍾鼎。1994。人類疾病實驗動物模式。國家實驗動物繁殖及研究中心簡訊(第一卷第四期)。
鄭欣怡。2002。以肝病相關轉錄因子活性建立藥物搜尋系統之研究。中國醫藥學院醫學研究所碩士論文。pp. 27-32。
Alan WB, Cormac TT, David JB. Non-antibiotic anti-diarrhoeal drugs: factors affecting oral bioavailability of berberine and loperamide in intestinal tissue. Adv Drug Deliv Rev 23: 111-120, 1997.

Anis KV, Rajeshkumar NV, Kuttan R. Inhibition of chemical carcinogenesis by berberine in rats and mice. J Pharm Pharmacol 53: 763-768, 2001.
Blum HE. Treatment of hepatocellular carcinoma. Best Pract Res Clin Gastroenterol 19: 129-145, 2005.
Bosma GC, Custer RP, Bosma MJ. A severe combined immunodeficiency mutation in the mouse. Nature 301: 527-530, 1983.
Brewster M, Bodor N. Improved delivery through biological membranes XVI. Enhancement of the brain-specificity of a dihydropyridine formed from pyridinium salt drug delivery system by controlled infusion. J Parenter Sci Technol 37:159-164, 1983.
Chen CM, Chang HC. Determination of berberine in plasma, urine and bile by high-performance liquid chromatography. J Chromatogr B Biomed Appl 665: 117-123, 1995.
Chen JC, Chung JG, Chen LD. Gypenoside induces apoptosis in human Hep3B and HA22T tumour cells. Cytobios 100: 37-48, 1999.
Chi CW, Chang YF, Chao TW, Chiang SH, P'eng FK, Lui WY, Liu TY. Flowcytometric analysis of the effect of berberine on the expression of glucocorticoid receptors in human hepatoma HepG2 cells. Life Sci 54: 2099-2107, 1994.
Franca AV, Elias Junior J, Lima BL, Martinelli AL, Carrilho FJ. Diagnosis, staging and treatment of hepatocellular carcinoma. Braz J Med Biol Res 37: 1689-1705, 2004.
Fukuda K, Hibiya Y, Mutoh M, Koshiji M, Akao S, Fujiwara H. Inhibition of activator protein 1 activity by berberine in human hepatoma cells. Planta Med 65: 381-383, 1999.
Fukuda K, Hibiya Y, Mutoh M, Koshiji M, Akao S, Fujiwara H. Inhibition by berberine of cyclooxygenase-2 transcriptional activity in human colon cancer cells. J Ethnopharmacol 66: 227-233, 1999.
Futakuchi M, Ogawa K, Tamano S, Takahashi S, Shirai T. Suppression of metastasis by nuclear factor kappaB inhibitors in an in vivo lung metastasis model of chemically induced hepatocellular carcinoma. Cancer Sci 95: 18-24, 2004.
Haginiwa J, Harada M. Pharmacological studies on crude drugs. Comparison of berberine type alkaloid-containing plants on their components and several pharmacological actions. Jpn J Pharmacol 82: 726-731, 1962.
Hoshi A, Ikekawa T, Ikeda Y, Shirakawa S, Iigo M. Antitumor activity of berberrubine derivatives. Gann 67: 321-325, 1976.
Hosnuter M, Babuccu O, Kargi E, Altinyazar C, Babuccu B, Isikdemir A. Rat skin surface measurement: a practical formula. Plast Reconstr Surg 112: 1486-1487, 2003.
Hsu SH, Tsai TR, Lin CN, Yen MH, Kuo KW. Solamargine purified from Solanum incanum Chinese herb triggers gene expression of human TNFR I which may lead to cell apoptosis. Biochem Biophys Res Commun 229: 1-5, 1996.
Hung H. Treatment modalities for hepatocellular carcinoma. Curr Cancer Drug Targets 5: 131-138, 2005.
Hwang JM, Wang CJ, Chou FP, Tseng TH, Hsieh YS, Lin WL, Chu CY. Inhibitory effect of berberine on tert-butyl hydroperoxide-induced oxidative damage in rat liver. Arch Toxicol 76: 664-670, 2002 .
Kettmann V, Kosfalova D, Jantova S, Cernakova M, Drimal J. In vitro cytotoxicity of berberine against HeLa and L1210 cancer cell lines. Pharmazie 59: 548-551, 2004.
Kowalewski Z, Mrozikiewicz A, Bobkiewicz T, Drost K, Hladon B. Toxicity of berberine sulfate. Acta Pol Pharm 32: 113-120, 1975.
Kulkarni SK, Dandiya PC, Varandani NL. Pharmacological investigations of berberine sulphate. Jpn J Pharmacol 22: 11-16, 1972.
Kuo CL, Chi CW, Liu TY. Modulation of apoptosis by berberine through inhibition of cyclooxygenase-2 and Mcl-1 expression in oral cancer cells. In Vivo 19: 247-252, 2005.
Lin HL, Liu TY, Lui WY, Chi CW. Up-regulation of multidrug resistance transporter expression by berberine in human and murine hepatoma cells. Cancer 85: 1937-1942, 1999.
Lin S, Tsai SC, Lee CC, Wang BW, Liou JY, Shyu KG. Berberine inhibits HIF-1alpha expression via enhanced proteolysis. Mol Pharmacol 66: 612-619, 2004.
Llovet JM, Schwartz M, Mazzaferro V. Resection and liver transplantation for hepatocellular carcinoma. Semin Liver Dis 25: 181-200, 2005.
Llovet JM. Updated treatment approach to hepatocellular carcinoma. J Gastroenterol 40: 225-235, 2005.
Long Y, Lin XT, Zeng KL, Zhang L. Efficacy of intramuscular matrine in the treatment of chronic hepatitis B. Hepatobiliary Pancreat Dis Int 3: 69-72, 2004.
Mabed M, El-Helw L, Shamaa S. Phase II study of viscum fraxini-2 in patients with advanced hepatocellular carcinoma. Br J Cancer 90: 65-69, 2004.
Mardikar BR, Moholkar AL, Joglekar GV. A report on the behavioural studies in albino mice with berberine sulphate. Indian J Med Sci 27: 540-544, 1973.
McCullough KD, Coleman WB, Smith GJ, Grishan JW. Age-dependent regulation of the tumorigenic potential of neoplastically transformed rat liver epithelial cells by the liver microenvironment. Cancer Res 54: 3668- 3671, 1994.
Ng KK, Vauthey JN, Pawlik TM, Lauwers GY, Regimbeau JM, Belghiti J, Ikai I, Yamaoka Y, Curley SA, Nagorney DM, Ng IO, Fan ST, Poon RT; International Cooperative Study Group on Hepatocellular Carcinoma. Is hepatic resection for large or multinodular hepatocellular carcinoma justified? Results from a multi-institutional database. Ann Surg Oncol 12: 364-373, Epub, 2005.
Nishino H, Kitagawa K, Fujiki H, Iwashima A. Berberine sulfate inhibits tumor-promoting activity of teleocidin in two-stage carcinogenesis on mouse skin. Oncology 43: 131-134, 1986.
Oh SH, Lee BH. Induction of apoptosis in human hepatoblastoma cells by tetrandrine via caspase-dependent Bid cleavage and cytochrome c release. Biochem Pharmacol 66: 725-731, 2003.
Okuda K, Ohtsuki T, Obata H, Tomimatsu M, Okazaki N, Hasegawa H, Nakajima Y, Ohnishi K. Natural history of hepatocellular carcinoma and prognosis in relation to treatment. Study of 850 patients. Cancer 56: 918- 928, 1985.
O'Reilly EM, Stuart KE, Sanz-Altamira PM, Schwartz GK, Steger CM, Raeburn L, Kemeny NE, Kelsen DP, Saltz LB. A phase II study of irinotecan in patients with advanced hepatocellular carcinoma. Cancer 91: 101-105, 2001.
Palmer DH, Hussain SA, Johnson PJ. Gene- and immunotherapy for hepatocellular carcinoma. Expert Opin Biol Ther 5: 507-523, 2005.
Pikarsky E, Porat RM, Stein I, Abramovitch R, Amit S, Kasem S, Gutkovich-Pyest E, Urieli-Shoval S, Galun E, Ben-Neriah Y. NF-kappaB functions as a tumour promoter in inflammation-associated cancer. Nature 23: 461-466, 2004.
Rahman KM, Sugie S, Watanabe T, Tanaka T, Mori H. Chemopreventive effects of melatonin on diethylnitrosamine and phenobarbital-induced hepatocarcinogenesis in male F344 rats. Nutr Cancer 47: 148-155, 2003.
Rui-Chuan C, Jin-Hua S, Gao-Liang O, Ke-Xia C, Jin-Quan L, Xiao-Guang X. Induction of differentiation in human hepatocarcinoma cells by isoverbascoside. Planta Med 68: 370-372, 2002.
Schmeller T, Latz-Bruning B, Wink M. Biochemical activities of berberine, palmatine and sanguinarine mediating chemical defence against microorganisms and herbivores. Phytochemistry 44: 257-266, 1997.
Simile MM, Pagnan G, Pastorino F, Brignole C, De Miglio MR, Muroni MR, Asara G, Frau M, Seddaiu MA, Calvisi DF, Feo F, Ponzoni M, Pascale RM. Chemopreventive N-(4-hydroxyphenyl) retinamide (fenretinide) targets deregulated NF-kappaB and Mat1A genes in the early stages of rat liver carcinogenesis. Carcinogenesis 26: 417-227, 2005.
Tsai PL, Tsai TH. Hepatobiliary excretion of berberine. Drug Metab Dispos 32: 405-412, 2004.
Wartenberg M, Budde P, De Marees M, Grunheck F, Tsang SY, Huang Y, Chen ZY, Hescheler J, Sauer H. Inhibition of tumor-induced angiogenesis and matrix-metalloproteinase expression in confrontation cultures of embryoid bodies and tumor spheroids by plant ingredients used in traditional chinese medicine. Lab Invest 83: 87-98, 2003.
Yang FC, Zheng SS, Jiang TA. A modified rat model for hepatocellular carcinoma. Hepatobiliary Pancreat Dis Int 3: 585-587, 2004.
Zhang M, Chen H, Huang J, Li Z, Zhu C, Zhang S. Effect of lycium barbarum polysaccharide on human hepatoma QGY7703 cells: inhibition of proliferation and induction of apoptosis. Life Sci 76: 2115- 2124, 2005.
Zhang MF, Shen YQ. Antidiarrheal and anti-inflammatory effects of berberine. Zhongguo Yao Li Xue Bao 10: 174-176, 1989.
Zhang RX, Dougherty DV, Rosenblum ML. Laboratory studies of berberine used alone and in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea to treat malignant brain tumors. Chin Med J (Engl) 103: 658-665, 1990.