本研究發現，中醫方劑促進部分肝切除後肝再生方面，不管是從肝 重/體重比、PCNA GF、PCNA LI 與 BrdU LI 都可發現，中醫補氣代 表方劑四君子湯具有促進大鼠部分肝切除後肝再生的傾向。另外，此 促進作用應會在肝臟重量逐漸恢復後減緩。
而中醫補血方劑四物湯在PCNA LI 與 BrdU LI 雖可觀察到有促進 部分肝切除後肝再生的傾向，但在肝重/體重比與 PCNA GF 部份則顯 出相反結果。因此，補血方劑四物湯是否具有促進肝再生的作用尚需 進一步研究。
本研究利用部分肝切除、PCNA 與 BrdU 之免疫組織化學染色法 等，成功地建立了中醫藥對肝再生影響的研究模式。運用這個研究模 式將能快速且方便地評估中醫藥方劑對肝臟再生能力的影響。
參考文獻
1. 行政院衛生署：民國 93 年衛生統計(二)生命統計年報，行政院衛生 署 2005: pp. 30-62.
2. Pahlavan PS, Feldmann RE, Zavos C, Kountouras J. Prometheus' challenge: molecular, cellular and systemic aspects of liver regeneration. J Surg Res. 2006;134(2):238-251.
3. Haga S, Ogawa W, Inoue H, Terui K, Ogino T, Igarashi R, Takeda K, Akira S, Enosawa S, Furukawa H, Todo S, Ozaki M. Compensatory recovery of liver mass by Akt-mediated hepatocellular hypertrophy in liver-specific STAT3-deficient mice. J Hepatol. 2005;43(5):799-807.
4. David Zakim, Thomas D. Boyer. Hepatology: a textbook of liver disease.
Saunders, Philadelphia 2003; 1: 31
5. Michalopoulos G K, Defrances M C. Liver regeneration. Science.
1997;276(5309):60-66.
6. Skarpen E, Oksvold MP, Grøsvik H, Widnes C, Huitfeldt HS. Altered regulation of EGF receptor signaling following a partial hepatectomy.
J Cell Physiol. 2005;202(3):707-716
7. Nelson Fausto, Jean S. Campbell, Kimberly J. Riehle. Liver Regeneration. Hepatology. 2006;43:S45-S53.
8. Alison MR. Liver regeneration with reference to stem cells. Semin Cell Dev Biol. 2002;13(6):385-387.
9. Michalopoulos GK. Liver regeneration: molecular mechanisms of growth control. FASEB J. 1990;4(2):176-187.
10. Ping C, Xiaoling D, Jin Z, Jiahong D, Jiming D, Lin Z. Hepatic sinusoidal endothelial cells promote hepatocyte proliferation early after partial hepatectomy in rats. Arch Med Res. 2006;37(5):576-583.
11. Nakashima H, Inui T, Habu Y, Kinoshita M, Nagao S, Kawaguchi A,
Miura S, Shinomiya N, Yagita H, Seki S. Activation of mouse natural killer T cells accelerates liver regeneration after partial hepatectomy.
Gastroenterology. 2006;131(5):1573-1583.
12. Krähenbühl L, Feodorovici M, Renzulli P, Schäfer M, Abou-Shady M, Baer HU. Laparoscopic partial hepatectomy in the rat: a new resectional technique. Dig Surg. 1998;15(2):140-144.
13. Marissa Rabelo TarláI, Fernando Ramalho, Leandra Naira Zambelli Ramalho, Tiago Castro e Silva, Daniel Ferracioli Brandão, Juliana Ferreira, Orlando Castro e Silva, Sérgio Zucoloto. Cellular aspects of liver regeneration. Acta Cirurgica Brasileira. 2006,21(1):63-66
14. Assy N, Minuk GY. Liver regeneration: methods for monitoring and their applications. J Hepatol. 1997;26(4):945-952.
15. Yin XY, Lai PB, Lee JF, Lau JW. Effects of hepatic blood inflow occlusion on liver regeneration following partial hepatectomy in an experimental model of cirrhosis. Br J Surg. 2000;87(11):1510-1515.
16. Assy N, Gong Y, Zhang M, Pettigrew NM, Pashniak D, Minuk GY.
Use of proliferating cell nuclear antigen as a marker of liver regeneration after partial hepatectomy in rats. J Lab Clin Med.
1998;131(3):251-256.
17. Foley J, Ton T, Maronpot R, Butterworth B, Goldsworthy TL.
Comparison of proliferating cell nuclear antigen to tritiated thymidine as a marker of proliferating hepatocytes in rats. Environ Health Perspect. 1993;101 Suppl 5:199-205.
18. Fairman MP. DNA polymerase δ/PCNA: actions and interactions. J Cell Sci. 1990;95:1-4
19. Al-Nafussi AI, Klys HS, Rebello G, Kelly C, Kerr G, Cowie V. The assessment of proliferating cell nuclear antigen (PCNA) immunostaining in the uterine cervix and cervical squamous neoplasia.
Int J Gynecol Cancer. 1993;3(3):154-158.
20. Levan Muskhelishvili, John R. Latendresse, Ralph L. Kodell, Eric B.
Henderson. Evaluation of cell proliferation in rat tissues with BrdU, PCNA, Ki-67(MIB-5) immunohistochemistry and in situ hybridization for histone mRNA. J Histochem Cytochem.
2003;51(12):1681-1688.
21. Lardelli P, Perentes E, Meier G, Navarro N, Ettlin RA. Quantification of hepatocytic proliferation in the laboratory mouse. A comparative study using immunohistochemical detection of bromodeoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression. Exp Toxicol Pathol. 1994;46(2):95-100.
22. Lee KS, Yang WI. Comparison of brain tumor growth kinetics by proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) labeling. Yonsei Med J. 1992;33(3):265-271.
23. Alison M, Chaudry Z, Baker J, Lauder I, Pringle H. Liver regeneration:
a comparison of in situ hybridization for histone mRNA with bromodeoxyuridine labeling for the detection of S-phase cells. J Histochem Cytochem. 1994;42(12):1603-1608.
24. Eldrige SR, Butterworth BE, Goldsworthy TL. Proliferating cell nuclear antigen: a marker for hepatocellular proliferation in rodents.
Environ Health Perspect. 1993;101 Suppl 5:211-218.
25. Ueda J, Saito H, Watanabe H, Evers BM. Novel and quantitative DNA dot-blotting method for assessment of in vivo proliferation. Am J Physiol Gastrointest Liver Physiol. 2005;288(4):G842-G847.
26. Soames AR, Lavender D, Foster JR, Williams SM, Wheeldon EB.
Image analysis of bromodeoxyuridine (BrdU) staining for measurement of S-phase in rat and mouse liver. J Histochem Cytochem. 1994;42(7):939-944.
27. 清 汪昂：醫方集解，文光圖書有限公司 1986;pp. 28.
28. 清 汪昂：醫方集解，文光圖書有限公司 1986;pp. 144.
29. 清 吳謙：刪補名醫方論，新文豐出版社 1985;pp. 5.
30. 游士勳、張錦清：實用中醫方劑學，樂群文化公司 1992;pp. 369.
31. 楊靖、詹向紅、孫曄、李秀昌、李厚剛：四君子湯對 D-半乳糖衰 老模型小鼠心、肝、腦組織 MDA 含量及端粒酶活性的影響。中 國中西醫結合雜誌 2005;6(25):531-533.
32. 陳麗艷、劉君星、施曉光：中藥四君子湯抗衰老的實驗研究。黑 龍江醫藥 2006;19(5):363-364.
33. 岑榮光、李溥、莫興菊、韋紹英、張靈、謝洪書：四君子湯對家 兔下丘腦神經遞質-甲狀腺軸的調節作用及機理研究。放射免疫學 雜誌 2005;1(18):43-44.
34. 葉富強、陳蔚文、李茹柳、張曼、徐頌芬、潘懷耿：四君子湯提 取物對大鼠胃腸活動的影響。中藥藥理與臨床 2002;18(3):2-4.
35. 蔣靈芝、熊平、蔣任才：四君子湯修復燒傷後腸黏膜屏障的超微 結構變化。中醫藥臨床雜誌 2004;1(16):80-81.
36. 李海燕、董禮陽、楊景云：四君子湯對急性肝損傷伴發菌群失調 及內毒素血癥的保護作用。中國微生態學雜誌 2005;2(17):100-101.
37. 劉紅林、程牛亮、趙建濱、牛勃、覃秀桃、楊濤：中藥復方補益 劑 對 不 同 細 胞 生 長 影 響 的 實 驗 研 究 。 山 西 醫 科 大 學 學 報 2005;3:312-314.
38. 趙寧、張皖東、賈紅偉、吳志鵬、呂愛平：四君子湯對利血平所 致脾虛大鼠腸道黏膜 TGF-β和 TNF-α表達的影響。中國中醫基 礎醫學雜誌 2007;13(1):44-46.
39. 章梅、夏天、張仲海、靳風爍、顏真、崔大祥：四君子湯對脾虛 患者血漿細胞因子的影響。第四軍醫大學學報 2000;21(4):411-413.
40. 鞠寶玲、畢蕾、楊景云：四君子湯改善抗生素脫污染小鼠腸道菌
44. 何永亮、王紅星、霍祥富、方素華、廖世平、鐘振東：四物湯對 小 鼠 急 性 失 血 性 貧 血 的 治 療 作 用 。 四 川 省 生 理 科 學 雜 誌 1998;20(2):40- 42.
45. 范啟蘭、康光忠、葉金花、賴日勇、甘祝軍、許春鵑：四物湯對 貧 血小 鼠 腎 促紅 細 胞 生成 素 基 因表 達 的 影響 。 中 國臨 床 康 復 2005 ;9(3):168-169.
46. 郭平、王升啟：四物湯及其單味藥補血作用的研究進展。山東中 醫雜誌 2005;24(2):121-123.
47. 譚洪玲、高月、馬增春、陶來寶、趙永紅：四物湯刺激 Peyer's Patch 促進骨髓造血的研究。中藥藥理與臨床 2001;17(2):1-3.
48. 李鷹飛、佟麗、梁乾德、王升啟：四物湯化學成分組合對人骨髓 實驗性肝損傷的保護作用。中藥藥理與臨床 1997;13(4):11-12.
53. ONISHI Yasuharu, YAMAURA Takeshi, TAUCHI Katsunori, SAKAMOTO Takashi, TSUKADA Kazuhiro, NUNOME Shinyu, KOMATSU Yasuhiro, SAIKI Ikuo. Expression of the anti-metastatic effect induced by Juzen-taiho-to is based on the content of Shimotsu-to constituents, Biol Pharm Bull. 1998;21(7):761-765
54. 周坤福、王明艷、陳全良、馬健、王興華、張旭、吳海濤：四物 湯抗變作用的實驗研究。南京中醫藥大學學報1998;14(1):23-24.
55. ZENGLIN LIANA, NIWA Kenji, JINGCHUN GAO, TAGAMI Keiko, HASHIMOTO Midori, YOKOYAMA Yasuhiro, MORI Hideki, TAMAYA Teruhiko. Shimotsu-to is the agent in Juzen-taiho-to responsible for the prevention of endometrial carcinogenesis in mice.
Cancer Lett. 2002;182(1):19-26.
56. Tagami K, Niwa K, Lian Z, Gao J, Mori H, Tamaya T. Preventive effect of Juzen-taiho-to on endometrial carcinogenesis in mice is based on Shimotsu-to constituent. Biol Pharm Bull.
2004;27(2):156-161.
57. 鄭欽岳、曹尉尉、曹穎瑛、林文：四物湯增加小鼠脾細胞分泌 IL-6 及 促 進 IL-6 mRNA 的 表 達 。 第 二 軍 醫 大 學 學 報 1998;19(3):290-292.
58. 張玉杰、張建明、宋敏：四物湯對小鼠免疫功能的影響。中國麻 風皮膚病雜誌 2004;20(6):546-547.
59. 曹尉尉、鄭欽岳、王洪斌：四物湯對小鼠淋巴細胞增殖及巨噬細 胞產生白細胞介素 1 的影響。第二軍醫大學學報 1998;19(1):91-92.
60. 曹蔚蔚、鄭欽岳、楊延莉：四物湯對小鼠脾細胞分泌 IL-3 和 IL-2 的促進作用。第二軍醫大學學報 2004;25:558-559.
61. H. Waynforth, Paul Flecknell: Experimental and surgical technique in the rat, second edition. Academic press 1992: pp. 241-246.
62. Dako 使用說明書 63. Abcam 使用說明書 64. Zymed 使用說明書
65. 李旺祚編譯：蓋統生理學，合計圖書出版社 1991:pp. 1061-1063.
66. Raza Malik, Neil Mellor, Clare Selden, Humphrey Hodgson.
Triiodothyronine enhances the regenerative capacity of the liver following partial hepatectomy. Hepatology. 2003;37:79-86.
67. V. Kirimlioglu, H. Kirimlioglu, S. Yilmaz, D. Ozgor, S. Coban, N.
Karadag, and S. Yologlu. Effect of fish oil, olive oil, and vitamin E on liver pathology, cell proliferation, and antioxidant defense system in
rats subjected to partial hepatectomy. Transplantation Proceedings.
2006;38:564-567.
68. Silva RL, de Melo GB, de Melo VA, Antoniolli AR, Michellone PR, Zucoloto S, Picinato MA, Franco CF, de Assis Mota G, de Castro e Silva O. Effect of the aqueous extract of Sida cordifolia on liver regeneration after partial hepatectomy. Acta Cir Bras.
2006;21(1):37-39.
69. Melo GB, Silva RL, Melo VA, Antoniolli AR, Michellone PR, Zucoloto S, de Souza ME, Gomes MC, Correia RB, Castro-e-Silva O.
Proliferative effect of the aqueous extract of Hyptis pectinata on liver regeneration after partial hepatectomy in rats. Acta Cir Bras. 2006;21 Suppl 1:33-36.
70. S.C. Pradhan, C. Girish. Hepatoprotective herbal drug, silymarin from experimental pharmacology to clinical medicine. Indian Journal of Medical Research. 2006;124(5):491-504.
71. Reinhard Saller, Remy Meier, Reto Brignoli. The use of silymarin in the treatment of liver diseases. Drugs. 2001;61(14):2035-2063.
72. K. Taira, S. Hiroyasu, M. Shiraishi, Y. Muto, T. Koji. Role of the fas system in liver regeneration after a partial hepatectomy in rats.
European Surgical Research. 2001;33:334-341.
73. 黃嘉文編譯：Saint-Frances 各科門診醫學-評估、診斷、檢查與治 療，合計圖書出版社 2005:pp. 246 -247.
74. 李旺祚編譯：蓋統生理學，合計圖書出版社 1991:pp. 1064.
75. 李旺祚編譯：蓋統生理學，合計圖書出版社 1991:pp. 1065-1067.
英文摘要
The liver diseases including viral hepatitis, drug induced hepatitis, cirrhosis and liver cancer is common in Taiwan. According the recent scientific literature, the regeneration of liver cell acts the important role to repair chemistry and biological injury, especially in liver cirrhosis, cancer, operation excision and transplant.
In order to understand the role of traditional chinese medicine in liver regeneration. This study drafts to explain the effects of qi-tonic and blood-tonic Chinese medicine recipes in liver regeneration.
120 rats were divided into five groups: sham, control, silymarin, si jun zi tang and si wu tang groups. All animals were submitted to oral administration of distilled water, silymarin and the aqueous extract of chinese medicine herbs, respectively. After a week, they underwent 2/3 partial hepatectomy. 3, 72 and 168 hours later, livers of each group were removed. Hepatic regeneration was assessed by liver/body weight ratio and immunohistochemical staining for PCNA and BrdU.
These results show that the qi-tonic chinese medicine si jun zi tang at oral doses of 1 g/kg causes a high liver regeneration rate in rats submitted to partial hepatectomy.
謝辭
感謝指導教授蔡金川老師以及林昭庚老師悉心的指導，並感謝本所 侯庭鏞老師與中興大學獸醫病理所廖俊旺老師在實驗方面的協助。亦 感謝李采娟老師在統計方面的指導與幫忙，最後還要感謝參與本研究 的所有研究室成員。
謝謝你們！