在台灣、亞洲及南非地區肝細胞癌是最嚴重的癌症。由於肝癌無論是手術治療或非手術治療，其治療效果均與腫瘤期別有密切關係，因此早期診斷、早期治療是非常重要的。然而高達80％的原發性肝細胞癌病人於五年內會再併發復發性肝細胞癌，其再復發的機制至今仍不十分清楚。因此對於肝癌之形成機轉、復發原因與預防或治療方式之探究是至為重要的。
隨著不同時期和程度的肝損傷，過度的活性氧(reactive oxygen species, ROS) 如O2˙-、H2O2 以及NO會造成脂質過氧化(lipid peroxidation)、蛋白質氧化(protein oxidation)、DNA損傷(DNA damage) 與突變產生(mutagenesis)。這些活性氧(reactive oxygen species, ROS)d可能主要來自肝細胞之粒線體、活化的巨噬細胞(activated macrophages, 或稱Kupffer cells)與入侵之中性球(infiltrating neutrophils)。活性氧(reactive oxygen species, ROS)會啟動細胞核因子nuclear factor-kappa B (NF-kB) 與activator protein-1 (AP-1) 移位至細胞核而造成許多發炎細胞激素的釋放引起發炎。過量活性氧(reactive oxygen species, ROS)透過Kupffer cell內的 NADPH oxidase 或是肝細胞之cytochrome P4502E1 (CYP2E1) 會促進肝臟內之stellate cells 活化而造成肝纖維化與肝癌。
肝損傷時肝臟內許多細胞受原位細胞或發炎細胞氧化壓力增加引起Bax/Bcl-2 family啟動細胞凋亡 (apoptosis)、caspase 1/IL-1調節之發炎性細胞死亡(pyroptosis)或Beclin-1/LC-3β調節之細胞自噬 (autophagy)造成肝功能損傷進而形成肝腫瘤。間葉幹細胞(MSC)具有極佳自我更新（self-renewal）及增生（proliferation）能力，MSC同時也具有旁分泌（paracrine）的功能，其培養基(CM)中可能含有高量的growth factors、cytokines及prostaglandins。這些物質會增加細胞增生、存活及血管生成而提升組織修復。
為探究MSC之治療效益，我們利用以N-nitrosodiethylamine (DEN)刺激肝發炎、硬化與腫瘤之大鼠動物模式，評估MSC給予對DEN誘發肝發炎、硬化與腫瘤之病理生理效應。研究發現靜脈給予MSC後，其主要匯集於受傷的肝臟組織。另外，MSC的給予改善DEN引起之肝發炎、硬化與腫瘤等病理特徵，包括降低Masson染色之損傷指標、細胞凋亡、細胞自噬，發炎性細胞死亡表現，並降低肝功能AST與ALT之數值。綜合研究結果，MSC有相當之潛力發展成為對肝損傷患者之細胞治療製劑。

Hepatocellular carcinoma (HCC) is the most severe cancer in Taiwan, Asia, and South Africa. The therapeutic strategy to treat HCC, either operative or non-operative methods, is related to the tumor stages. Therefore, an earlier diagnosis and treatment of HCC is important. However, 80% of the patients with primary HCC will be accompanied by relapsing HCC in five years. The mechanism of HCC recurrence is not clearly understood yet. Based on the information, it is necessary to investigate the pathophysiologic mechanisms of HCC formation and recurrence and to discover novel techniques and methods to prevent and cure HCC.
Hepatogenesis begins in the setting of chronic liver inflammation and hepatic fibrosis, which are possibly caused by hepatotoxins. Increased oxidative stress, inflammatory cytokines and growth factors subsequently lead to hepatocyte proliferation. Prolonged cell damage by chronic inflammation is critical in cancer development. Overproduction of reactive oxygen species (ROS) including O2˙-, H2O2 and NO, can cause lipid peroxidation, protein oxidation, DNA damage and mutagenesis associated with various stages of liver injury. ROS can trigger translocation of nuclear factor-kappa B (NF-B) and activator protein-1 (AP-1) to nucleus and activation of several inflammatory cytokines and adhesion molecules that contribute to further production of ROS and consecutive cell death. The increased ROS can enhance Bax/Bcl-2 ratio, caspase 3 (CPP32) expression, and poly-(ADP-ribose)-polymerase (PARP) fragments subsequently resulting in apoptotic cell death, enhance the expression of the autophagy-promoting protein Beclin-1 expression, leading to autophagy and caspase 1/IL-1 mediated pyroptosis.
Mesenchymal stem cells (MSC) provide excellent self-renewal and proliferation ability. In addition, MSC may release high levels of growth factors, cytokines and prostaglandins in the microenvironment or condition medium (CM) by the paracrine mechanism to enhance cell proliferation, survival and angiogenesis to repair injured tissue.
To investigate the therapeutic efficiency of MSC treatment, we used N-nitrosodiethylamine (DEN) as an inducer to develop hepatocellular inflammation, fibrosis and carcinoma on rats, and evaluated the pathophysiological effects of MSC treatment on these rat models. MSC application improves DEN-induced hepatocellular inflammation, fibrosis and carcinoma. The degree of fibrosis by Masson staining was also decreased. Furthermore, the marker of apoptosis, pyroptosis, and autophagy was enhanced by DEN injury and was decreased by MSC treatment. The increased level of AST and ALT activity by DEN injury was also reduced by MSC treatment. In conclusion, MSC has a considerable potential to be a cell therapy for patients with liver injury and HCC.

1. Ahn, B., Han, B.S., Kim, D.J. & Ohshima, H. (1999) Immunohistochemical localization of inducible nitric oxide synthase and 3-nitrotyrosine in rat liver tumors induced by N-nitrosodiethylamine. Carcinogenesis 20(7): p. 1337-1344
2. Albanis, E., Safadi, R. & Friedman, S.L. (2003) Treatment of hepatic fibrosis: almost there. Current gastroenterology reports 5(1): p. 48-56
3. Babior, B., Lambeth, J. & Nauseef, W. (2002) The neutrophil NADPH oxidase. Archives of biochemistry and biophysics 397(2): p. 342-344
4. Bataller, R., Ginès, P., Nicolás, J.M., Görbig, M.N., Garcia-Ramallo, E., Gasull, X., Bosch, J., Arroyo, V. & Rodés, J. (2000) Angiotensin II induces contraction and proliferation of human hepatic stellate cells. Gastroenterology 118(6): p.1149-1156
5. Blum, H.E. (2002) Molecular targets for prevention of hepatocellular carcinoma. Digestive Diseases 20(1): p. 81-90
6. Boitier, E., Merad-Boudia, M., Guguen-Guillouzo, C., Defer, N., Ceballos-Picot, I., Leroux, J.P. & Marsac, C. (1995) Impairment of the mitochondrial respiratory chain activity in diethylnitrosamine-induced rat hepatomas: possible involvement of oxygen free radicals. Cancer research 55(14): p. 3028-3035
7. Casini, A., Ceni, E., Salzano, R., Biondi, P., Parola, M., Galli, A., Foschi, M., Caligiuri, A., Pinzani, M. & Surrenti, C. (1997) Neutrophil-derived superoxide anion induces lipid peroxidation and stimulates collagen synthesis in human hepatic stellate cells: role of nitric oxide. Hepatology 25(2): p. 361-367
8. Chien, C.T., Yu, H.J., Cheng, Y.J., Wu, M.S., Chen, C.F. & Hsu, S.M. (2000) Reduction in renal haemodynamics by exaggerated vesicovascular reflex in rats with acute urinary retention. The Journal of physiology 526(2): p. 397-408
9. Chien, C.T., Lee, P.H., Chen, C.F., Ma, M.C., Lai, M.K. & Hsu, S.M. (2001) De novo demonstration and co-localization of free-radical production and apoptosis formation in rat kidney subjected to ischemia/reperfusion. Journal of the American Society of Nephrology 12(5): p. 973-982
10. Chien, C.T., Yu, H.J., Lin, T.B., Lai, M.K. & Hsu, S.M. (2003) Substance P via NK1 receptor facilitates hyperactive bladder afferent signaling via action of ROS. American Journal of Physiology-Renal Physiology 284(4): p. 840-851
11. Chen, W.C., Hayakawa, S., Shimizu, K., Chien, C.T. & Lai, M.K. (2004) Catechins prevents substance P-induced hyperactive bladder in rats via the downregulation of ICAM and ROS. Neuroscience letters 367(2): p. 213-217
12. Yu, H.J., Lin, B.R., Lee, H.S., Shun, C.T., Yang, C.C., Lai, T.Y., Chien, C.T. & Hsu, S.M. (2005) Sympathetic vesicovascular reflex induced by acute urinary retention evokes proinflammatory and proapoptotic injury in rat liver. American Journal of Physiology-Renal Physiology 288(5): p. 1005-1014
13. Coker, H., Thomas, A. & Akintonwa, A. (1991) Determination of the total level of nitrosamines in select consumer products in Lagos area of Nigeria. Bulletin of environmental contamination and toxicology 47(5): p. 706-710
14. Corpechot, C., Barbu, V., Wendum, D., Kinnman, N., Rey, C., Poupon, R., Housset, C. & Rosmorduc, O. (2002) Hypoxia‐induced VEGF and collagen I expressions are associated with angiogenesis and fibrogenesis in experimental cirrhosis. Hepatology 35(5): p. 1010-1021
15. Greenwel, P., Domínguez‐Rosales, J.A., Mavi, G., Rivas‐Estilla, A.M. & Rojkind, M. (2000) Hydrogen peroxide: A link between acetaldehyde‐elicited α1 (i) collagen gene up‐regulation and oxidative stress in mouse hepatic stellate cells. Hepatology 31(1): p. 109-116
16. Hellwig-Bürgel, T., Rutkowski, K., Metzen, E., Fandrey, J. & Jelkmann, W. (1999) Interleukin-1β and Tumor Necrosis Factor Stimulate DNA Binding of Hypoxia-Inducible Factor-1. Blood 94(5): p. 1561-1567
17. Hsu, I.C., Metcalf, R.A., Sun, T., Welsh, J.A., Wang, N.J. & Harris, C.C. (1991) Mutational hot spot in the p53 gene in human hepatocellular carcinomas Nature 350(6317): p. 427-428
18. Yang, J.C., Hung, C.Y., Shun, C.T., Wang, T.H., Chien, C.T. & Kao, Y. (2013) Catechins and sialic acid attenuate Helicobacter pylori-triggered epithelial caspase-1 activity and eradicate Helicobacter pylori infection. Evidence-Based Complementary and Alternative Medicine 2013, 248585
19. Chung, S.D., Lai, T.Y., Chien, C.T. & Yu, H.J. (2012)
Activating Nrf-2 signaling depresses unilateral ureteral obstruction-evoked mitochondrial stress-related autophagy, apoptosis and pyroptosis in kidney PloS One 7(10): e47299
20. Al-Sowayan, B., Keogh, R.J., Abumaree, M., Georgiou, H.M. & Kalionis, B. (2017) The effect of endothelial cell activation and hypoxia on placental chorionic mesenchymal stem/stromal cell migration. Placenta S0143-4004(17): p. 30637-30639
21. Shin, J.H., Jeon, H.J., Park, J. & Chang, M.S. (2016) Epigallocatechin-3-gallate prevents oxidative stress-induced cellular senescence in human mesenchymal stem cells via Nrf2. International journal of molecular medicine 38(4): p. 1075-1082
22. Chani, B., Puri, V., Sobti, R.C. & Puri1, S. (2016) Epigallocatechin Gallate Inhibits Mouse Mesenchymal Stem Cell Differentiation to Adipogenic Lineage. Journal of stem cells & regenerative medicine 12(1): p. 16-24
23. Kim, Y.S., Farrar, W., Colburn, N.H. & Milner, J.A. (2012) Cancer stem cells: potential target for bioactive food components. The Journal of nutritional biochemistry 23(7): p. 691-698
24. Kuo, C.L., Chen, T.S., Liou, S.Y. & Hsieh, C.C. (2014) Immunomodulatory effects of EGCG fraction of green tea extract in innate and adaptive immunity via T regulatory cells in murine model. Immunopharmacology and immunotoxicology 36(5): p. 364-370
25. Chen, T.S., Liou, S.Y., Wu, H.C., Tsai, F.J., Tsai, C.H., Huang, C.Y. & Chang, Y.L. (2011) Efficacy of epigallocatechin-3-gallate and Amla (Emblica officinalis) extract for the treatment of diabetic-uremic patients. Journal of medicinal food 14(7-8): p. 718-723
26. Nunes, A.R., Alves, M.G., Moreira, P.I., Oliveira, P.F & Silva, B.M. (2014) Can Tea Consumption be a Safe and Effective Therapy Against Diabetes Mellitus-Induced Neurodegeneration? Current neuropharmacology 12(6): p. 475-489
27. Jiang, W., Gao, M., Sun, S., Bi, A., Xin, Y., Han, X., Wang, L., Yin Z. & Luo, L. (2012) Protective effect of L-theanine on carbon tetrachloride-induced acute liver injury in mice. Biochemical and biophysical research communications 422(2): p. 344-350
28. Li, G., Ye, Y., Kang, J., Yao, X., Zhang, Y., Jiang, W., Gao, M., Dai, Y., Xin, Y., Wang, Q., Yin, Z. & Luo, L. (2012) L-Theanine prevents alcoholic liver injury through enhancing the antioxidant capability of hepatocytes. Food and chemical toxicology 50(2): p. 363-372
29. Kakuda, T. (2011) Neuroprotective effects of theanine and its preventive effects on cognitive dysfunction. Pharmacological Research 64(2): p. 162-168
30. Kwon, K.J., Kim, J.N., Kim, M.K., Lee, J., Ignarro, L.J., Kim, H.J., Shin, C.Y. & Han, S.H. (2011) Melatonin synergistically increases resveratrol‐induced heme oxygenase‐1 expression through the inhibition of ubiquitin‐dependent proteasome pathway: a possible role in neuroprotection. Journal of pineal research 50(2): p. 110-123
31. Tiniakos, D. (2009) Liver biopsy in alcoholic and non-alcoholic steatohepatitis patients. Gastroenterologie clinique et biologique 33(10-11): p. 930-939
32. Erba, D., Riso, P., Bordoni, A., Foti, P., Biagi, P.L. & Testolin, G. (2005) Effectiveness of moderate green tea consumption on antioxidative status and plasma lipid profile in humans. The Journal of nutritional biochemistry 16(3): p. 144-149
33. O'Brien, T., Simsiman, R. & Boutwell, R. (1975) Induction of the polyamine-biosynthetic enzymes in mouse epidermis and their specificity for tumor promotion. Cancer research 35(9): p. 2426-2433
34. Laurie, S.A., Miller, V.A., Grant, S.C., Kris, M.G. & Ng, K.K. (2005) Phase I study of green tea extract in patients with advanced lung cancer. Cancer chemotherapy and pharmacology 55(1): p. 33-38
35. Cheng, K.W., Wong, C.C, Chao, J., Lo, C., Chen, F., Chu, I.K., Che, C.M., Ho, C.T. & Wang, M. (2009) Inhibition of mutagenic PhIP formation by epigallocatechin gallate via scavenging of phenylacetaldehyde. Molecular nutrition & food research 53(6): p. 716-725
36. Koo, M.W. & Cho, C.H. (2004) Pharmacological effects of green tea on the gastrointestinal system. European journal of pharmacology 500(1-3): p. 177-185
37. Li, C.F., Zhu, Y., Yu, Y., Zhao, Q.Y., Wang, S.J., Wang, X.C., Yao, M.Z., Luo, D., Li, X., Chen, L. & Yang Y.J. (2015) Global transcriptome and gene regulation network for secondary metabolite biosynthesis of tea plant (Camellia sinensis). BMC genomics 16(1): p. 560
38. Kimura, K., Ozeki, M., Juneja, L.R. & Ohira, H. (2007) L-Theanine reduces psychological and physiological stress responses. Biological psychology 74(1): p. 39-45
39. Egashira, N., Hayakawa, K., Mishima, K., Kimura, H., Iwasaki, K. & Fujiwara, M. (2004) Neuroprotective effect of γ-glutamylethylamide (theanine) on cerebral infarction in mice. Neuroscience letters 363(1): p. 58-61
40. Juneja, L.R., Chu, D.C., Okubo, T., Nagato, Y. & Yokogoshi, H. (1999) L-theanine—a unique amino acid of green tea and its relaxation effect in humans. Trends in Food Science & Technology 10(6-7): p. 199-204
41. Hsu, S.P., Wu, M.S., Yang, C.C., Huang, K.C., Liou, S.Y., Hsu, S.M. & Chien, C.T. (2007) Chronic green tea extract supplementation reduces hemodialysis-enhanced production of hydrogen peroxide and hypochlorous acid, atherosclerotic factors, and proinflammatory cytokines. The American journal of clinical nutrition 86(5): p. 1539-1547
42. Celso, C.L., Fleming, H.E., Wu, J.W., Zhao, C.X., Miake-Lye, S., Fujisaki, J., Côté, D., Rowe, D.W., Lin, C.P. & Scadden, D.T. (2009) Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche. Nature 457(7225): p. 92-96
43. Rockey, D.C. (2000) The cell and molecular biology of hepatic fibrogenesis: clinical and therapeutic implications. Clinics in liver disease 4(2): p. 319-355
44. Wang, J., Cen, P., Chen, J., Fan, L., Li, J., Cao, H., & Li, L. (2017) Role of mesenchymal stem cells, their derived factors, and extracellular vesicles in liver failure. Stem Cell Research & Therapy 8(1): p. 137
45. Caplan, A.I. & Correa, D. (2011) The MSC: an injury drugstore. Cell stem cell 9(1): p. 11-15
46. Christ, B., Brückner, S. & Winkler, S. (2015) The therapeutic promise of mesenchymal stem cells for liver restoration. Trends in molecular medicine 21(11): p. 673-686
47. Trounson, A. & McDonald, C. (2015) Stem cell therapies in clinical trials: progress and challenges. Cell Stem Cell 17(1): p. 11-22
48. Agarwal, R. & Mukhtar, H. (1993) Oxidative stress in skin chemical carcinogenesis. Oxidative Stress in Dermatology 102(1): p. 207-241
49. Hung, G.D., Li, P.C., Lee, H.S., Chang, H.M., Chien, C.T. & Lee, K.L. (2012) Green tea extract supplementation ameliorates CCl 4-induced hepatic oxidative stress, fibrosis, and acute-phase protein expression in rat. Journal of the Formosan Medical Association 111(10): p. 550-559
50. D'Alessandro, T., Prasain, J., Benton, M.R., Botting, N., Moore, R., Darley-Usmar, V., Patel, R. & Barnes, S. (2003) Polyphenols, inflammatory response, and cancer prevention: chlorination of isoflavones by human neutrophils. The Journal of nutrition 133(11): p. 3773-3777
51. Kim, H.J., Jeon, S.M., Lee, M.K., Jung, U.J., Shin, S.K. & Choi, M.S. (2009) Antilipogenic effect of green tea extract in C57BL/6J‐Lepob/ob mice. Phytotherapy Research 23(4): p. 467-471
52. Carlson, A.L., Fujisaki, J., Wu, J., Runnels, J.M., Turcotte, R., Spencer, J.A., Celso, C.L., Scadden, D.T., Strom, T.B. & Lin, C.P. (2013) Tracking single cells in live animals using a photoconvertible near-infrared cell membrane label. PloS one 8(11): e69257