臺灣博碩士論文加值系統

　多能性胚胎幹細胞具有分化成三個胚層的能力，因此在臨床上具有很大的應用價值。然而在胚胎幹細胞的培養上需要添加leukemia inhibitory factor (LIF)，透過LIF來開啟Jak2/Stat3 signaling pathway以維持胚胎幹細胞的自我更新及多能性。然而LIF卻是一種很昂貴的藥劑，因此在本篇研究當中，便想找出傳統中草藥取代LIF並且維持胚胎幹細胞之多能性。根據先前本實驗室初篩的結果顯示酒精萃取牛樟芝 (ethanol extract Antrodia Camphorata, EEAC) 可以增加老鼠胚胎纖維母細胞(Mouse embryonic fibroblast, MEF) Oct4及Sox2的基因表現，因此我們推測EEAC具有潛力取代LIF培養胚胎幹細胞。而我們利用了鹼性磷酸酶及免疫螢光染色來檢測處理了EEAC的ES cells是否依舊有胚胎幹細胞的特性，結果顯示處理了EEAC的ES cells仍然具有胚胎幹細胞的特性，像是鹼性磷酸酶、Nanog及SSEA1，尤其是在0.8 μg ml-1的濃度最有效果。接下來便用含有0.8 μg ml-1濃度的EEAC取代LIF對ES cells連續培養六代並且使用EB formation來檢測其多能性。而分化的結果直接證明了EEAC所培養的ES cells確實可以分化成三胚層，像是Tuj1、α-SMA及Gata4。最後我們有興趣想要去探討EEAC透過哪一條pathway來維持ES cells的pluripotency。藉由西方墨點法、Q-PCR及ELISA 的結果顯示EEAC 會活化 Jak2/Stat3 pathway並且增加cytokine的基因表現使得胚胎幹細胞得以維持自我更新及多能性。結論是EEAC透過活化Jak2/Stat3 signaling pathway並且維持胚胎幹細胞的多能性。

　　Embryonic stem (ES) cells are pluripotent stem cells, that have the ability to differentiate into all types of cells. For this reason, ES cells have the potential to clinical application. Cultivation of the ES cells need leukemia inhibitory factor (LIF) to maintain ES cells self-renewal by activating Jak2/Stat3 signaling pathway. However, LIF is an expensive reagent. The goal in this study is to find out a traditional Chinese medicine extract which can replace LIF to maintain the pluripotency of ES cells. In our previous study, we found that Ethanol Extracts Antrodia Camphorata (EEAC) could increase the gene expression leves of Oct4 and Sox2, the genes that could maintain the stemness of stem cells. For this reason, AC has the potential to replace LIF in ES cells cultivation. ES cells were treated with different concentrations of EEAC and identified the stemness of ES cells by Alkaline phosphatease (AP) and immunofluorescent staining. We observed these cells expressed the characteristic of ES cells, including AP, Nanog, and SSEA1 at the concentration of 0.8 μg ml-1. Furthermore, the ES cells were passaged for six generation by the culture medium containing EEAC 0.8 μg ml-1, then used embryoid body formation to identify the pluripotency of ES cells. The results showed that the ES cells still could differentiate to the three germ layer including Tuj1, α-SMA, and Gata4. Finally, we wanted to know why EEAC could maintain the stem cell pluripotency and find the major pathway. By the data of western blot, Q-PCR and ELISA, EEAC activated Jak2/Stat3 pathway and increased the gene expression of related cytokines resulting in maintaining the ES cells self-renewal and pluripotency. In summary, we demonstrated that EEAC could maintain ES pluripotency by activing Jak2/Stat3 signaling pathway.  

目錄 I
圖表目錄 III
縮寫對照表 V
Abstract VII
中文摘要 VIII
壹、緒論 1
一、 幹細胞簡介 1
二、 胚胎幹細胞 2
三、 Oct4 3
四、 Sox2 4
五、 Nanog 5
六、 SSEA1 6
七、 LIF 6
八、 鹼性磷酸酶 7
九、 Jak2/Stat3 pathway 7
十、 牛樟芝 8
貳、研究動機與實驗架構 9
参、實驗材料與方法 10
一、實驗材料 10
二、實驗藥品配製 13
三、實驗設備 22
四、實驗方法 24
肆、實驗結果 32
一、 Antrodia Camphorata對老鼠胚胎纖維母細胞的毒性測試 32
二、 Antrodia Camphorata增加老鼠胚胎纖維母細胞的Oct4及Sox2基因表現 32
三、 Antrodia Camphorata 維持胚胎幹細胞之特性 33
四、 利用Antrodia Camphorata 繼代培養胚胎幹細胞及自發性分化 34
五、 Antrodia Camphorata維持多能胚胎幹細胞自我更新之訊息路徑 35
六、 Antrodia Camphorata對於LIF 接受器相關cytokines 之影響 36
七、 Antrodia Camphorat 維持胚胎幹細胞多能性之訊息路徑 36
伍、討論 37
陸、圖表 40
捌、參考文獻 64

1.Silva, J. and A. Smith, Capturing pluripotency. Cell, 2008. 132(4): p. 532-6.
2.Kolios, G. and Y. Moodley, Introduction to stem cells and regenerative medicine. Respiration, 2013. 85(1): p. 3-10.
3.Klimanskaya, I., Embryonic stem cells from blastomeres maintaining embryo viability. Semin Reprod Med, 2013. 31(1): p. 49-55.
4.Larijani, B., et al., Stem cell therapy in treatment of different diseases. Acta Med Iran, 2012. 50(2): p. 79-96.
5.Oron, E. and N. Ivanova, Cell fate regulation in early mammalian development. Phys Biol, 2012. 9(4): p. 045002.
6.Martin, G.R. and M.J. Evans, Differentiation of clonal lines of teratocarcinoma cells: formation of embryoid bodies in vitro. Proc Natl Acad Sci U S A, 1975. 72(4): p. 1441-5.
7.Furukawa, Y., et al., Monitoring neural stem cell differentiation using PEDOT-PSS based MEA. Biochim Biophys Acta, 2013.
8.Torres, J., et al., Efficient differentiation of embryonic stem cells into mesodermal precursors by BMP, retinoic acid and Notch signalling. PLoS One, 2012. 7(4): p. e36405.
9.Touboul, T., et al., Generation of functional hepatocytes from human embryonic stem cells under chemically defined conditions that recapitulate liver development. Hepatology, 2010. 51(5): p. 1754-65.
10.Shi, G. and Y. Jin, Role of Oct4 in maintaining and regaining stem cell pluripotency. Stem Cell Res Ther, 2010. 1(5): p. 39.
11.Wang, X. and J. Dai, Concise review: isoforms of OCT4 contribute to the confusing diversity in stem cell biology. Stem Cells, 2010. 28(5): p. 885-93.
12.Scholer, H.R., et al., New type of POU domain in germ line-specific protein Oct-4. Nature, 1990. 344(6265): p. 435-9.
13.Rosner, M.H., et al., A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo. Nature, 1990. 345(6277): p. 686-92.
14.Okamoto, K., et al., A novel octamer binding transcription factor is differentially expressed in mouse embryonic cells. Cell, 1990. 60(3): p. 461-72.
15.Lengner, C.J., et al., Oct4 expression is not required for mouse somatic stem cell self-renewal. Cell Stem Cell, 2007. 1(4): p. 403-15.
16.Herr, W. and M.A. Cleary, The POU domain: versatility in transcriptional regulation by a flexible two-in-one DNA-binding domain. Genes Dev, 1995. 9(14): p. 1679-93.
17.Chew, J.L., et al., Reciprocal transcriptional regulation of Pou5f1 and Sox2 via the Oct4/Sox2 complex in embryonic stem cells. Mol Cell Biol, 2005. 25(14): p. 6031-46.
18.Zhang, J., et al., Sall4 modulates embryonic stem cell pluripotency and early embryonic development by the transcriptional regulation of Pou5f1. Nat Cell Biol, 2006. 8(10): p. 1114-23.
19.Yang, J., et al., Genome-wide analysis reveals Sall4 to be a major regulator of pluripotency in murine-embryonic stem cells. Proc Natl Acad Sci U S A, 2008. 105(50): p. 19756-61.
20.Xu, N., et al., MicroRNA-145 regulates OCT4, SOX2, and KLF4 and represses pluripotency in human embryonic stem cells. Cell, 2009. 137(4): p. 647-58.
21.Tay, Y., et al., MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature, 2008. 455(7216): p. 1124-8.
 
22.Boyer, L.A., et al., Core transcriptional regulatory circuitry in human embryonic stem cells. Cell, 2005. 122(6): p. 947-56.
23.Bani-Yaghoub, M., et al., Role of Sox2 in the development of the mouse neocortex. Dev Biol, 2006. 295(1): p. 52-66.
24.Alatzoglou, K.S., D. Kelberman, and M.T. Dattani, The role of SOX proteins in normal pituitary development. J Endocrinol, 2009. 200(3): p. 245-58.
25.Dy, P., Y. Han, and V. Lefebvre, Generation of mice harboring a Sox5 conditional null allele. Genesis, 2008. 46(6): p. 294-9.
26.Avilion, A.A., et al., Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev, 2003. 17(1): p. 126-40.
27.Zhao, S., et al., SoxB transcription factors specify neuroectodermal lineage choice in ES cells. Mol Cell Neurosci, 2004. 27(3): p. 332-42.
28.Masui, S., et al., Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells. Nat Cell Biol, 2007. 9(6): p. 625-35.
29.Niwa, H., How is pluripotency determined and maintained? Development, 2007. 134(4): p. 635-46.
30.Fong, Y.W., et al., A DNA repair complex functions as an Oct4/Sox2 coactivator in embryonic stem cells. Cell, 2011. 147(1): p. 120-31.
31.Chambers, I., et al., Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell, 2003. 113(5): p. 643-55.
32.Mitsui, K., et al., The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell, 2003. 113(5): p. 631-42.
33.Golubovskaya, V.M., FAK and Nanog cross talk with p53 in cancer stem cells. Anticancer Agents Med Chem, 2013. 13(4): p. 576-80.
 
34.Theunissen, T.W. and J.C. Silva, Switching on pluripotency: a perspective on the biological requirement of Nanog. Philos Trans R Soc Lond B Biol Sci, 2011. 366(1575): p. 2222-9.
35.Chambers, I., et al., Nanog safeguards pluripotency and mediates germline development. Nature, 2007. 450(7173): p. 1230-4.
36.Saunders, A., F. Faiola, and J. Wang, Pursuing Self-Renewal and Pluripotency with the Stem Cell Factor Nanog. Stem Cells, 2013.
37.Mullin, N.P., et al., The pluripotency rheostat Nanog functions as a dimer. Biochem J, 2008. 411(2): p. 227-31.
38.Capela, A. and S. Temple, LeX is expressed by principle progenitor cells in the embryonic nervous system, is secreted into their environment and binds Wnt-1. Dev Biol, 2006. 291(2): p. 300-13.
39.Chodorowska, G., A. Glowacka, and M. Tomczyk, Leukemia inhibitory factor (LIF) and its biological activity. Ann Univ Mariae Curie Sklodowska Med, 2004. 59(2): p. 189-93.
40.Sims, N.A. and R.W. Johnson, Leukemia inhibitory factor: a paracrine mediator of bone metabolism. Growth Factors, 2012. 30(2): p. 76-87.
41.Cullinan, E.B., et al., Leukemia inhibitory factor (LIF) and LIF receptor expression in human endometrium suggests a potential autocrine/paracrine function in regulating embryo implantation. Proc Natl Acad Sci U S A, 1996. 93(7): p. 3115-20.
42.Taupin, J.L., et al., Leukemia inhibitory factor: part of a large ingathering family. Int Rev Immunol, 1998. 16(3-4): p. 397-426.
43.Zhu, H., H. Yang, and M.R. Owen, Combined microarray analysis uncovers self-renewal related signaling in mouse embryonic stem cells. Syst Synth Biol, 2007. 1(4): p. 171-81. 
44.Kim, E.E. and H.W. Wyckoff, Reaction mechanism of alkaline phosphatase based on crystal structures. Two-metal ion catalysis. J Mol Biol, 1991. 218(2): p. 449-64.
45.de Backer, M., et al., The 1.9 A crystal structure of heat-labile shrimp alkaline phosphatase. J Mol Biol, 2002. 318(5): p. 1265-74.
46.Yu Plisova, E., et al., A highly active alkaline phosphatase from the marine bacterium cobetia. Mar Biotechnol (NY), 2005. 7(3): p. 173-8.
47.Webb, D.A., Short Term Storage of Enzyme-Treated Cells. Vox Sang, 1964. 9: p. 510-1.
48.Shamblott, M.J., et al., Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci U S A, 1998. 95(23): p. 13726-31.
49.Chung, B.M., et al., Jak2 and Tyk2 are necessary for lineage-specific differentiation, but not for the maintenance of self-renewal of mouse embryonic stem cells. Biochem Biophys Res Commun, 2006. 351(3): p. 682-8.
50.Raz, R., et al., Essential role of STAT3 for embryonic stem cell pluripotency. Proc Natl Acad Sci U S A, 1999. 96(6): p. 2846-51.
51.Niwa, H., et al., Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev, 1998. 12(13): p. 2048-60.
52.Matsuda, T., et al., STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells. EMBO J, 1999. 18(15): p. 4261-9.
53.Gearing, D.P., et al., Leukemia inhibitory factor receptor is structurally related to the IL-6 signal transducer, gp130. EMBO J, 1991. 10(10): p. 2839-48.
 
54.Nakamura, T., et al., A selective switch-on system for self-renewal of embryonic stem cells using chimeric cytokine receptors. Biochem Biophys Res Commun, 1998. 248(1): p. 22-7.
55.Taga, T. and T. Kishimoto, Gp130 and the interleukin-6 family of cytokines. Annu Rev Immunol, 1997. 15: p. 797-819.
56.Chen, X., et al., Crystal structure of a tyrosine phosphorylated STAT-1 dimer bound to DNA. Cell, 1998. 93(5): p. 827-39.
57.Thompson, L.H., et al., A LIF/Nanog axis is revealed in T lymphocytes that lack MARCH-7, a RINGv E3 ligase that regulates the LIF-receptor. Cell Cycle, 2010. 9(20): p. 4213-21.
58.Lee, T.K., et al., CD24(+) liver tumor-initiating cells drive self-renewal and tumor initiation through STAT3-mediated NANOG regulation. Cell Stem Cell, 2011. 9(1): p. 50-63.
59.Bourillot, P.Y., et al., Novel STAT3 target genes exert distinct roles in the inhibition of mesoderm and endoderm differentiation in cooperation with Nanog. Stem Cells, 2009. 27(8): p. 1760-71.
60.Williams, R.L., et al., Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature, 1988. 336(6200): p. 684-7.
61.Geethangili, M. and Y.M. Tzeng, Review of Pharmacological Effects of Antrodia camphorata and Its Bioactive Compounds. Evid Based Complement Alternat Med, 2011. 2011: p. 212641.
62.Chen, Y.C., et al., New Anti-Inflammatory Aromatic Components from Antrodia camphorata. Int J Mol Sci, 2013. 14(3): p. 4629-39.
63.Lee, I.H., et al., Antrodia camphorata polysaccharides exhibit anti-hepatitis B virus effects. FEMS Microbiol Lett, 2002. 209(1): p. 63-7. 
64.Yue, P.Y., et al., Review of biological and pharmacological activities of the endemic Taiwanese bitter medicinal mushroom, Antrodia camphorata (M. Zang et C. H. Su) Sh. H. Wu et al. (higher Basidiomycetes). Int J Med Mushrooms, 2012. 14(3): p. 241-56.
65.Ao, Z.H., et al., Niuchangchih (Antrodia camphorata) and its potential in treating liver diseases. J Ethnopharmacol, 2009. 121(2): p. 194-212.
66.Lin, W.C., et al., Filtrate of fermented mycelia from Antrodia camphorata reduces liver fibrosis induced by carbon tetrachloride in rats. World J Gastroenterol, 2006. 12(15): p. 2369-74.
67.Lu, M.K., et al., Fermented Antrodia cinnamomea extract protects rat PC12 cells from serum deprivation-induced apoptosis: the role of the MAPK family. J Agric Food Chem, 2008. 56(3): p. 865-74.
68.Huang, N.K., et al., Antrodia camphorata prevents rat pheochromocytoma cells from serum deprivation-induced apoptosis. FEMS Microbiol Lett, 2005. 244(1): p. 213-9.
69.Liu, D.Z., et al., Antihypertensive activities of a solid-state culture of Taiwanofungus camphoratus (Chang-chih) in spontaneously hypertensive rats. Biosci Biotechnol Biochem, 2007. 71(1): p. 23-30.
70.Wang, G.J., et al., The vasorelaxation of Antrodia camphorata mycelia: involvement of endothelial Ca(2+)-NO-cGMP pathway. Life Sci, 2003. 73(21): p. 2769-83.
71.Lu, M.C., et al., Active extracts of wild fruiting bodies of Antrodia camphorata (EEAC) induce leukemia HL 60 cells apoptosis partially through histone hypoacetylation and synergistically promote anticancer effect of trichostatin A. Arch Toxicol, 2009. 83(2): p. 121-9.
 
72.Schmitz, J., et al., SOCS3 exerts its inhibitory function on interleukin-6 signal transduction through the SHP2 recruitment site of gp130. J Biol Chem, 2000. 275(17): p. 12848-56.
73.Takahashi, K. and S. Yamanaka, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006. 126(4): p. 663-76.
74.Liu, S.P., et al., Induced pluripotent stem (iPS) cell research overview. Cell Transplant, 2011. 20(1): p. 15-9.
75.Daley, W.P., S.B. Peters, and M. Larsen, Extracellular matrix dynamics in development and regenerative medicine. J Cell Sci, 2008. 121(Pt 3): p. 255-64.
76.Takase, O., et al., The role of NF-kappaB signaling in the maintenance of pluripotency of human induced pluripotent stem cells. PLoS One, 2013. 8(2): p. e56399.