發炎對類上皮子宮內膜幹細胞之影響與子宮內膜異位症形成之相關性

致謝 III 摘要 IV ABSTRACT VI ABBREVIATIONS VIII CHAPTER 1 INTRODUCTION 1 Human endometrium and menstrual cycle 1 Stem cells 4 Stem cells in human endometrium 5 Markers of endometrium stem cells 6 Stemness-related transcription factors in human endometrium 7 Stem cells in mouse endometrium 8 Endometrial stem cells and gynaecological disease 9 Relationship between inflammation and endometriosis 10 Relationship between inflammation and cancer 13 Epithelial-mesenchymal transition 15 Rationale and specific aims 16 CHAPTER 2 MATERIALS AND METHODS 17 Experimental animal 17 Cultivation of mouse epithelial-like endometrial stem cells in serum-free culture medium 17 Cultivation of mouse epithelial-like endometrial stem cells in serum-free culture medium under inflammatory conditioned medium 18 Preparation for inflammatory conditioned medium 18 RNA isolation and RT-PCR 18 Polymerase Chain Reaction 19 Real-time Polymerase Chain Reaction 19 Immunocytochemical staining 20 Human tissues 20 Immunohistochemical staining 21 Transfection 21 HA-Oct-4 plasmid 22 Western blotting 22 CHAPTER 3 RESULTS 24 Expression of pluripotent genes Nanog and Oct-4 in hyperplasia and endometriosis (adenomyosis and chocolate cysts) tissues in patients age of 25-35 by RT-PCR analysis 24 Expression of pluripotent genes Nanog and Oct-4 in hyperplasia and endometriosis (adenomyosis and chocolate cysts) tissues in patients age of 35-40 and over 40 by RT-PCR analysis 24 Expression of pluripotent transcription factor Oct-4 in endometriosis (chocolate cyst) tissue 25 Cultivation of epithelial-like endometrial stem cells (mEESCs) in serum-free culture condition 26 Identification of mouse epithelial-like endometrial stem cells (mEESCs) by immunochemical staining 26 Effect of inflammation on mouse epithelial-like endometrial stem cells (mEESCs) 26 Effect of inflammation on Promotor-Oct-4-EGFP RL95-2 cell line 27 Effect of inflammation on HA-Oct-4 RL95-2 cell line 28 CHAPTER 4 DISCUSSION 30 CHAPTER 5 FIGURES 34 Figure 1. Expression of Nanog and Oct-4 in hyperplasia and endometriosis (adenomyosis and chocolate cyst) tissues in patients age of 25-35. 34 Figure 2. Expression of Nanog and Oct-4 in hyperplasia and endometriosis (adenomyosis and chocolate cyst) tissues in patients age of 36-40. 35 Figure 3. Expression of Nanog and Oct-4 in hyperplasia and endometriosis (adenomyosis and chocolate cyst) tissues in patients age of over 40. 36 Figure 4. Expression of pluripotent transcription factor Oct-4 in endometriosis (chocolate cyst) tissue (immunohistochemistry staining). 37 Figure 5. Effect of coating materials on cultivation of mouse epithelial-like endometrial stem cells (mEESCs) in serum-free culture condition. 38 Figure 6. Identification of transcription factors on mouse epithelial-like endometrial stem cells (mEESCs) by immunocytochemical staining. 39 Figure 7. Effect of inflammation on mouse epithelial-like endometrial stem cells (mEESCs). 41 Figure 8. Effect of inflammation on Promotor-Oct-4-EGFP RL95-2 cell line. 43 Figure 9. Effect of inflammation on HA-Oct-4 RL95-2 cell line. 46 SUPPLEMENTS 49 Table 1. Expression of epithelial-like endometrial stem cells cell surface markers 49 Table 2. PCR primer and product size 50 Table 3. Real-time PCR primer 51 REFERENCES 52 1. Gargett, C.E. Uterine stem cells: What is the evidence. Hum Reprod Update 13, 87-101 (2007). 2. Gargett, C.E., Schwab, K.E., Zillwood, R.M., Nguyen, H.P. & Wu, D. Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium. Biol Reprod 80, 1136-45 (2009). 3. Gargett, C.E., Chan, R.W. & Schwab, K.E. Hormone and growth factor signaling in endometrial renewal: role of stem/progenitor cells. Mol Cell Endocrinol 288, 22-9 (2008). 4. Julet, M. Female reproductive physiology. Thomson 20, 770-80 (2004). 5. Du, H. & Taylor, H.S. Stem cells and female reproduction. Reprod Sci 16, 126-39 (2009). 6. Fuchs, E., Tumbar, T. & Guasch, G. Socializing with the neighbors: stem cells and their niche. Cell 116, 769-78 (2004). 7. Padykula, H.A. Regeneration in the primate uterus: the role of stem cells. Ann N Y Acad Sci 622, 47-56 (1991). 8. Chan, R.W., Schwab, K.E. & Gargett, C.E. Clonogenicity of human endometrial epithelial and stromal cells. Biol Reprod 70, 1738-50 (2004). 9. Taylor, H.S. Endometrial cells derived from donor stem cells in bone marrow transplant recipients. JAMA 292, 81-5 (2004). 10. Schwab, K.E. & Gargett, C.E. Co-expression of two perivascular cell markers isolates mesenchymal stem-like cells from human endometrium. Hum Reprod 22, 2903-11 (2007). 11. Wolff, E.F., Wolff, A.B., Hongling, D. & Taylor, H.S. Demonstration of multipotent stem cells in the adult human endometrium by in vitro chondrogenesis. Reprod Sci 14, 524-33 (2007). 12. Cho, N.H., Park, Y.K., Kim, Y.T., Yang, H. & Kim, S.K. Lifetime expression of stem cell markers in the uterine endometrium. Fertil Steril 81, 403-7 (2004). 13. Schwab, K.E., Hutchinson, P. & Gargett, C.E. Identification of surface markers for prospective isolation of human endometrial stromal colony-forming cells. Hum Reprod 23, 934-43 (2008). 14. Gotte, M., Wolf, M., Staebler, A., Buchweitz, O., Kelsch, R., Schuring, A. N. & Kiesel, L. Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma. J Pathol 215, 317-29 (2008). 15. Matthai, C., Horvat, R., Noe, M., Nagele, F., Radjabi, A., van Trotsenburg, M., Huber, J., Kolbus, A. Oct-4 expression in human endometrium. Mol Hum Reprod 12, 7-10 (2006). 16. Forte, A., Schettino, M. T., Finicelli, M., Cipollaro, M., Colacurci, N., Cobellis, L., Galderisi, U. Expression pattern of stemness-related genes in human endometrial and endometriotic tissues. Mol Med 15, 392-401 (2009). 17. Gargett, C.E., Chan, R.W. & Schwab, K.E. Endometrial stem cells. Curr Opin Obstet Gynecol 19, 377-83 (2007). 18. Chan, R.W. & Gargett, C.E. Identification of label-retaining cells in mouse endometrium. Stem Cells 24, 1529-38 (2006). 19. Cervello, I., Martinez-Conejero, J.A., Horcajadas, J.A., Pellicer, A. & Simon, C. Identification, characterization and co-localization of label-retaining cell population in mouse endometrium with typical undifferentiated markers. Hum Reprod 22, 45-51 (2007). 20. Du, H. & Taylor, H.S. Contribution of bone marrow-derived stem cells to endometrium and endometriosis. Stem Cells 25, 2082-6 (2007). 21. Giudice, L.C. & Kao, L.C. Endometriosis. Lancet 364, 1789-99 (2004). 22. Bulun, S.E. Endometriosis. N Engl J Med 360, 268-79 (2009). 23. Weiss, G., Goldsmith, L.T., Taylor, R.N., Bellet, D. & Taylor, H.S. Inflammation in reproductive disorders. Reprod Sci 16, 216-29 (2009). 24. Lebovic, D.I., Mueller, M.D. & Taylor, R.N. Immunobiology of endometriosis. Fertil Steril 75, 1-10 (2001). 25. Brenner, R.M., Nayak, N.R., Slayden, O.D., Critchley, H.O. & Kelly, R.W. Premenstrual and menstrual changes in the macaque and human endometrium: relevance to endometriosis. Ann N Y Acad Sci 955, 60-74 (2002). 26. Wu, M.Y. & Ho, H.N. The role of cytokines in endometriosis. Am J Reprod Immunol 49, 285-96 (2003). 27. Punnonen, J., Teisala, K., Ranta, H., Bennett, B. & Punnonen, R. Increased levels of interleukin-6 and interleukin-10 in the peritoneal fluid of patients with endometriosis. Am J Obstet Gynecol 174, 1522-6 (1996). 28. Ho, H.N., Wu, M. Y., Chao, K. H., Chen, C. D., Chen, S. U., Yang, Y. S. Peritoneal interleukin-10 increases with decrease in activated CD4+ T lymphocytes in women with endometriosis. Hum Reprod 12, 2528-33 (1997). 29. McLaren, J., Prentice, A., Charnock-Jones, D. S., Millican, S. A., Muller, K. H., Sharkey, A. M., Smith, S. K. Vascular endothelial growth factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian steroids. J Clin Invest 98, 482-9 (1996). 30. Taketani, Y., Kuo, T.M. & Mizuno, M. Comparison of cytokine levels and embryo toxicity in peritoneal fluid in infertile women with untreated or treated endometriosis. Am J Obstet Gynecol 167, 265-70 (1992). 31. Lebovic, D.I., Bentzien, F., Chao, V. A., Garrett, E. N., Meng, Y. G., Taylor, R. N. Induction of an angiogenic phenotype in endometriotic stromal cell cultures by interleukin-1beta. Mol Hum Reprod 6, 269-75 (2000). 32. Ray, P., Ghosh, S.K., Zhang, D.H. & Ray, A. Repression of interleukin-6 gene expression by 17 beta-estradiol: inhibition of the DNA-binding activity of the transcription factors NF-IL6 and NF-kappa B by the estrogen receptor. FEBS Lett 409, 79-85 (1997). 33. Yue, T.L., Yue, T. L., Wang, X., Sung, C. P., Olson, B., McKenna, P. J., Gu, J. L., Feuerstein, G. Z. Interleukin-8, a mitogen and chemoattractant for vascular smooth muscle cells. Circ Res 75, 1-7 (1994). 34. Ryan, I.P., Ryan, I. P., Tseng, J. F., Schriock, E. D., Khorram, O., Landers, D. V., Taylor, R. N. Interleukin-8 concentrations are elevated in peritoneal fluid of women with endometriosis. Fertil Steril 63, 929-32 (1995). 35. Arici, A., Tazuke, S.I., Attar, E., Kliman, H.J. & Olive, D.L. Interleukin-8 concentration in peritoneal fluid of patients with endometriosis and modulation of interleukin-8 expression in human mesothelial cells. Mol Hum Reprod 2, 40-5 (1996). 36. Wu, M.Y., Ho, H. N., Chen, S. U., Chao, K. H., Chen, C. D., Yang, Y. S.. Increase in the production of interleukin-6, interleukin-10, and interleukin-12 by lipopolysaccharide-stimulated peritoneal macrophages from women with endometriosis. Am J Reprod Immunol 41, 106-11 (1999). 37. Kennedy, M.K., Picha, K.S., Shanebeck, K.D., Anderson, D.M. & Grabstein, K.H. Interleukin-12 regulates the proliferation of Th1, but not Th2 or Th0, clones. Eur J Immunol 24, 2271-8 (1994). 38. Mori, H., Sawairi, M., Nakagawa, M., Itoh, N., Wada, K., Tamaya, T. Peritoneal fluid interleukin-1 beta and tumor necrosis factor in patients with benign gynecologic disease. Am J Reprod Immunol 26, 62-7 (1991). 39. Verit, F.F. & Aya