博士后学位论文. Importin 13: 一个新的角膜上皮前体细胞标志物. Importin 13:a Novel Potential Marker for Corneal Epithelial Progenitor Cells 指导教师 : 刘祖国 专业名称 : 眼科学

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1 学校编码 :10384 学号 :BH 博士后学位论文 Importin 13: 一个新的角膜上皮前体细胞标志物 Importin 13:a Novel Potential Marker for Corneal Epithelial Progenitor Cells 王华 指导教师 : 刘祖国 专业名称 : 眼科学 答辩日期 :2009 年 9 月

2 厦门大学学位论文原创性声明 本人呈交的学位论文是本人在导师指导下, 独立完成的研究成果 本人在论文写作中参考其他个人或集体已经发表的研究成果, 均在文中 以适当方式明确标明, 并符合法律规范和 厦门大学研究生学术活动规 范 ( 试行 ) 另外, 该学位论文为 ( ) 课题 ( 组 ) 的研究成果, 获得 ( ) 课题 ( 组 ) 经费或实验室的资助, 在 ( ) 实验室完成 ( 请在以上括号内填写课题或课题组负责人或实验室名称, 未有此项声明内容的, 可以不作特别声明 ) 声明人 ( 签名 ): 年月日

3 厦门大学学位论文著作权使用声明 本人同意厦门大学根据 中华人民共和国学位条例暂行实施办法 等规定保留和使用此学位论文, 并向主管部门或其指定机构送交学位 论文 ( 包括纸质版和电子版 ), 允许学位论文进入厦门大学图书馆及其数 据库被查阅 借阅 本人同意厦门大学将学位论文加入全国博士 硕士 学位论文共建单位数据库进行检索, 将学位论文的标题和摘要汇编出版, 采用影印 缩印或者其它方式合理复制学位论文 本学位论文属于 : ( )1. 经厦门大学保密委员会审查核定的保密学位论文, 于年月日解密, 解密后适用上述授权 ( )2. 不保密, 适用上述授权 ( 请在以上相应括号内打 或填上相应内容 保密学位论文应是已经厦门大学保密委员会审定过的学位论文, 未经厦门大学保密委员会审定的学位论文均为公开学位论文 此声明栏不填写的, 默认为公开学位论文, 均适用上述授权 ) 声明人 ( 签名 ): 年月日

4 摘要 Importin13(IPO13) 是 importinβ 家族中最新发现的新成员, 是该家族中唯一具有双向转运功能的受体蛋白, 它可介导一些与细胞发育和生理功能密切相关的重要蛋 白质的核输入和核输出 本研究通过免疫学染色和 RT-PCR 检测 IPO13 在人角膜组织 角膜上皮细胞原代培养和克隆培养中的特异性表达 ; 并且利用人角膜边缘上皮细 胞克隆培养和小鼠角膜 / 边缘上皮前体细胞系 TKE2 细胞 (the murine limbal/corneal epithelium-derived progenitor cell line) 为研究对象, 并以 IPO13 抑制剂 ---- Ran G19V/Q69L 处理, 或转换培养基 (TKE2) 研究 IPO13 对角膜上皮前体细胞增殖与分化的调控 IPO13 主要在人角膜缘上皮基底细胞的核内高表达, 在胞质内弱表达, 但在角膜缘其他细胞层和中央角膜上皮的全层均无表达 在角膜缘上皮细胞克隆形成早期,IPO13 主要表达于克隆内的上皮细胞 ; 随克隆培养时间的延长, 阳性表达 IPO13 的细胞逐渐减少并局限于克隆的边缘区域 培养于 KSFM 培养基中的 TKE2 细胞阳性表达 IPO13;TKE2 细胞转培养于加有 FBS 的 DMEM 培养基中的方法可促进 TKE2 细胞的分化, 此时 IPO13 表达转为阴性 以不同浓度 IPO13 抑制剂 ---Ran G19V/Q69L 处理人角膜边缘上皮细胞克隆培养和 TKE2 细胞后,IPO13 在两者中的表达明显下调, 人角膜边缘上皮细胞的克隆形成率和克隆生长能力 TKE2 细胞增殖能力明显降低, 并促进人角膜边缘上皮克隆内干细胞和 TKE2 细胞向成熟角膜上皮细胞分化 我们的研究结果首次证实 :IPO13 在角膜上皮干细胞中特异性表达, 并在维持角膜上皮干细胞表型 低分化状态和高增殖潜能中起到重要的调控作用 ;IPO13 可作为一个新的角膜上皮前体细胞标志物 关键词 :Importin13; 干细胞 ; 标志物 ; 角膜 ; 上皮

5 Abstract Importin13 (IPO13), the newest member of importin β family discovered recently, is a unique nucleus-cytoplasm bidirection transport receptor protein. In this study, IPO13 expression in human corneal tissue, limbal epithelial primary explant and clonal cultures was evaluated by immunostaining and RT-PCR. IPO13 function was evaluated in the corneal epithelial cultures treated with IPO13 inhibitor, or FBS-containing DMEM medium by colony-forming efficiency (CFE), clone growth capacity, MTT, immunostaining and Western blotting assay. IPO13 protein was expressed mainly in nuclei of limbal epithelial basal cells, but not in the other cell layers of limbus and full thickness of corneal epithelia. IPO13 expressed in majority of epithelial cells in early stage clone and in the margin of late stage clone. IPO13 was positively expressed in mouse TKE2 progenitor cells cultured in KSFM, while it became negative in FBS-containing DMEM, which promoted TKE2 cell differentiation. At the presence of IPO13 inhibitor, IPO13 expression and the proliferative capacity decreased in human limbal epithelial clones and mouse TKE2 cells, which were accompanied with the cell differentiation. In conclusion, our findings demonstrate for the first time that IPO13 is uniquely expressed by human limbal basal epithelial cells, and plays an important role in maintaining the phenotype, high proliferative potential and less differentiation of corneal epithelial progenitor cells, suggesting that IPO13 could serve as a novel potential marker for corneal epithelial progenitor cells. Keywords: importin13corneaepitheliumprogenitor celllimbal stem cellmarker

6 参考资料 1 Tavazoie M, Van der Veken L, Silva-Vargas V et al Cell Stem Cell 2008;3: Kim K, Lee KM, Han DJ et al. Adult Stem Cell-like the Kidney. Int J Clin Exp Pathol 2008;1: Tavazoie M, Van der Veken L, Silva-Vargas V et al Cell Stem Cell 2008;3: Roh JK, Jung KH, Chu K. Adult stem cell transplan Cell Res Ther 2008;3: Sotiropoulou PA, Candi A, Blanpain C. The Majorit Their Genome By Asymmetrical Chromosome Segregation. 6 Müller FJ, Laurent LC, Kostka D et al. Regulatory lines. Nature 2008; 455: Loebinger MR, Sage EK, Janes SM.Mesenchymal stem 2008; 5: Schermer A, Galvin S, Sun TT. Differentiation-rel culture suggests limbal location of corneal epitheli 9 Tseng SC. Concept and application of limbal stem 10 Thoft RA, Wiley LA, Sundarraj N.The multipotentia 11 Blau HM, Brazelton TR, Weimann JM. The evolving 2001;105: Pajoohesh-Ganji A,Stepp MA.In search of markers Cell,2005;97(4): Lavker RM, Sun TT. Epidermal stem cells: proper 2000;97: Schlötzer-Schrehardt U, Kruse FE. Identifi Res 2005;81: Chee KY, Kicic A, Wiffen SJ. Limbal stem cells: 2006;34: depaiva CS, Chen Z, Corrales RM et al. ABCG2 Tra Limbal Epithelial Cells. Stem Cells 2005;23: Chen Z, Howard EW, Stephen C et al. Gap Junction Stem Cell-Containing Population of Human Limbal Epit 18 Pellegrini G, Dellambra E, Golisano O et al. p ;98: Sun TT, Lavker RM. Corneal epithelial stem cells 2004;9: Qi H, Li DQ, Shine HD,et al. Nerve growth factor human corneal epithelial progenitor cells. Exp Eye R 21 Hong Qi, Eliseu Yung Chuang, Kyung-Chul Yoon, et receptors in human limbal and corneal regions. Molec 22 Du Y, Funderburgh ML, Mann MM.,et al. Multipoten cells2005, 23: Chen Z, Cintia SP, Luo L, et al. Characterizatio Stem cells2004, 22: Zhang C, Sweezey NB, Gagnon S, et al. A novel Kar

7 regulated in fetal lung. Am J Respir Cell Mol Biol, 25 Yoneda Y. Nucleocytoplasmic protein traffic and Kim HS, Jun S, X, de Paiva CS et al. Phenotypic ex vivo from limbal explant and single cell cultures 27 Tseng SC, Kruse FE, Merritt J et al. Comparison b clonal culture systems: evidence showing that epithe conditioned media. Curr Eye Res 1996;15: Kawakita T, Shimmura S, Hornia A et al. Stratifi corneal/limbal progenitor cell. J Cell Mol Med 2008; 29 Mingot JM, Kostka S, Kraft R et al. Importin 13: 2001;20: Ploski JE., Shamsher MK, Radu A. Paired-Type Hom Nucleus by Karyopherin 13. Molecular And Cellular Bi 31 Tao T, Lan J, Lukacs GL et al. Importin 13 regul epithelial cells. Am J Respir Cell Mol Biol. 2006,35 32 Tao T, Lan J, John F. Presley et al. Nucleocytopl Lung. Am. J. Respir. Cell Mol. Biol. 2004; 30: Yamaguchi YLi, Tanaka SS, Yasuda K et al. Stage-s in the mouse. Dev Biol 2006 ;297: Rondanino C, Bousser MT, Monsigny M et al. Sugar living cells. Glycobiology 2003;13: Liang J, Ke G, You W et al. Interaction between pathway for myopodin. Mol Cell Biochem 2008;307: Hideki Yamasaki, Toshihiro Sekimoto, Tadashi Ohk nuclear localization signal for importin -mediated n 37 Shoubridge C, Cloosterman D, Parkinson-Lawerence tract mutations in the Aristaless-related homeobox g 38 Quan Yu, Ji Z, Wang X et al. Evolutionary and tr proteins. Mol Cell Proteomics2008;7: Whitehurst AW, Robinson FL, Moore MS et al. The entry of ERK2 by inhibiting required interactions. J 40 Tomoyuki Ohba, Takashi Seki, Yoshiaki Azuma, et a of RCC1 Is Inhibited by GTP- and GTPγS-Ran, but Not 41 Majo F, Rochat A, Nicolas M et al. Oligopotent s surface. Nature2008; 456:

8 Degree papers are in the Xiamen University Electronic Theses and Dissertations Database. Full texts are available in the following ways: 1. If your library is a CALIS member libraries, please log on and submit requests online, or consult the interlibrary loan department in your library. 2. For users of non-calis member libraries, please mail to etd@xmu.edu.cn for delivery details.

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