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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 許書豪(SHU-HAO HSU) | |
dc.contributor.author | Jia-Jheng Jhuang | en |
dc.contributor.author | 莊佳錚 | zh_TW |
dc.date.accessioned | 2021-06-08T02:59:59Z | - |
dc.date.copyright | 2017-09-08 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-07-27 | |
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Semin Cell Dev Biol, 2013. 24(4): p. 347-56. 16. Margall-Ducos, G., et al., Liver tetraploidization is controlled by a new process of incomplete cytokinesis. J Cell Sci, 2007. 120(Pt 20): p. 3633-9. 17. Celton-Morizur, S., et al., The insulin/Akt pathway controls a specific cell division program that leads to generation of binucleated tetraploid liver cells in rodents. J Clin Invest, 2009. 119(7): p. 1880-7. 18. Winter, J., et al., Many roads to maturity: microRNA biogenesis pathways and their regulation. Nat Cell Biol, 2009. 11(3): p. 228-34. 19. Sempere, L.F., et al., Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation. Genome Biol, 2004. 5(3): p. R13. 20. Hsu, S.H., et al., MicroRNA-122 regulates polyploidization in the murine liver. Hepatology, 2016. 64(2): p. 599-615. 21. Zhou, X., et al., Induction of hepatocyte-like cells from human umbilical cord-derived mesenchymal stem cells by defined microRNAs. J Cell Mol Med, 2017. 21(5): p. 881-893. 22. Gatfield, D., et al., Integration of microRNA miR-122 in hepatic circadian gene expression. Genes Dev, 2009. 23(11): p. 1313-26. 23. Zhao, Y., et al., MicroRNA-194 acts as a prognostic marker and inhibits proliferation in hepatocellular carcinoma by targeting MAP4K4. Int J Clin Exp Pathol, 2015. 8(10): p. 12446-54. 24. Bao, C., et al., NF-kappaB signaling relieves negative regulation by miR-194 in hepatocellular carcinoma by suppressing the transcription factor HNF-1alpha. Sci Signal, 2015. 8(387): p. ra75. 25. Zhao, H.J., et al., MiR-194 deregulation contributes to colorectal carcinogenesis via targeting AKT2 pathway. Theranostics, 2014. 4(12): p. 1193-208. 26. Li, P., et al., MiR-194 functions as a tumor suppressor in laryngeal squamous cell carcinoma by targeting Wee1. J Hematol Oncol, 2017. 10(1): p. 32. 27. Morimoto, A., et al., An HNF4alpha-microRNA-194/192 signaling axis maintains hepatic cell function. J Biol Chem, 2017. 292(25): p. 10574-10585. 28. Jung, K.H., et al., MicroRNA Regulates Hepatocytic Differentiation of Progenitor Cells by Targeting YAP1. Stem Cells, 2016. 34(5): p. 1284-96. 29. Sundaram, P., et al., p53-responsive miR-194 inhibits thrombospondin-1 and promotes angiogenesis in colon cancers. Cancer Res, 2011. 71(24): p. 7490-501. 30. DeLaForest, A., et al., HNF4A is essential for specification of hepatic progenitors from human pluripotent stem cells. Development, 2011. 138(19): p. 4143-53. 31. Li, J., G. Ning, and S.A. Duncan, Mammalian hepatocyte differentiation requires the transcription factor HNF-4alpha. Genes Dev, 2000. 14(4): p. 464-74. 32. Tanami, S., et al., Dynamic zonation of liver polyploidy. Cell Tissue Res, 2017. 368(2): p. 405-410. 33. Su, K.C., T. Takaki, and M. Petronczki, Targeting of the RhoGEF Ect2 to the equatorial membrane controls cleavage furrow formation during cytokinesis. Dev Cell, 2011. 21(6): p. 1104-15. 34. Yuce, O., A. Piekny, and M. Glotzer, An ECT2-centralspindlin complex regulates the localization and function of RhoA. J Cell Biol, 2005. 170(4): p. 571-82. 35. Kim, H., et al., Centralspindlin assembly and 2 phosphorylations on MgcRacGAP by Polo-like kinase 1 initiate Ect2 binding in early cytokinesis. Cell Cycle, 2014. 13(18): p. 2952-61. 36. Celton-Morizur, S. and C. Desdouets, Liver physiological polyploidization: MicroRNA-122 a key regulator. Clin Res Hepatol Gastroenterol, 2017. 41(2): p. 123-125. 37. Li, J., et al., MicroRNA-194 promotes osteoblast differentiation via downregulating STAT1. Biochem Biophys Res Commun, 2015. 460(2): p. 482-8. 38. Xu, J., et al., MiR-194 regulates chondrogenic differentiation of human adipose-derived stem cells by targeting Sox5. PLoS One, 2012. 7(3): p. e31861. 39. Hino, K., T. Fukao, and M. Watanabe, Regulatory interaction of HNF1-alpha to microRNA-194 gene during intestinal epithelial cell differentiation. Nucleic Acids Symp Ser (Oxf), 2007(51): p. 415-6. 40. Hino, K., et al., Inducible expression of microRNA-194 is regulated by HNF-1alpha during intestinal epithelial cell differentiation. RNA, 2008. 14(7): p. 1433-42. 41. Gupta, S., Hepatic polyploidy and liver growth control. Semin Cancer Biol, 2000. 10(3): p. 161-71. 42. Pandit, S.K., B. Westendorp, and A. de Bruin, Physiological significance of polyploidization in mammalian cells. Trends Cell Biol, 2013. 23(11): p. 556-66. 43. Stoker, M.G. and H. Rubin, Density dependent inhibition of cell growth in culture. Nature, 1967. 215(5097): p. 171-2. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/20714 | - |
dc.description.abstract | 末期肝臟疾病的治療方式往往受限肝臟移植手術,然而可移植之活體肝臟遠低於等待移植的患者,因而造成病患極高的死亡率,所以我們需要更深入了解基礎肝臟生物學,進而發展新的治療方法促進末期肝病患者之肝臟修復及再生。肝臟細胞的特徵之一為染色體非整倍數體,其可提供肝細胞之間基因組的變異並有助於肝臟慢性肝臟損傷維持再生能力。而非整倍數體肝細胞產自多倍體化(Polyploidization) 之過程:意指『單核二倍體肝細胞(mononucleate diploid hepatocytes)』經由細胞分裂過程中的胞質分裂失敗(Cytokinesis failure)形成『雙核多倍體肝細胞(binucleate diploid hepatocytes)』的過程。迄今已有許多基因被證實會調控胞質分裂過程,包括Myc、P53還有Akt。然而非編碼核醣核酸(non-coding RNA)的作用仍不清楚。微核醣核酸(microRNAs, 或簡稱miR),為長度約20~25個核苷酸(nucleotide)所組成的單股非編碼核醣核酸,可通過與目標3’端的非轉譯區(3’-untranslated region, 簡稱3’UTR)的序列特異性相互作用達到負調控蛋白質編碼基因。先前研究顯示小鼠在出生後肝臟發育的過程中,肝細胞染色體從雙核轉變為多倍體伴隨著miR-194高度表現並且持續增加,這個研究結果使我們推測miR-194會藉由抑制胞質分裂基因促使幼年肝臟發育時期肝細胞的雙核化。 為證實此假說,我們利用微RNA目標基因資料庫 (例如TargetScan 和miRNADA)發現兩個重要胞質分裂基因Racgap1和Ect2可能為miR-194有效作用的目標基因。進一步抑制小鼠(Hepa 1-6 cell line)及人類(Huh7 cell line)肝癌細胞株中的miR-194表現時,Racgap1及Ect2 mRNA 表現量皆顯著上升;相反的,利用人類子宮頸癌細胞株(Hela cell line)和小鼠肝癌細胞株(Hepa 1-6 cell line)高度表現miR-194時,Racgap1及Ect2 mRNA 表現量皆下降。同時螢光素酶報告分析方法(luciferase reporter assay)的結果,進一步證實miR-194可透過Ect2的3’UTR促進其RNA之降解。接著我們分析出生後24天大小鼠肝細胞的基因表現,發現相較於二倍體的肝細胞,四倍體肝細胞的miR-194表現量顯著下降而Ect2基因表現量顯著上升,顯示miR-194的上升為雙核化過程的上游事件。最後,我們發現人類肝前驅細胞株HepaRG經分化刺激之後會表現miR-194且產生細胞雙核化現象,而miR-194的過度表現可抑制Ect2的表現且促進細胞雙核化。綜合以上結果,我們推論miR-194可能透過調控Racgap1及Ect2基因而促使正常肝臟細胞雙核化現象的發生。 | zh_TW |
dc.description.provenance | Made available in DSpace on 2021-06-08T02:59:59Z (GMT). No. of bitstreams: 1 ntu-106-R04446004-1.pdf: 2690514 bytes, checksum: 4d6f154c45cf7f533ddc844f83881726 (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | 致謝 i
中文摘要 ii 英文摘要 iv 目錄 vi 第一章 緒論 1 1.1 引言 1 1.2 肝臟的構造與功能 2 1.3 肝細胞多倍體化現象 3 1.4 肝細胞多倍體化的關鍵過程-胞質分裂失敗(Cytokinesis Failure) 4 1.5 微核醣核酸的生成與功能 5 1.6 微核醣核酸在肝臟的功能 5 1.7 研究動機 6 第二章 實驗材料 8 2.1 實驗儀器 8 2.2 實驗材料與試劑 8 2.2.1 實驗動物均來自本院實驗動物中心 8 2.2.2 細胞株 8 2.2.3 細胞培養 9 2.2.4 細胞計量 9 2.2.5 核醣核酸萃取(RNA) 9 2.2.6 細胞轉染 10 2.2.7 反轉錄聚合酶連鎖反應(Reverse transcription polymerase chain reaction ,RT-PCR) 10 2.2.8 即時定量聚合酶連鎖反應(Quantitative real-time,q-PCR) 11 2.2.9 西方墨點法(Western blot assay) 11 2.2.10 免疫螢光染色 12 2.2.11 螢光激活細胞分選儀之細胞染色 12 2.2.12 質體製作 13 2.2.13 螢光素酶報告分析(Dual-Luciferase Reporter Assay System) (Promega) 13 2.2.14 肝臟細胞分離試驗 13 2.3 實驗溶液配置 14 2.3.1 細胞培養液 14 2.3.2 西方墨點法之溶液 14 2.3.3 螢光激活細胞分選儀之細胞溶液 15 第三章 實驗方法 17 3.1 肝臟免疫組織染色 17 3.2 核醣核酸(RNA)萃取 18 3.3 反轉錄聚合酶連鎖反應(Reverse Transcription-Polymerase Chain Reaction) 19 3.3.1 反轉錄成一般cDNA的步驟: 19 3.3.2 反轉錄微型核醣核酸的Taqman probe cDNA步驟 20 3.4 即時定量聚合酶連鎖反應(Real-time Quantitative PCR, q-PCR) 20 3.4.1 非專一性化學物質: 20 3.4.2 專一性化學物質: 21 3.5 細胞株轉染實驗 21 3.6 小鼠初級肝細胞分離實驗 22 3.6.1 初級肝細胞培養及轉染: 23 3.6.2 細胞分選儀-分群單核雙倍體(2C)或雙核四倍體(4C)肝細胞: 23 3.7 製作並放大帶有目標基因-3’UTR psiCHECK2質體 24 3.7.1 設計質體 24 3.7.2 質體放大 24 3.8 螢光素酶報告分析(Dual-luciferase reporter assay) 25 3.9 人類肝前驅細胞的分化 25 第四章 實驗結果 28 4.1 離乳初期小鼠(C57BL/6)肝臟細胞產生雙核化的現象 28 4.2 小鼠(C57BL/6)肝臟組織中的微核醣核酸(miR-194)含量急遽上升並調控胞質分裂相關基因(Racgap1、Ect2)的表現 28 4.3 人類及小鼠細胞株中miR-194可以調控RACGAP1、ECT2的表現量 29 4.4 miR-194/Ect2/Racgap1表現量在小鼠單核雙倍體以及雙核四倍體肝細胞中的變化 30 4.5 Ect2基因為miR-194之直接調控基因 31 4.6 miR-194調控人類肝前驅細胞(HepaRG cell)中ECT2及RACGAP1基因的表現及雙核化現象的發生 32 4.7 過度表達或是調降miR-194於未分化的HepaRG細胞會調控細胞ECT2/RACGAP1基因的表現及雙核現象的發生 33 第五章 討論 34 5.1 肝細胞多倍化現象於小鼠幼年期肝臟發育階段大量發生 34 5.2 miR-194調控與中央紡錘絲(cetralspindlin)形成的相關基因(Racgap1、Ect2) 34 5.3 單核二倍體細胞及雙核四倍體細胞之間miR-194表現量關係 35 5.4 miR-194參與細胞分化及再生的過程 36 第六章 結論 38 第七章 附圖 39 參考文獻 56 | |
dc.language.iso | zh-TW | |
dc.title | MiR-194在肝細胞雙核化所扮演的角色 | zh_TW |
dc.title | The Role of MircoRNA-194 in Liver Binucleation | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳慧玲(HUEY-LING CHEN),陳玉怜(YUH-LIEN CHEN) | |
dc.subject.keyword | 多倍體化,非整倍數體,胞質分裂失敗,miR-194,Ect2,Racgap1,肝細胞雙核化, | zh_TW |
dc.subject.keyword | Polyploidization,Aneuploidy,Cytokinesis failure,miR-194,Ect2,Racgap1,Liver binucleation, | en |
dc.relation.page | 58 | |
dc.identifier.doi | 10.6342/NTU201702045 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2017-07-27 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 解剖學暨細胞生物學研究所 | zh_TW |
顯示於系所單位: | 解剖學暨細胞生物學科所 |
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