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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳宏文(Hung-Wen Chen) | |
| dc.contributor.author | Sih-Han Chen | en |
| dc.contributor.author | 陳思涵 | zh_TW |
| dc.date.accessioned | 2021-06-16T22:59:22Z | - |
| dc.date.available | 2022-12-31 | |
| dc.date.copyright | 2012-08-28 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-08 | |
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Jiang, G. F. Alberts, M. Costa, L. Lu, J. A. Winkles and W. Dai (2008). 'Polo-like kinase 3 functions as a tumor suppressor and is a negative regulator of hypoxia-inducible factor-1 alpha under hypoxic conditions.' Cancer Res 68(11): 4077-4085. Yata, K., J. Lloyd, S. Maslen, J. Y. Bleuyard, M. Skehel, S. J. Smerdon and F. Esashi (2012). 'Plk1 and CK2 act in concert to regulate Rad51 during DNA double strand break repair.' Mol Cell 45(3): 371-383. Yu, C., K. Shen, M. Lin, P. Chen, C. Lin, G. D. Chang and H. Chen (2002). 'GCMa regulates the syncytin-mediated trophoblastic fusion.' J Biol Chem 277(51): 50062-50068. Zimmerman, W. C. and R. L. Erikson (2007). 'Finding Plk3.' Cell Cycle 6(11): 1314-1318. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64790 | - |
| dc.description.abstract | 人類 glial cell missing-1 (GCM1) 蛋白質為專一性表現在胎盤的細胞滋養層細
胞(cytotrophoblast) 和融合滋養層細胞(syncytiotrophoblast) 與絨毛外胎盤細胞 (extravillous trophoblast) 的轉錄因子，可調控syncytin-1，syncytin-2 與MFSD2A 蛋白質表現，促進細胞滋養層細胞產生細胞融合，進而分化成融合滋養層細胞； 也會調控HtrA4 蛋白質表現，促進絨毛外胎盤細胞的移動與侵入子宮內膜組織。 GCM1 透過調控樹狀絨毛與絨毛外兩類型胎盤細胞分化的路徑，以調控胎盤結構 的正常發育，使胎兒和母體間養分與氣體等物質交換達到最大效率。細胞滋養層 細胞為融合滋養層細胞的前驅細胞，具有細胞增生與分化的能力，而GCM1 轉錄 活性是否受到細胞週期所調控目前仍屬未知。過去實驗室利用蛋白質體研究GCM1 結合蛋白，其中發現polo-like kinase 1 (PLK1) 此一在細胞週期中扮演非常重要角 色的serine/threonine 磷酸激酶。 為了進一步了解 PLK1 是否調控GCM1 活性，首先利用免疫沉澱實驗確定了 GCM1 和PLK1 在細胞中的確有交互作用，而免疫螢光染色也觀察到GCM1 和 PLK1 在metaphase 的表現會有部分重疊在中心體的位置。在細胞外磷酸化實驗發 現，全長的GCM1 和其transactivation domain 1 (TAD1) 會受到PLK1 磷酸化。特 別地，當GCM1 和PLK1 在細胞中共同表現時，可發現GCM1 與含有TAD1 的片 段會出現有較高分子量的變化，推測可能是PLK1 促進GCM1 受到某種轉譯後修 飾。而在細胞外去磷酸化實驗中，排除了此較高分子量之GCM1 為磷酸化修飾所 造成。在胎盤細胞株的細胞週期同步實驗中可觀察到，此較高分子量之GCM1 的 出現與細胞週期有關。利用luciferase 冷光報導基因實驗也觀察到，PLK1 在胎盤 細胞株會抑制內生性GCM1 的轉錄活性，然而不具有磷酸激酶活性的PLK1 K82M 突變也會抑制GCM1 轉錄活性。隨著GCM1 轉錄因子所調控的標的基因在近年來 陸續被發現，探討PLK1 調控GCM1 活性的詳細機制，將有助於了解GCM1 在調 控胎盤發育與分化上的功能。 | zh_TW |
| dc.description.abstract | Human glial cell missing-1 (GCM1) is a placenta-specific transcription factor,
which is expressed in the villous cytotrophoblasts, villous syncytiotrophoblasts and extravillous trophoblasts. GCM1 regulates the expression of syncytin-1, syncytin-2 and MFSD2A proteins and promotes cytotrophoblast differentiation into a syncytiotrophoblast layer by cell-cell fusion. GCM1 also facilitates invasion of extravillous trophoblasts by activating the expression of HtrA4 serine protease. It remains unknown whether GCM1 is regulated by cell cycle. We have recently identified polo-like kinase 1 (PLK1), which is a cell cycle-related serine/threonine protein kinase, as a GCM1-associated protein by tandem tag affinity purification coupled with mass spectrometry analysis. In this study, we confirmed the interaction of GCM1 and PLK1 by co-immunoprecipitation and demonstrated that PLK1 interacts with the transactivation domain 1 and -2 (TAD1 and TAD2) in GCM1. In vitro kinase assay indicated that PLK1 phosphorylates TAD1 in GCM1. We further observed that endogenous GCM1 and PLK1 colocalize at centrosomes in metaphase. Interestingly, GCM1 of higher molecular weight was detected by immunoblotting when GCM1 and PLK1 are co-expressed in cells, suggesting that PLK1 may facilitate post-translational modification of GCM1. In vitro alkaline phosphatase dephosphorylation assay indicated that the observed GCM1 of higher molecular weight is not due to hyperphosphorylation. Moreover, GCM1 of higher molecular weight was detected in synchronized JAR cells. We further demonstrated that GCM1 transcriptional activity is suppressed by PLK1 in 293T and JAR cells in a dose-dependent manner by luciferase reporter assay. Interestingly, the kinase-inactive PLK1 mutant (PLK1 K82M) did not suppress GCM1 activity in 293T cells; however, it did suppress GCM1 activity in JAR cells. As an increasing number of GCM1 target genes have been identified in recent years, regulation of GCM1 activity by PLK1 may help to better understand how GCM1 regulates placental development and function. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T22:59:22Z (GMT). No. of bitstreams: 1 ntu-101-R99b46006-1.pdf: 2670367 bytes, checksum: 19e51588dfa890b5166d212af5c841bd (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 目錄 ……………………………………………………………………………… I
圖表目錄………………………………………………………………………… III 中文摘要 ………………………………………………………………………… IV 英文摘要………………………………………………………………………… V 第一章 緒論 1.1 胎盤 ………………………………………………………… 1 1.2 GCM 轉錄因子…………………………………………… 3 1.3 PLK1 磷酸激酶…………………………………………… 8 1.4 研究動機………………………………………………… 18 第二章 材料與方法 2.1 重組質體構築……………………………………………… 19 2.2 重組蛋白表現……………………………………………… 22 2.3 細胞株培養與轉染………………………………………… 22 2.4 細胞蛋白質萃取…………………………………………… 23 2.5 SDS 聚丙烯醯胺凝膠電泳與西方墨點法………………… 25 2.6 共同免疫沉澱法…………………………………………… 26 2.7 免疫螢光染色……………………………………………… 26 2.8 細胞外 PLK1 磷酸化實驗………………………………… 27 2.9 細胞外去磷酸化實驗……………………………………… 28 2.10 細胞週期同步實驗………………………………………… 28 2.11 Luciferase 冷光報導基因活性檢測……………………… 29 第三章 實驗結果 3.1 PLK1 磷酸激酶與GCM1 轉錄因子的交互作用……… 30 3.2 PLK1 磷酸激酶與GCM1 的結合片段………………… 31 3.3 PLK1 磷酸激酶對GCM1 的磷酸化作用與片段……… 31 3.4 PLK1 磷酸激酶促進GCM1 的轉譯後修飾……………… 32 3.5 PLK1 與GCM1 在胎盤細胞株的表現位置……………… 34 3.6 細胞週期對 GCM1 轉譯後修飾之影響………………… 35 3.7 PLK1 磷酸激酶降低GCM1 轉錄因子的轉錄活性……… 36 第四章 討論與總結…………………………………………………………… 38 第五章 圖表…………………………………………………………………… 44 第六章 參考文獻……………………………………………………………… 57 | |
| dc.language.iso | zh-TW | |
| dc.subject | 細胞週期 | zh_TW |
| dc.subject | Glial cell missing-1 | zh_TW |
| dc.subject | Polo-like kinase 1 | zh_TW |
| dc.subject | 胎盤細胞滋養層細胞的分化 | zh_TW |
| dc.subject | Cell cycle | en |
| dc.subject | Glial cell missing-1 | en |
| dc.subject | Polo-like kinase 1 | en |
| dc.subject | Cytotrophoblast differentiation | en |
| dc.title | 探討PLK1磷酸激酶對GCM1轉錄活性之調控 | zh_TW |
| dc.title | Regulation of GCM1 transcriptional activity by polo-like kinase 1 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李明亭(Ming-Ting Lee),張震東(Geen-Dong Chang),張茂山(Mau-Sun Chang),張功耀(Kung-Yao Chang) | |
| dc.subject.keyword | Glial cell missing-1,Polo-like kinase 1,細胞週期,胎盤細胞滋養層細胞的分化, | zh_TW |
| dc.subject.keyword | Glial cell missing-1,Polo-like kinase 1,Cell cycle,Cytotrophoblast differentiation, | en |
| dc.relation.page | 69 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-08-08 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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