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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張明富(Ming-Fu Chang) | |
dc.contributor.author | Cing-Ruei Sie | en |
dc.contributor.author | 謝清瑞 | zh_TW |
dc.date.accessioned | 2021-06-15T16:08:38Z | - |
dc.date.available | 2018-09-25 | |
dc.date.copyright | 2015-09-25 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-08-19 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52147 | - |
dc.description.abstract | 原發性自體隱性畸形小頭症(primary autosomal recessive microcephaly, MCPH)是一種遺傳疾病,患者的大腦皮質有明顯萎縮,神經發育緩慢,最常導致此種疾病的原因即為Aspm (abnormal spindle–like, microcephaly associated) 的基因突變。ASPM 蛋白質在物種間具有保守性,例如哺乳類、果蠅以及線蟲。除了表現在腦部神經前驅細胞外,ASPM蛋白質在分裂組織中也會表現。同時在某些癌症中,例如卵巢癌、子宮頸癌、胰臟癌等,其表現量均有上升現象。人類ASPM蛋白質在有絲分裂的過程中表現於紡錘體相關結構中,調控有絲分裂的進行,此現象與果蠅中的Asp同源蛋白質相同,但詳細的功能與機制尚未清楚。本實驗室先前的研究指出,C型肝炎病毒非結構性蛋白質NS5A,透過PKR-p38的訊號傳遞路徑,抑制ASPM蛋白質表現,導致細胞週期G2/M階段延遲。此外,在細胞中表現N端ASPM蛋白質,ASPM(1-666)也會使細胞週期於G2/M有延遲現象,同時造成紡錘體極失去極性、染色體於分裂板異常排列等現象。本研究承接先前的推測,首先著手探討CDK1是否會磷酸化ASPM蛋白質。以免疫螢光染色法,觀察人類肝癌細胞株Huh 7在有絲分裂中ASPM蛋白質的分佈情形,結果發現其於中期會位在紡錘體極,後期會位在紡錘體極與中心微小管之負極端,末期則會位在midzone。顯示ASPM蛋白質參與有絲分裂之進行,座落在紡錘體的各種結構。當以CDK1抑制劑處理細胞時,發現有絲分裂中期、末期之中心微小管失去雙極性 (bipolarity)。進一步探討雙極性現象與ASPM磷酸化程度相關,發現ASPM之磷酸化程度隨著CDK1抑制劑濃度升高而顯著下降。推測ASPM是CDK1蛋白質下游磷酸化受質。再者,本研究期望進一步研究ASPM N端subdomains對於細胞有絲分裂的影響,分別建構帶有TAT-domain之ASPM (1-204)或ASPM (186-670)之質體,轉入RosettaTM (DE3)大腸桿菌中表現融合蛋白質,再以親和性管柱層析的方式純化出此融合蛋白質,TAT-ASPM (1-204)和TAT-ASPM (186-670)。以免疫螢光染色法觀察,發現兩段TAT融合蛋白質於轉導30分鐘時已進入細胞核且至60分鐘時有持續累積的現象。為了觀察TAT- ASPM轉導蛋白質對有絲分裂的影響,以nocodazole讓細胞同步化在G2/M階段,偵測到TAT-ASPM (1-204)或TAT-ASPM (186-670)均和紡錘體微小管有共位現象,且prophase之多極化比率分別為23.3% 以及17%;metaphase分別為30%以及16%。此結果證實外源性轉導之ASPM microtubule binding domain會佔據內生性ASPM於微小管的結合位,並造成有絲分裂在前期與中期紡錘體有多極化之顯著性負面影響現象。 | zh_TW |
dc.description.abstract | Primary autosomal recessive microcephaly (MCPH) is a genetic disorder. Patients with MCPH show a small cerebral cortex and mild to moderate mental retardation. One of most common causes of MCPH is mutations in the Aspm (Abnormal spindle- like microcephaly associated protein) gene. ASPM is conserved among mammals, Drosophila and nematodes. Besides brain, ASPM is widely expressed in proliferating tissues and upregulated in ovarian, uterine and pancreatic cancers. Human ASPM localizes to the spindle poles during mitosis, similar to its Drosophila homolog abnormal spindle protein (Asp). Asp mutants exhibit a mitotic metaphase checkpoint arrest with abnormal spindle pole organization. However, molecular mechnisms involved in the biological function of ASPM are not fully understood. Our previous studies demonstrated that hepatitis C virus non-structural 5A protein down-regulated ASPM expression and induced G2/M cell cycle delay through PKR-p38 signaling pathway. In addition, overexpression of the N-terminal ASPM, ASPM(1-666), induced cell cycle delay at G2/M phase, disrupted spindle polarity and resulted in chromosome misalignment. In this study, whether ASPM is a downstream target of CDK1 was firstly validated. Results from immunofluorescence staining indicated that ASPM is involved in mitosis and associated with spindle structures in Huh 7 cells. In addition, cells treated with CDK1 inhibitor purvalanol A showed loss of spindle bi-polarity in metaphase and anaphase.Further studies demonstrated a decline of the phosphorylation status with ASPM, suggesting that ASPM is a downstream target of CDK1 and the loss of bi-polarity is associated with the phosphorylation status of ASPM. Results showed that increasing concentration of purvalanol A was associated with phosphor- rylation status decline of ASPM, supporting the previous inference that ASPM might be a downstream target of CDK1. Towards understanding the effects of the N-terminal ASPM subdomains on the progression of mitosis, expression plasmids encoding TAT-ASPM (1-204) and TAT-ASPM (186-670) were constructed, expressed in E.coli RosettaTM (DE3) and purified by nickel affinity chromatography. Thirty minutes after incubation with Huh 7 cells, the partially purified TAT-ASPM (1-204) and TAT-ASPM (186-670) were detected in the nucleus continueand accumulation was observed at 60 min. For observing the effects of the TAT-ASPM proteins on mitosis, huh 7 cells were treated with nocodazole that synchronized cells at G2/M phase. Immunofluorescent staining detected colocalization of the TAT-ASPM proteins with spindle microtubule. In addition, transduction of TAT-ASPM (1-204) or TAT-ASPM (186-670) resulted in 23.33% and 16.67% multipolar spindle in prophase and 30% and 16% in metaphase, respectively. These results indicate that the ecotopically expressed microtubule binding domain of ASPM can disrupt function of the endogenous ASPM in a dominant-negative manner. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T16:08:38Z (GMT). No. of bitstreams: 1 ntu-104-R01442022-1.pdf: 3182078 bytes, checksum: 27034b243e3d590e9704c9cbc514b8a1 (MD5) Previous issue date: 2015 | en |
dc.description.tableofcontents | 中文摘要…………………………………………………………………………………I
英文摘要………………………………………………………………………….……Ⅲ 縮寫表………………………………………………………………………………….Ⅴ 一、緒論………………………………………………………………………………….1 1.1有絲分裂…………………………………………………………………………..1 1.1.1 簡介………………………………………………………………………...1 1.1.2 有絲分裂之調控---SAC (spindle assembly checkpoint)………………...1 1.1.2.1 SAC簡介……………………………………………………………..1 1.1.2.2 SAC分子機制………………………………………………………..3 1.1.3 CDK1在有絲分裂期中扮演的角色及其調控……………………………4 1.2 MCPH (Autosomal Recessive Primary Microcephaly)疾病簡介………………………5 1.3 Aspm (abnormal spindle-like microcephaly-associated)基因……………………..6 1.4 ASPM (abnormal spindle-like microcephaly-associated) 蛋白質之功能…..8 1.5 CPPs(Cell Penetrating Peptides)簡介、分類與適用性……………………...10 1.5.1 定義與簡介……………………………………………………………...10 1.5.2 分類……………………………………………………………………...11 1.5.3 適用性與發展…………………………………………………………...11 1.6 細胞攝入CPP過程中之結構需求 ………………………………………….11 1.6.1 精胺酸組成對於細胞攝入之重要性……………………………….......11 1.6.2 疏水性胺基酸組成以及Tryptophan對於細胞攝入之重要性……….12 1.7 TAT簡介、特性、機制、應用……………………………………………….13 1.7.1 TAT簡介…………………………………………………………………13 1.7.2 機制…...…………………………………………………………………13 1.7.3 應用……………………………………………………………………...13 1.7.3.1 TAT融合蛋白之適用性…………………………………………...13 1.7.3.2 轉導蛋白質功能性存在與否……………………………………..14 研究主題……………………………………………………………………………….15 二、實驗材料來源…………………………………………………………………….16 三、實驗方法………………………………………………………………………….22 四、結果……………………………………………………………………….………39 五、討論……………………………………………………………………………….46 圖表(含附圖)………………………………………………………….……..…………50 六、參考文獻…..………………………………………………………………………72 | |
dc.language.iso | zh-TW | |
dc.title | ASPM胺基端之紡錘體微小管結合區域參與細胞有絲分裂之過程 | zh_TW |
dc.title | The Microtubule Binding Domains at the N-terminus of ASPM Involve in the Cell Mitosis | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 李明學(Ming-Syue Li),蔡有光(Yeou-Guang Tsai),張富雄(Fu-Syong Chang) | |
dc.subject.keyword | 紡錘體微小管結合區域, | zh_TW |
dc.subject.keyword | Microtubule binding domain, | en |
dc.relation.page | 78 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2015-08-19 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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