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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳宏文(Hung-Wei Chen) | |
dc.contributor.author | Yu-Hsuan Pao | en |
dc.contributor.author | 包鈺玄 | zh_TW |
dc.date.accessioned | 2021-06-16T13:28:38Z | - |
dc.date.available | 2025-08-11 | |
dc.date.copyright | 2020-08-13 | |
dc.date.issued | 2020 | |
dc.date.submitted | 2020-07-29 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/62116 | - |
dc.description.abstract | 滋養層細胞(trophoblast cell)的分化是早期胎盤發育最關鍵的過程。滋養層細胞經由細胞融合分化為融合滋養層細胞(syncytiotrophoblast),形成絨毛結構協助養分的交換,或者分化為具有細胞侵入能力的絨毛外滋養層細胞(extravillous trophoblast),重塑子宮螺旋動脈。GCM1 (glial cell missing 1)轉錄因子在滋養層細胞分化的過程扮演重要的角色,在胎盤專一性的表現,藉由調控HtrA4和syncytin等基因,促進滋養層細胞的細胞侵入與細胞融合能力。 長鏈非編碼RNA (long non-coding RNA, lncRNA)的定義指超過200個核苷酸,且不轉譯蛋白質產物的RNA。近期的研究證實了長鏈非編碼RNA調控細胞分化的過程,然而關於滋養層細胞的分化,長鏈非編碼RNA扮演何種角色還是未知的。在本篇研究,我們分析了Genbank資料庫,觀察到長鏈非編碼RNA――LINC-PLA的表現量和其他滋養層分化相關的基因呈現正相關的關係。接著我們利用原位雜交技術觀察LINC-PLA在胎盤組織的分布,發現主要分布在妊娠早、中期的融合滋養層細胞。確認LINC-PLA僅在分化後的融合滋養層細胞表達後,我們想確認LINC-PLA和GCM1的關聯。使用毛喉素(forskolin)誘導人類絨毛癌細胞株BeWo細胞中GCM1的表現,發現LINC-PLA的表現量增加。接著我們證明了RNA干擾LINC-PLA使GCM1的mRNA表現量下降。更進一步,透過交聯免疫沉澱法及RNA交互作用實驗,證實LINC-PLA和GCM1存在直接的交互作用。而後我們利用冷光報導基因實驗證明LINC-PLA調控GCM1的轉錄活性及自身調節能力。最後,我們證明RNA干擾LINC-PLA導致GCM1介導的滋養層細胞融合能力下降。綜上所述,我們確認在滋養層分化中存在新的長鏈非編碼RNA――LINC-PLA作為GCM1轉錄因子的正調控因子,藉由和GCM1直接結合以促進GCM1轉錄因子的活性。 | zh_TW |
dc.description.abstract | Trophoblast differentiation is a crucial process for early placental development. Trophoblast cells differentiate into syncytiotrophoblasts (STB) by cell fusion forming the outermost layer of placental villus to facilitate nutrient exchange, and into extravillous trophoblasts (EVT) capable of invasion into uterus and remodeling spiral arteries. GCM1 (glial cell missing 1), which is a placenta-specific transcription factor, plays an essential role in trophoblast differentiation by regulation of trophoblast cell invasion and fusion through transcriptional activation of HtrA4 and syncytin genes, respectively. The long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides that do not translate into proteins. Recent studies have suggested that lncRNAs can regulate cell differentiation. However, the role of lncRNAs in trophoblast cell differentiation is still unknown. Here we analyzed Genbank databases and observed that the expression of LINC-PLA lncRNA is positively correlated with the expression of trophoblast differentiation-related genes. Next, we performed in situ hybridization to detect LINC-PLA transcripts in the first- and second-trimester syncytiotrophoblasts, suggesting that LINC-PLA is expressed in the differentiated syncytiotrophoblasts. To study functional and physical interaction between LINC-PLA and GCM1, we first demonstrated that forskolin (FSK) upregulates LINC-PLA and GCM1 expression in the BeWo choriocarcinoma cell line. Also, LINC-PLA knockdown impairs GCM1 expression. Importantly, we revealed direct interaction between LINC-PLA and GCM1 by cross-linking immunoprecipitation and RNA pull-down assays. We further performed luciferase reporter assays to verify that LINC-PLA upregulates GCM1 transcriptional activity and showed that LINC-PLA knockdown decreases GCM1-mediated trophoblast cell fusion. In summary, we identify a novel lncRNA LINC-PLA as a positive regulator of GCM1 in trophoblast differentiation through direct interaction with GCM1. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T13:28:38Z (GMT). No. of bitstreams: 1 U0001-1806202008481500.pdf: 3454710 bytes, checksum: 9518000abc734b66cb0dff47a8d65fe8 (MD5) Previous issue date: 2020 | 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 1.1 胎盤 1 1.2 GCM1轉錄因子 4 1.3 長鏈非編碼RNA 7 1.4 研究動機 11 第二章 材料與方法 12 2.1 重組質體構築 12 2.2 細胞株培養及轉染 16 2.3 RNA干擾及慢病毒感染 18 2.4 原位雜交技術 18 2.5 交聯免疫沉澱法 20 2.6 即時定量聚合酶連鎖反應 22 2.7 RNA交互作用實驗 24 2.8 SDS-聚丙烯醯胺凝膠電泳與西方墨點法 26 2.9 冷光報導基因活性檢測 27 2.10 細胞融合能力實驗 28 第三章 實驗結果 30 3.1 比對三個RNA定序資料庫找到候選lncRNA——LINC-PLA 30 3.2 篩選LINC-PLA在胎盤細胞的剪接變異體 30 3.3 LINC-PLA在胎盤細胞的分布 31 3.4 LINC-PLA在人類胎盤組織的分布 32 3.5 LINC-PLA與GCM1轉錄因子的互相調控 32 3.6 LINC-PLA與GCM1轉錄因子的交互作用 34 3.7 LINC-PLA維持GCM1的自身調節 35 3.8 LINC-PLA影響GCM1轉錄因子的下游基因表現 36 3.9 LINC-PLA促進胎盤細胞的融合能力 37 3.10 LINC-PLA在人類滋養層幹細胞的表達 37 第四章 討論與總結 38 第五章 圖表 43 參考文獻 62 | |
dc.language.iso | zh-TW | |
dc.title | 長鏈非編碼RNA LINC-PLA調控胎盤融合滋養層細胞分化機制之探討 | zh_TW |
dc.title | Regulation of Placental Syncytiotrophoblast Differentiation by Long Non-coding RNA LINC-PLA | en |
dc.type | Thesis | |
dc.date.schoolyear | 108-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 張震東(Geen-Dong Chang),張功耀(Kung-Yao Chang),黃娟娟(Jiuan-Jiuan Hwang) | |
dc.subject.keyword | 長鏈非編碼RNA,胎盤,滋養層,融合滋養層細胞,GCM1, | zh_TW |
dc.subject.keyword | Long non-coding RNA,Placenta,Trophoblast,Syncytiotrophoblast,GCM1, | en |
dc.relation.page | 70 | |
dc.identifier.doi | 10.6342/NTU202000928 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2020-07-29 | |
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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