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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭建興(Jiann-Shing Jeng) | |
| dc.contributor.author | Ming-Tung Lien | en |
| dc.contributor.author | 連銘銅 | zh_TW |
| dc.date.accessioned | 2021-06-08T01:19:27Z | - |
| dc.date.copyright | 2020-08-26 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-13 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18687 | - |
| dc.description.abstract | 腦中風可分為缺血性中風和出血性中風兩種，而出血性中風的死亡率較缺血性中風高出許多。出血性中風以自發性腦出血 （spontaneous intracerebral hemorrhage，ICH）為主，一個月內死亡率可高達50-60%，三個月內死亡率約40%。雖然緊急移除血塊的手術治療可減少血塊擴大造成進一步壓迫周遭正常腦組織、水腦症等引發嚴重的腦水腫和顱內壓上升外，但6個月內造成殘障的機率仍高達80%，進而造成永久之失能。 近年來基因體醫學的進展迅速，目前已經發現真核生物基因體的轉錄程度遠超越過去生物學家的認知；過去認為真核生物的基因主要轉錄為信使核醣核酸 (messeger RNA，mRNA)及微核醣核酸 (microRNA，miRNA)，其實只佔所有轉錄基因數量約1%，大於90%的真核基因體轉錄為長鏈非編碼核醣核酸 (long noncoding RNA，lncRNA)。lncRNA是由一群異質性高，長度大於200核苷酸的非編碼RNA所組成。許多lncRNA已經被發現在調節生理及病理機轉上具有重要性，他們雖然不能轉錄蛋白質，但卻能經由改變表觀基因體(epigenetic)、基因體轉錄及核酸轉譯的機制來調節生理功能及疾病發生。在癌症及退化性神經疾病的領域，已經有許多研究證明lncRNA的異常表現是導致人體癌症及神經元變化的重要機轉。雖然lncRNA在人類疾病的研究發展十分迅速，但目前針對lncRNA在ICH而導致神經損傷及預後的研究仍非常稀少，lncRNA在自發性腦出血的病態生理上的角色也仍不清楚。因此，我們研究的目標為尋找與ICH病人相關之lncRNA，並探討和ICH的神經細胞受損及預後之關係。 我們一共招募ICH病人15位(年齡58.7±11.0，男性占53%)和年齡及性別相符之非中風對照組受試者9位(年齡61.7±13.9，男性占44%)，ICH個案收集腦出血後24小時內的周邊血液並追蹤三個月的臨床預後，對照組則收集一次的周邊血液，這些血液經離心後進行RNA萃取。利用次世代高速核酸定序法（RNA sequencing）比較ICH病人和非中風對照組受試者的血中lncRNA的表現形態差異，分析這些與ICH相關的lncRNA在ICH病人中的疾病嚴重度及臨床預後之關係，進一步探詢ICH的神經細胞損傷之機轉。最後運用Quantitative real-time polymerase chain reaction方法來確認我們所找尋的lncRNA的正確性。 經由RNA seq實驗比較ICH病人和非中風對照組受試者的血中lncRNA及mRNA的表現形態差異，結果發現兩組表現有顯著差異的lncRNA共有10個，mRNA共有60個。且相較對照組，ICH病人都呈現down regulation的現象。由於lncRNA常藉由順式調控(cis-regulation)來發揮其表觀遺傳學調控(epigenetic regulation)的功能，所以我們也尋找這10個lncRNA附近的mRNA(cis-mRNA)) ，但發現這些cis-mRNA並未出現在這60個mRNA中，因此推測這些lncRNA可能藉由其他機轉來發揮它們的功能，需再進一步研究它們的調控機轉。 接著，我們進一步分析這10個lncRNA和60個mRNA與ICH疾病嚴重度和臨床預後之間的關係。疾病嚴重度則以入院時NIHSS分數和ICH出血量作為指標，臨床預後以出血後三個月的modified Rankin Scale (mRS)作為指標，發現這10個lncRNA在疾病嚴重度與臨床預後上均無明確相關。我們亦針對mRNA作ICH病嚴重度與臨床預後之分析，發現在臨床疾病嚴重度有1個mRNA (PRPF40A)達到統計上顯著差異(p < 0.05)，在臨床預後有2個mRNA (RNA28SN5、RNA28SN3)達到統計上顯著差異(p < 0.05)，接著再進一步分析這些lncRNA附近的mRNA (cis-mRNA))及mRNA所牽涉之病理生理機轉及相關路徑，包含可能與endoplasmic reticulum membrane、transmembrane receptor protein tyrosine phosphatase signaling pathway、ribosomal mRNA and mRNA processing factor及 platelet aggregation / activation等有相關。 我們也建立ICH細胞傷害模式探討lncRNA在致病機轉之角色，培養人類SH-SY5Y細胞株，並將血液溶血後的產物氯化血紅素(hemin)加至細胞培養液當成ICH細胞傷害模式，藉由前述找出在ICH病人所具有的潛力lncRNA及mRNA作為標的基因(target gene)，運用Quantitative real-time polymerase chain reaction方法驗證以hemin為ICH細胞傷害模式的SH-SY5Y細胞是否有相同表現。我們從前述10個與ICH有意義的lncRNA選出其中4個，從60個與ICH有意義的mRNA選出其中6個，用qRT-PCR的方式對我們收集的細胞檢體作驗證，實驗結果發現隨著hemin-treated時間增加，這些lncRNA與mRNA的變化大多呈現上升現象(upregulation) ，其中mRNA中的TLN1在hemin-treated 9小時與24小時有達到統計上顯著差異。 總結來說，我們這個研究完整分析ICH發生後，血中的lncRNA及mRNA在發病後24小時內的變化，並且探討ICH病人血中的lncRNA及mRNA表現與其疾病嚴重度和臨床預後之間的關係。結果發現相較於非ICH對照組，ICH病人血中的特定lncRNA及mRAN有明顯down regulation的現象。回溯過去的文獻，相關的研究結果多來自於缺血性中風病患或者腦出血動物實驗，本研究的初步成果提供了具潛力的與ICH發生後的病生理機制及臨床生物標誌的未來可以努力的目標及方向。 | zh_TW |
| dc.description.abstract | Background. Stroke can be divided into ischemic stroke and hemorrhagic stroke (ICH). Hemorrhagic stroke is characterized by high mortality rate. The probability of causing disability within 6 months is still as high as 80% even though emergently surgical treatment of removal of hematomas. Until now, it remained unclear how long non-coding RNA (lncRNA) regulation contributes to ICH such as neuronal injury and prognosis.
Objectives. This study aimed to look for ICH associated lncRNAs and try to explore these ICH-specific lncRNAs in the role of neuronal damage, disease severity and prognosis. Methods. 15 ICH patients and 9 non-ICH control subjects (NIC) of matched age and gender were enrolled. We draw blood from the ICH patient within 24 hours after onset of ICH and followed clinical prognosis until three months later. The blood was collected once in the NSC group. By exploiting next-generation sequencing technology, we compared the differences of lncRNAs in plasma between ICH and NIC subjects. Furthermore, we tried to explore lncRNAs and relationship between neuronal damage, disease severity and prognosis in ICH. Finally, we utilized Quantitative real-time polymerase chain reaction to confirm the accuracy of the lncRNAs which we looked for. Results. A total of 10 lncRNAs were significantly down regulated in ICH group in analysis of 1,715 lncRNAs. No statisctially significant differences were found in disease severity and prognosis of ICH group. A total of 60 mRNAs were significantly down regulated in ICH group in analysis of 4.266 mRNAs. Two mRNA (RNA28SN5、RNA28SN3) in prognosis and one mRNA (PRPF40A) in disease severity of ICH group reached statisctially significant differences. No nearby mRNAs (cis-mRNAs) were found at mRNAs. The pathogensis and pathway analysis by Gene ontology and KEGG showed probable association with endoplasmic reticulum membrane, transmembrane receptor protein tyrosine phosphatase signaling pathway, ribosomal mRNA, mRNA processing factor, and platelet aggregation/activation. ICH cell model showed upregulation of ICH-specific lncRNAs and mRNAs. Conclusions. This was the first study to investigate human circulating lncRNAs between ICH and non-ICH subjects. Conflicted results between circulating ICH-specific lncRNAs/mRNAs and cell model might imply time point of blood-drawing less than 24 hours from onset and no successive circulating lncRNA monitoring in ICH group was performed due to limited budget. We needed to enroll large number of cases to investigate the pathogenesis of ICH by successive circulating lncRNA tests and narrowing spectrum. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T01:19:27Z (GMT). No. of bitstreams: 1 U0001-1108202012234800.pdf: 2834834 bytes, checksum: a2ee41d81f55abe25b6c6882a1ce0957 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 口試委員會審定書…………………………………….………………………………… i 誌謝…………………………………………………………….…………………………. ii 中文摘要……………………………………………………………….………………… iii 英文摘要……………………………………………………………………….………… vi 碩士論文內容 第一章 緒論……………………………………………………………………………1 第二章 研究方法與材料………………………………………………………………8 第三章 結果………………………………………………………………….11 第四章 討論……….………………………………………………………………….14 第五章 展望…………………………………………………………………….…….26 第六章 論文英文簡述 (summary)…………………………………………………….28 第七章 參考文獻…………………………………………………………………30 第八章 圖表……………………………………………………………………38 圖一、中風次分類圖示…………………………………………………………………38 圖二、中風次分類比例圖示……………………………………………………………38 圖三、自發性腦出血導致的神經細胞受損機轉 ……… …… ………………………39 圖四、人類的基因組的轉錄產物中lncRNA………………………………………39 圖五、人類lncRNA調控基因表現的機轉…………………………………………40 圖六、LncRNA與自發性腦出血之關係………………………………………40 圖七、人類中樞神經系統受損之相關下游分子(downstream molecules) … …41 圖八、LncRNA在中樞神經系統調控之功能及相關分子………………………41 圖九、研究方法與進行步驟……………………………………………………42 圖十、於台大醫院第三共同研究室完成之細胞實驗………………………………42 圖十一、ICH病人與對照組lncRNA表現之hierarchical clustering heat map………43 圖十二、ICH病人與對照組的mRNA表現之volcano plot……………………43 圖十三、ICH病人與對照組的mRNA表現之hierarchical clustering heat map……44 圖十四、以qRT-PCR驗證細胞實驗結果……………………………………………44 圖十五、ICH病人cis-mRNA相關之病理生理機轉及路徑分析………………45 圖十六、ICH病人mRNA相關之病理生理機轉及路徑分析……………………46 表1、ICH病人和對照組之流行病學資料………………………………………47 表2、ICH病人以預後(mRS)分析之流行病學資料……………………………47 表3、ICH病人有顯著差異之lncRNA鄰近cis-mRNA的表現…………48 表4、ICH病人和對照組間表現有顯著差異之lncRNA其附近之mRNA 所牽涉之致病機轉…………………………………………………………48 表5、ICH病人和對照組間表現有顯著差異之mRNA的表現…………………50 表6、ICH病人mRNA與臨床預後和疾病嚴重度之關係………………………51 表7、ICH病人和對照組表現有顯著差異之mRNA所牽涉之致病機轉………51 | |
| dc.language.iso | zh-TW | |
| dc.title | 探討長鏈非編碼核糖核酸在自發性腦出血的神經損傷及預後的角色 | zh_TW |
| dc.title | The role of long non-coding RNA in neuronal injury and prognosis of spontaneous intracerebral hemorrhage | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 湯頌君(Sung-Chun Tang) | |
| dc.contributor.oralexamcommittee | 楊偉勛(Wei-Shuing Yang),楊鎧鍵(Kai-Chien Yang) | |
| dc.subject.keyword | 自發性腦出血,長鏈非編碼核糖核酸,血漿生物標記物,次世代高速核酸定序法,人類神經母細胞瘤細胞株, | zh_TW |
| dc.subject.keyword | spontaneous intracerebral hemorrhage,long non-coding RNA,plasma biomarker,next-generation sequencing,SH-SY5Y cell line, | en |
| dc.relation.page | 55 | |
| dc.identifier.doi | 10.6342/NTU202002918 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2020-08-13 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 臨床醫學研究所 | zh_TW |
| 顯示於系所單位： | 臨床醫學研究所 | |
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