以重複序列擴增班馬魚和細胞疾病模式探討天然抗氧化合物和解旋酶的作用

中山醫學大學機構典藏 CSMUIR > 醫學科技學院 > 生物醫學科學學系暨碩士班 > 博碩士論文 > Item 310902500/19702

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题名:	以重複序列擴增班馬魚和細胞疾病模式探討天然抗氧化合物和解旋酶的作用 Investigate the effects of natural anti-oxidative compounds and helicase on the zebrafish and cell models of trinucleotide repeat disease
作者:	林冠宇 Lin, Guan-Yu
贡献者:	中山醫學大學:生物醫學科學學系碩士班;潘惠錦
关键词:	polyQ疾病;薑黃素;白藜蘆醇;DM1疾病;解旋? polyQ disease;Curcumin;Resveratrol;myotonic dystrophy;helicase
日期:	2018
上传时间:	2019-01-04T04:53:41Z (UTC)
摘要:	第一部分摘要: 多麩醯胺酸疾病(polyQ)為 CAG 三聯核酸重複序列位於基因的轉譯區內異常擴增，造成蛋白質形成長鏈的麩醯胺酸（polyglutamine），突變的蛋白會產生不正常折疊而改變神經細胞的功能，造成細胞凋亡等，最終造成神經退化性疾病。目前對於這類疾病並沒有預防或治療的方法，只能藉由減緩其症狀來達到改善的效果。先前實驗室已建立能表達polyQ蛋白的轉殖基因斑馬魚和N2a細胞。在polyQ轉殖魚中，我們觀察到有發育遲緩、脫卵膜率和生存率下降的情況。我們也發現polyQ蛋白擴增對於斑馬魚逃脫反應和游動行為是有影響的。在N2a細胞中，我們可觀察到polyQ蛋白的聚集，以及神經細胞分化異常的現象。近期的研究指出polyQ蛋白的擴增會造成氧化自由基(ROS)的上升，因此，我們測試兩種具有抗氧化能力的天然化合物薑黃素(Curcumin)和白藜蘆醇(Resveratrol)，觀察其是否在斑馬魚和細胞中能改善polyQ疾病表型。在薑黃素和白藜蘆醇分別處理後，我們發現斑馬魚胚胎的生存率和脫卵膜率、逃脫反應和行為分析有明顯的回復，對於神經細胞突觸分化也有改善的效果。利用dihydroethidium (DHE) 染色，可觀察到薑黃素和白藜蘆醇皆能降低斑馬魚和細胞在氧化壓力下所產生的ROS。此結果證實薑黃素和白藜蘆醇對於polyQ疾病表型有改善的作用，也顯示我們所建立的斑馬魚模式適合作為藥物篩選的平台。第二部分摘要: 強直型肌肉萎縮症第一型(myotonic dystrophy,DM1)是一種顯性遺傳疾病，由DMPK基因3端非轉譯區內的CTG重複序列擴增所引起。轉錄出來的CUG擴增RNA，會與一些蛋白如MBNL結合，並在細胞核內形成RNA foci，進而造成RNA 剪切和基因轉錄異常。先前以DM1線蟲疾病模式和RNAi library篩選找到一些疾病的修飾基因，其中之一為Werner syndrome解旋?(WRN)，它屬於RecQ解旋?家族成員的DNA解旋?，在DNA修補和DNA結構穩定上扮演重要的角色。以即時定量PCR分析C2C12小鼠肌纖維母細胞內生性WRN表現量，發現CUG200的細胞內WRN表現量相較於對照組明顯增多。為了探討WRN和CUG擴增的交互作用和機制，我們利用WRN shRNA的慢病毒進行WRN弱化，發現在感染shWRN後可以改善因CUG RNA毒性所造成肌肉分化異常的現象、提高MHC蛋白質表現量。另外，我們觀察到在弱化WRN後CUG200的細胞EGFP蛋白質和RNA表現量大幅下降，暗示WRN可能可解開擴增CUG重複序列的結構並促進其表現。我們也利用siRNA方式弱化DM1病人細胞的WRN基因，同樣地發現DMPK表現量下降，且細胞核內的RNA 聚集減少。這些結果指出WRN對擴增的CUG重複序列所導致的毒性效應是必須的，而弱化WRN的表現可能可以成為治療DM1疾病的一條途徑。 Part 1: Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders caused by an abnormal expansion of a coding trinucleotide (CAG) repeat, which is translated into an elongated glutamine (Q) tract in the respective mutant proteins. It leads to protein misfolding and aggregation, altering the function of neuronal cells and eventually resulting in neurodegeneration. Currently there is no treatment to prevent or cure the disease, only symptomatic treatments exist. We have established transgenic zebrafish and N2a cell lines expressing polyQ proteins. The polyQ zebrafish showed growth retardation, delayed hatching and increased mortality rate. The escape response and swimming pattern were also affected. In N2a cells, polyQ protein aggregation in the cytoplasm was observed and expanded polyQ interrupts branching of neuronal processes upon differentiation. Since ROS is increased in cells expressing polyQ proteins, we tested the effects of curcumin and resveratrol, both known as antioxidative compounds, in rescuing the phenotypes in the polyQ fish and cell models. After treating with curcumin and resveratrol, the mortality rate, hatching rate and escapes response were all improved significantly. In addition, both compounds decreased ROS production in the polyQ fish and cell models. The results demonstrated that curcumin and resveratrol are beneficial for the polyQ diseases and that the zebrafish model is a feasible platform for polyQ disease drug test. Part 2: Myotonic dystrophy type1 (DM1) is an autosomal dominant disorder caused by the expansion of a CTG trinucleotide repeat in the 3′-UTR of the dystrophia myotonica-protein kinase (DMPK) gene. The transcripts of CUG repeats accumulate in nucleus and disrupt a variety of cellular regulatory pathways including splicing and transcription by sequestration of MBNL proteins. Previous study using a DM1 C. elegans model and RNAi library screening has revealed several modifier genes that could mitigate the disease phenotype. One of the candidate genes is WRN (Werner syndrome helicase), a DNA helicase of the RecQ family that plays a critical role in repairing DNA and maintaining the structure of DNA. The endogenous expression of WRN in C2C12-CUG200 cells and DM1 cells are higher than in control. To investigate the role of WRN in DM1 disease, we used lentiviral shRNA of WRN to knock down its expression in C2C12-CUG200 cells. Decreased expression of WRN could rescue cell differentiation defect caused by the CUG repeats. In addition, knockdown of WRN reduced the expression of EGFP in CUG200 cells, suggesting that WRN could modify the CUG repeats and facilitate its expression at the RNA level. We also used siRNA to suppress WRN expression in human DM1 cells. Similarly, knockdown of WRN decreased the expression of DMPK gene and RNA foci in the nucleus. These results indicate that WRN is required for the toxic effect caused by expended CUG repeats and down-regulation of WRN may provide an avenue for treatment of DM1 disease.
URI:	https://ir.csmu.edu.tw:8080/ir/handle/310902500/19702
显示于类别:	[生物醫學科學學系暨碩士班] 博碩士論文

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