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系統識別號 U0026-2208201622313600
論文名稱(中文) 探討polycystin-1蛋白之細胞質端在多囊性腎病中的分子機轉
論文名稱(英文) Study on the molecular mechanism of the cytoplasmic tail of polycystin-1 in polycystic kidney disease
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) Department of Biochemistry and Molecular Biology
學年度 104
學期 2
出版年 105
研究生(中文) 王藝潔
研究生(英文) Yi-Chieh Wang
學號 S16034039
學位類別 碩士
語文別 中文
論文頁數 62頁
口試委員 指導教授-吳昭良
口試委員-蕭璦莉
口試委員-王明誠
口試委員-歐弘毅
口試委員-李哲欣
中文關鍵字 多囊性腎臟病  PC-1蛋白  OGT 
英文關鍵字 Polycystic Kidney Disease  PC1  OGT 
學科別分類
中文摘要 多囊性腎臟病 (polycystic kidney disease, PKD)是常見的遺傳性疾病之一,疾病特色為腎臟會出現囊泡,囊泡不斷增生擴大影響到腎臟的正常組織功能,在疾病末期導致腎衰竭的發生。多囊性腎臟病可能是PKD1基因或者是PKD2基因發生突變所造成,大部分的患者屬於PKD1基因突變型,PKD1基因表現PC-1蛋白 (polycystin-1),目前相關的研究已發現到PC-1蛋白會被γ-secretase進行剪切出一段細胞質端CTT (C-terminal cytoplasmic tail),被切出來CTT片段會進一步入核並且參與活化調節許多的訊息路徑,且多囊性腎臟病患者腎臟會有CTT異常累積的情形,其中詳細的機制目前尚未清楚。有研究發現單獨過量表現CTT片段就足以導致斑馬魚胚胎有囊泡形成,不過若是抑制了PKD1的表達則會有發育生長異常的情形,此時若再給予CTT則可以挽救這些異常狀況,所以適量表現CTT蛋白能夠有效抑制多囊性腎臟病相關的生長死亡、發育型態等異常情況。關於多囊性腎臟病大家已熟知mTOR會失去控制過度活化,而有文獻指出mTOR會增加OGT (O-GlcNAc transferase)蛋白的穩定性,所以我們想要探討多囊性腎臟病之中OGT是否會扮演著什麼角色。在本研究中我們發現OGT會對CTT蛋白的穩定性及功能產生影響,過量表現的OGT可以讓CTT蛋白穩定性增加,且降低了CTT活化的訊息路徑表現,而將OGT進行抑制得到了反證結果。給予細胞藥物加強O-GlcNAc修飾作用之後,發現CTT蛋白表現位置從細胞核更進一步往細胞核仁集中。從動物實驗中發現Thiamet-G藥物可以減緩多囊性腎臟病小鼠的病症。本研究主要提出OGT會對CTT蛋白進行修飾,且造成CTT穩定性及功能發生改變,並且經由加強O-GlcNAc修飾作用的策略確實可以緩解多囊性腎臟病小鼠的病症。
英文摘要 Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder characterized by presence of cysts in the kidney and other organs and eventual renal failure. Mutations of PKD1 (PC1) or PKD2 (PC2) are responsible for ADPKD. PC1 undergoes multiple cleavages that produce fragments. One of these cleavages is the C-terminal cytoplasmic tail (CTT) of PC1, which intervenes in several signaling pathways through nuclear translocation. Recent results show that CTT overexpression in the renal tissues of PKD patients is sufficient to rescue the dorsal body curvature phenotype in zebrafish embryos resulting from suppression of Pkd1 expression, suggesting that CTT is necessary for the physiological functions of kidney morphogenesis. However, the mechanisms of trafficking and protein stability are largely unknown. Here we show that O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) is required for altering CTT protein stability and function. Overexpression of OGT up-regulated the protein stability of CTT through increasing O-GlcNAcylation of CTT, but down-regulated CTT-mediated signaling pathways. Meanwhile, OGT knockdown specifically up-regulated CTT-mediated transcriptional regulation. Treatment of cells with the OGA inhibitor PUGNAc increased OGT activity and altered CTT localization from the cytoplasm to the nucleolus, resulting in decreased CTT-mediated transcriptional regulation. Inhibition of OGA reversed disease progression in the ADPKD mouse model, suggesting that OGA inhibitors may be potential therapeutic agents for ADPKD. Taken together this study not only uncovers a functional posttranslational modification of CTT but also reveals a unique role of OGT in the development of ADPKD.
論文目次 中文摘要 Ⅰ
英文延伸摘要 Ⅲ
縮寫 ⅩⅣ
緒論
1.多囊性腎臟病 (Polycystic kidney disease ; PKD)
1-1.簡介 1
1-2.ADPKD致病成因 2
1-3.診斷與治療 2
1-4.ADPKD小鼠模型 4
2.C-terminal tail (CTT) of polycystin-1
2-1.簡介 4
2-2.CTT與PKD 5
3.O-GlcNAcylation
3-1.簡介 6
3-2.OGT與PKD 7
研究動機 9

材料與方法
一.材料
1.質體(plasmid)
1-1.表現載體(expression vector)10
1-2.RNA干擾質體(short hairpin RNA , shRNA)10
2.引子(primer) 11
3.細胞株 (cell line)11
4.抗體(antibody)
4-1.一級抗體(primary antibody)11
4-2.二級抗體(secondary antibody)12
5.實驗動物 12
6.菌株 13
7.試劑
7-1.細菌培養液 14
7-2.緩衝液
7-2-1.細胞緩衝液 14
7-2-2.蛋白質膠體電泳緩衝液 15
7-2-3.報導基因檢測法之緩衝液 (reporter assay buffer)16
8.藥物
8-1.Cycloheximide 17
8-2.PUGNAc 18
8-3.Thiamet G 18
8-4.Dimethyl sulfoxide (DMSO) 19
二.實驗方法
1.細胞培養:293T、293細胞 19
2.質體轉染transfection 19
3.慢病毒lentivirus
3-1.慢病毒生產 production of lentivirus 20
3-2.慢病毒感染細胞 20
4.RNA萃取 21
5.反轉錄(reverse transcription) 21
6.即時半定量反轉錄酶聚合連鎖反應(real-time PCR )21
7.報導基因檢測法 (reporter assay)22
8.細胞免疫螢光染色 (immunofluorescence staining ;IF)22
9.免疫沉澱分析法(Immunoprecipitation; IP)23
10.西方墨點法(western blot)23
11.動物實驗
11-1.Pkd小鼠基因型確認 (genotyping) 24
11-2.組織固定與包埋 24
11-3.H&E染色 24
11-4.免疫組織化學染色法(Immunohistochemistry) 25
11-5.老鼠藥模式 26
11-6.小鼠腎臟比重計算方法 26
11-7.cyst index分析計算 26
11-8.統計分析 27
結果
一.CTT在Pkd小鼠中的表現量相較WT小鼠明顯加 28
二.OGT與O-GlcNAc在Pkd小鼠中的表現量相較WT小鼠明顯加 28
三.在cell中發現抑制Pkd1會造成OGT與O-GlcNAc的表現增加 29
四.OGT會對CTT進行O-GlcNAcylation修飾 29
五.O-GlcNAcylation會增加CTT的蛋白穩定性 30
六.OGT的表現確實會影響到CTT表現 32
七.CTT入核活化相關路徑的能力反而因為OGT而減弱 33
八.O-GlcNAcylation會造成CTT的localization改變 35
九.Thiamet-G藥物可以減緩Pkd小鼠病症 36
結論 38
討論 40
參考文獻 45
圖表 49
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