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系統識別號 U0026-2608201915101400
論文名稱(中文) 腎臟特定腎小管Wdr19基因之破壞可導致小鼠多囊腎病的發生
論文名稱(英文) Targeted Disruption of Wdr19 in Renal Tubules Causes Polycystic Kidney Disease in Mice
校院名稱 成功大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Institute of Clinical Medicine
學年度 107
學期 2
出版年 108
研究生(中文) 游上萱
研究生(英文) Shang-Shiuan Yu
學號 S98991021
學位類別 博士
語文別 英文
論文頁數 101頁
口試委員 指導教授-邱元佑
指導教授-蔣思澈
指導教授-許育祥
口試委員-王家義
口試委員-林清淵
口試委員-田祐霖
中文關鍵字 Wdr19  鞭毛內運輸  初級纖毛  腎囊泡  多囊腎病 
英文關鍵字 Wdr19  intraflagellar transport  primary cilium  renal cyst  polycystic kidney disease 
學科別分類
中文摘要 Wdr19蛋白是鞭毛內運輸(IFT)系統中運輸蛋白的一成員,而初級纖毛這胞器的組成與功能與鞭毛內運輸系統是息息相關,若此胞器出了問題則引發纖毛病變。先前的研究報導指出具有Wdr19基因突變的患者顯示骨骼異常或腎囊性疾病,而N-乙基-N-亞硝基脲(ENU)誘發的Wdr19基因突變小鼠因神經或骨骼異常造成早期胚胎致死。然而,時至今日Wdr19基因在腎臟中的生理角色仍未清楚地被闡釋。在本研究中,我們利用外顯子4編碼序列直至末端的Wdr19 cDNA融合紅色螢光蛋白替換小鼠中相對應的54kb基因組片段作為條件性剔除等位基因。結合腎臟專一表達cre基因轉殖小鼠與Wdr19基因標的小鼠可獲得腎臟專一Wdr19條件性基因剔除小鼠(Wdr19f/-::Cdh16-creTg/0)。在此小鼠中,我們發現數千個逐漸生長的腎囊泡分佈在擴大的腎臟約71%區域,且存活不到一個月。另外,在囊泡大小上,中型腎囊泡約佔52%,而在囊泡數目上,小型腎囊泡約佔57%。有趣的是,腎囊泡襯裡上皮細胞表現出較少的增殖和更多的細胞凋亡,形成相對較小的腎囊泡且疾病進展較慢,與常見的腎臟專一Pkd1條件性基因剔除小鼠(Pkd1f/-::Cdh16-creTg/0)造成的多囊腎病(PKD)不同。此外,腎囊泡襯裡上皮細胞中幾乎沒有初級纖毛存在,與具有功能失調的初級纖毛但缺失Pkd1基因引發的腎囊泡顯著不同。另外,在腎臟專一Wdr19誘導式基因剔除小鼠(Wdr19f/-::Cdh16-cre/ERT2Tg/0)中,Wdr19基因的關鍵發育轉換時間點大約是在出生後第11天。我們的研究結果表明,主要在逆行鞭毛內運輸的Wdr19蛋白對於初級纖毛構建至關重要並且影響腎小管發育。腎囊泡發生的潛在分子機制可能在無初級纖毛細胞和含有初級纖毛結構的細胞之間有所不同,應進一步研究釐清。這些新穎地Wdr19基因剔除小鼠對於在發育階段的其他組織和器官之未來功能研究將是有價值的。
英文摘要 The Wdr19 protein was a member of trafficking proteins in the intraflagellar transport (IFT) machinery which was closely related to the formation and function of the primary cilium. Defects in this organelle brought about the ciliopathy. Previous studies reported that patients with mutant Wdr19 displayed skeletal anomalies or renal cystic diseases; whereas N-ethyl-N-nitrosourea (ENU)-derived mutant Wdr19 mice expressed early embryonic lethality due to neurological or skeletal abnormalities. However, the physiological role of the Wdr19 gene in kidneys remained unclear until now. In this study, we used a loxP-flanked C-terminal RFP-tagged Wdr19 cDNA coding from exon4 till the end to replace corresponding 54 kb genomic fragments in mice as a conditional knockout allele. Combining kidney-specific cre transgenic mice with heterozygous Wdr19 deficient (Wdr19+/-::Cdh16-creTg/0) and homozygous Wdr19 targeted mice (Wdr19f/f) obtained kidney-specific Wdr19 conditional knockout mice (Wdr19f/-::Cdh16-creTg/0). In these mice, we found that thousands of progressively growing cysts were distributed in approximately 71% areas of enlarged kidneys and survived for less than one month. Additionally, in cystic sizes, medium-sized cysts occupied for about 52%, while in cystic numbers, small-sized cysts occupied for about 57%. Interestingly, cyst-lining epithelia showed less proliferative and more apoptotic than those of the most common polycystic kidney disease (PKD) causing gene Pkd1 conditional knockout mice by the same cre transgene (Pkd1f/-::Cdh16-creTg/0), resulting in relatively smaller cysts with the slower disease progression. Moreover, the primary cilium was almost absent in the cyst-lining epithelium and was dramatically different from dysfunctional primary cilium in Pkd1-knockout cysts. Furthermore, a critical developmental switch time point of the Wdr19 gene was at postnatal day 11 (P11) in kidney-specific Wdr19 inducible knockout mice (Wdr19f/-::Cdh16-cre/ERT2Tg/0). Our results indicated that the Wdr19 protein primarily proposed for retrograde IFT is crucial for the primary cilium construction and affected the renal tubule development. The underlying molecular mechanism of the renal cystogenesis likely differs between primary cilium-free cells and Pkd1-knockout cells, which contain the primary cilium structure, and should be further investigated. These novel Wdr19 knockout mice will be valuable for future functional studies in other tissues and organs under variable development stages.
論文目次 中文摘要 I
Abstract II
Acknowledgement III
Table of Contents IV
List of Tables VII
List of Figures VIII
List of Abbreviations X
Chapter 1 Introduction 1
1.1 Cilium 1
1.2 Intraflagellar transport (IFT) 1
1.3 Ciliopathy 2
1.4 Wdr19 3
1.5 Animal models 5
1.6 Hypothesis 5
1.7 Aims 6
Chapter 2 Materials and Methods 7
2.1 Targeting Construct 7
2.2 Mouse Lines 7
2.3 DNA Extraction 7
2.4 Polymerase Chain Reaction (PCR) Analysis 7
2.5 Agarose Gel Electrophoresis 7
2.6 RNA Extraction 8
2.7 Reverse Transcription Polymerase Chain Reaction (RT-PCR) Analysis 8
2.8 Western Blot (WB) Analysis 8
2.9 Blood Chemistry Analysis 8
2.10 Histological Analysis 9
2.11 Cystic Index and Fibrotic Index Quantification 9
2.12 Immunohistochemistry (IHC) Analysis 9
2.13 Terminal dUTP Nick-End Labeling (TUNEL) Assay 10
2.14 Proliferative Index and Apoptotic Index Quantification 10
2.15 Lectin Analysis 11
2.16 Transmission Electron Microscopy (TEM) Analysis 11
2.17 Drug Induction Analysis 11
2.18 Statistical Analysis 11
Chapter 3 Results 13
3.1 Generation of Wdr19 floxed mice 13
3.2 Phenotype of Wdr19 conventional knockout mice 13
3.3 Characterization of kidney-specific Wdr19 conditional knockout mice 14
3.4 Phenotype of kidney-specific Wdr19 conditional knockout mice 15
3.5 Renal function of kidney-specific Wdr19 conditional knockout mice 15
3.6 Progression of renal cysts in kidney-specific Wdr19 conditional knockout mice 16
3.7 Progression of proliferation in the renal cyst-lining epithelium in kidney-specific Wdr19 conditional knockout mice 16
3.8 Progression of apoptosis in the renal cyst-lining epithelium in kidney-specific Wdr19 conditional knockout mice 16
3.9 Progression of interstitial fibrosis in kidney-specific Wdr19 conditional knockout mice 16
3.10 Renal tubule markers of renal cysts in kidney-specific Wdr19 conditional knockout mice 17
3.11 Ultrastructures of the primary cilium in kidney-specific Wdr19 conditional knockout mice 17
3.12 Induction time of renal cysts in kidney-specific Wdr19 inducible knockout mice 17
3.13 Phenotype of kidney-specific Pkd1 conditional knockout mice 17
3.14 Renal function of kidney-specific Pkd1 conditional knockout mice 18
3.15 Progression of renal cysts in kidney-specific Pkd1 conditional knockout mice 18
3.16 Classification of renal cysts in kidney-specific Wdr19- or Pkd1- conditional knockout mice 19
3.17 Progression of proliferation and apoptosis in the renal cyst-lining epithelium in kidney-specific Pkd1 conditional knockout mice 19
3.18 Renal tubule markers of renal cysts in kidney-specific Pkd1 conditional knockout mice 19
3.19 Ultrastructures of the primary cilium in kidney-specific Pkd1 conditional knockout mice 19
3.20 Phenotype of kidney-specific Wdr19 and Pkd1 conditional double knockout mice 20
3.21 Progression of renal cysts in kidney-specific Wdr19 and Pkd1 conditional double knockout mice 20
3.22 Conclusion 20
3.23 Summary 21
Chapter 4 Discussion 22
4.1 Wdr19 conventional knockout mice 22
4.2 Kidney-specific Wdr19 conditional knockout mice 22
4.3 Kidney-specific Wdr19 inducible knockout mice 22
4.4 Comparison of kidney-specific Wdr19- or Pkd1- conditional knockout mice 23
4.5 Kidney-specific Wdr19 and Pkd1 conditional double knockout mice 24
Table Legends 26
Figure Legends 27
Tables 32
Figures 41
Chapter 5 References 96
Curriculum Vitae 101
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