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系統識別號 U0026-0309201223391300
論文名稱(中文) Wwox基因剔除小鼠生理與神經系統異常情形之分析
論文名稱(英文) Physiological and neurological alterations in Wwox knockout mice
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 100
學期 2
出版年 101
研究生(中文) 程雅筠
研究生(英文) Ya-Yun Cheng
學號 T36994064
學位類別 碩士
語文別 英文
論文頁數 59頁
口試委員 指導教授-徐麗君
口試委員-張南山
口試委員-蔡坤哲
口試委員-黃相碩
口試委員-詹明修
中文關鍵字 含雙色胺酸功能區氧化還原酶  基因剔除  生長遲緩  腫瘤生成 
英文關鍵字 WW domain-containing oxidoreductase  gene knockout  growth retardation  tumorigenesis 
學科別分類
中文摘要 含雙色胺酸功能區氧化還原酶 (WW domain-containing oxidoreductase) 被命名為WWOX、FOR或是小鼠WOX1,因其蛋白質的表現量在許多病人的癌細胞中都有下降的趨勢,所以被視為是腫瘤抑制蛋白質 (tumor suppressor)。先前文獻指出,WWOX/WOX1蛋白質參與神經發育及退化的過程,然而WWOX/WOX1在生理功能方面的角色迄今所知仍極其有限。為了要探討WWOX/WOX1蛋白質在動物體內的功能,我們實驗室利用基因標的 (gene targeting) 的技術,製造出將Wwox基因exon 1- (WD1) 、或是exon 2/3/4- (WD234) 剔除的小鼠。研究中發現,我們實驗室所生產的Wwox基因剔除小鼠 (Wwox-/-)。Wwox-/-小鼠出生後往往無法存活超過21天。在外觀上,Wwox-/-小鼠體重顯著低於Wwox+/+與Wwox+/-小鼠,呈現生長上的遲緩。進一步為了探討WWOX/WOX1蛋白質在腫瘤的形成的過程中所扮演的角色,我們觀察並統計Wwox+/+與Wwox+/-小鼠其自發性腫瘤生成的情形。結果顯示Wwox+/+與Wwox+/-小鼠之間,其自發性腫瘤生成的比例並無顯著的差異,表示單一Wwox對偶基因的缺失並不會促進腫瘤的生成。總結以上的實驗結果,我們證明WWOX/WOX1蛋白質在胚胎的發育及產後的存活至關重要。
英文摘要 WW domain-containing oxidoreductase, designated as WWOX, FOR, or murine WOX1, has been identified as a tumor suppressor because of its downregulation in many human cancers. Previous studies have shown that WWOX/WOX1 participates in neural development and neuronal damage. However, the physiological functions of WWOX/WOX1 are still largely unknown. To investigate the functional roles of WWOX/WOX1 in vivo, whole-body exon 1- (WD1) or exon 2/3/4-targeting (WD234) deletion of Wwox gene in mice was performed in our laboratory. High embryonic or postnatal lethality was observed in our developed homozygous Wwox gene knockout mice (Wwox-/-). All Wwox-/- mice died by 3 weeks of age. Compared with their control littermates, Wwox-/- mice showed growth retardation along with a significant reduction in the body weight. Furthermore, to investigate the role of WWOX/WOX1 in tumorigenesis, we monitored the incidence of spontaneous tumor formation in aged Wwox+/+ and Wwox+/- mice. Our results showed that the incidence of spontaneous tumor formation in Wwox+/- mice was comparable to Wwox+/+ mice, suggesting that loss of one allele of Wwox gene may not lead to tumorigenesis. In conclusion, our results suggest that WWOX/WOX1 is crucial for postnatal survival.
論文目次 Chinese Abstract I
English Abstract II
Acknowledgement III
Contents IV
Figure index VI
Introduction 1
Materials and Methods 6
A.Materials 6
A-1 Chemical 6
A-2 Antibodies 7
A-3 Instruments 7
B. Methods 8
B-1 Generation of Wwox knockout mice 8
B-2 Genotyping 8
B-2-1 Genomic DNA extraction 8
B-2-2 Polymerase chain reaction (PCR) 9
B-3 Measurement of brain water content 11
B-4 Behavioral tests 11
B-4-1 Rotarod tests 12
B-4-2 Footprint analysis 12
B-5 Histological analysis 12
B-5-1 Hematoxylin and eosin (H&E) staining 13
B5-2 Nissl staining 13
B-6 Immunohistochemistry staining 14
B-7 TUNEL assay 16
B-8 Electron microscopy 18
B-9 Immunoelectron microscopy 19
B-10 Induction of seizure 20
B-11 Statistical analysis 21
Results 22
Generation of Wwox gene knockout mice 22
Phenotypical analysis of the Wwox-/- mice 22
Incidence of spontaneous tumor formation in Wwox+/+ and Wwox+/- mice 23
Behavior disorders in Wwox-/- mice 24
Aberrant cerebellum morphology in Wwox-/- mice 25
Peripheral nerve degeneration of Wwox-/- mice 26
Brain malformation and epileptic seizures in Wwox-/- mice 26
Cellular alternations in the hippocampus of Wwox-/- mice 27
Discussion 29
References 32
Figures 41
Tables 58
Appendix 59
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