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研究生:陳筑毓
研究生(外文):Chu Yu Chen
論文名稱:第八型遺傳型顯性帕金森氏症(PARK8)的基因轉殖鼠動物模式
論文名稱(外文):Generation of familial type 8 of Parkinson’s disease (PARK8) transgenic mouse animal model
指導教授:王鴻利
指導教授(外文):H.L.Wang
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
論文頁數:54
中文關鍵詞:帕金森氏症基因轉殖鼠
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第八型家族遺傳性帕金森氏症患者為體染色體顯性遺傳(autosomal dominant inheritance) 且患者表現晚發性的帕金森氏症症候群症狀。近期研究指出Leucine-rich repeat kinase 2 (第二型多白胺酸性磷酸酶; LRRK2) 的誤義突變(missense mutations) 會導致第八型遺傳型顯性帕金森氏症(PARK8),而LRRK2 亦為導致晚發性顯性遺傳帕金森氏症最常見的基因。此外, LRRK2的突變亦在偶發性帕金森氏症佔有顯著的比例。具有LRRK2 突變的帕金森氏症病患具有與偶發性帕金森氏症患者相似的晚發性臨床特徵,顯示第八型遺傳型顯性帕金森氏症患者和偶發性帕金森氏症患者在發病過程上具有共有的分子機制。目前普遍認為第八型遺傳型顯性帕金森氏症突變是屬於一個獲得功能的突變(gain-of-function) 並且具有神經毒性的特性,導致了黑質-紋狀體多巴胺神經通路的退化以及帕金森氏症症候群症狀。為研究突變LRRK2 導致黑質-紋狀體神經系統的退化以及帕金森氏症症候群症狀的細胞及分子機轉,目前我們藉由產生野生型以及突變型(G2019S) LRRK2 基因轉殖鼠建立第八型遺傳型顯性帕金森氏症的活體動物系統。
在本實驗中我們使用PDGF-chain promoter作為表現控制以產生野生型以及突變型 (G2019S) LRRK2 基因轉殖鼠。野生型以及突變型 (G2019S) LRRK2 基因轉殖鼠的產生是透過原核顯微注射(Pronuclei microinjection)轉殖基因至受精卵母細胞的雄性原核中。之後以南方墨點法 ( southern blotting ) 加以篩選得到初代小鼠,且其表現大於五個表現 ( > 5 copies) 的初代小鼠將用於建立野生型以及突變型 (G2019S) LRRK2 基因轉殖鼠表現群組。西方墨點法證實不論野生型或是突變型(G2019S) LRRK2 基因轉殖鼠在大腦皮質、黑質、海馬迴以及紋狀體等部位皆有野生型或突變型 (G2019S) LRRK2蛋白的表現。我們使用多種動物行為模式測驗,包含rotarod test, locomotor activity test 以及 pole test, 評估野生型以及突變型 (G2019S) LRRK2 基因轉殖鼠的運動功能。突變型 (G2019S) LRRK2 基因轉殖鼠於五個月大時出現運動功能障礙的症狀,並且神經學上的表徵隨著時間增加在接下來數月有明顯的進展。突變型 (G2019S) LRRK2 基因轉殖鼠表現顯著 PARK8 症狀,包含活動力低下以及動作協調以及表現障礙;相較而言,野生型 LRRK2 基因轉殖鼠並無表現出症狀以及運動功能障礙。目前研究顯示突變型 (G2019S) LRRK2 基因轉殖鼠明顯表現出 PARK8 神經學上的症狀,並且可以被用於作為 PARK8 的活體動物模式。此外野生型以及突變型(G2019S) LRRK2 基因轉殖鼠也可以用於探討LRRK2 在腦中的生理功能。
Patients with familial type 8 of Parkinson’s disease (PARK8) exhibit late-onset parkinsonism symptoms and autosomal dominant inheritance. Recent studies identified missense mutations of leucine-rich repeat kinase 2 (LRRK2) as the cause of PARK8. LRRK2 is the most frequent causative gene in late-onset autosomal dominant PD. Furthermore, LRRK2 mutations are also detected in a significant fraction of sporadic PD patients. Late-onset clinical features of PD patients with LRRK2 mutation are similar to those of sporadic PD patients, suggesting that common molecular mechanisms are involved in the pathogenesis of both PARK8 and sporadic PD. It is generally believed that PARK8 mutation confers a gain-of-function and neurotoxic property on mutant LRRK2, which causes the degeneration of nigrostriatal dopaminergic pathway and parkinsonism. To investigate the cellular and molecular mechanism of mutant LRRK2-induced degeneration of nigrostriatal system and resulting parkinsonism, in the present study we prepared in vivo animal model of PARK8 by generating transgenic mice expressing PARK8 mutant (G2019S) LRRK2.
The expression of human wild-type or mutant (G2019S) LRRK2 in transgenic mice was under the transcriptional control of PDGF-chain promoter. Transgenic mice expressing wild-type or mutant (G2019S) LRRK2 were generated by microinjecting transgene DNA into the male pronucleus of fertilized oocytes. Then, Southern blot analysis was performed to identify founder animals, and founder mice with a high copy number (> 5 copies) were used to establish stable lines of LRRK2 or (G2019S) LRRK2 transgenic mice. Western blot analysis demonstrated that wild-type or mutant (G2019S) LRRK2 protein was expressed in various brain regions of transgenic mouse, including cerebral cortex, substantia nigra , hippocampus, and striatum. Various behavioral tests, including the rotarod test, locomotor activity test and pole test, were used to evaluate the motor function of wild-type or (G2019S) LRRK2-expressing transgenic mice. Transgenic mice expressing mutant (G2019S) LRRK2 displayed the symptoms of motor dysfunction with an onset age of about 5 months, and the severity of neurological phenotypes progressively increased in the following months. (G2019S) LRRK2 transgenic mice exhibited several PARK8 symptoms, including hypoactivity, impaired motor coordination and performance. In contrast, transgenic mice expressing wild-type LRRK2 failed to display the symptom of motor dysfunction. The present study clearly shows that mutant (G2019S) LRRK2-expressing transgenic mice exhibit neurological phenotypes of PARK8 and can be used as an in vivo PARK8 animal model. Furthermore, transgenic mice expressing wild-type or mutant (G2019S) LRRK2 can also be utilized to study the physiological function(s) of LRRK2 in the brain.
Abstract (English) v
Abstract (Chinese) viii
Contents ix
I. Introduction 1
II. Specific Aim and Significance 10
III. Experimental Procedures 11
3.1 Construction of point mutant LRRK2 11
3.2 Generation of transgenic mice expressing PARK8 mutant
(G2019S) LRRK2 11
3.3 Identification of transgenic animals by Southern blotting
of tail DNA 12
3.4 Behavioral tests 12
3.5 Western blot analysis 13
3.6 Immunocytochemical staining 14
IV. Results 15
4.1 Transgene construction for transgenic mice expressing
wild-type or mutant (G2019S) LRRK2 15
4.2 Generation of transgenic mice expressing wild-type or
mutant (G2019S) LRRK2 16
4.3 Transgenic mice expressing mutant (G2019S) LRRK2
exhibit PARK8 neurological phenotypes 17
V. Discussion 19
VI. References 26
VII. Figures and Figure Legends 35
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