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研究生:陳勢杰
研究生(外文):Shih-Chieh Chen
論文名稱:正腎上腺素接受器亞型基因Adra2c剔除小鼠之表現型分析
論文名稱(外文):Phenotype Characterization of Adra2c Knockout / lacZ Knock-in Mutant Mice
指導教授:張南驥錢嘉韻錢嘉韻引用關係
指導教授(外文):Nan-Chi ChangAlice Chien Chang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:86
中文關鍵詞:(正)腎上腺素受器(正)腎上腺素基因剔除小鼠基因標的五羥色胺鴉片類受器
外文關鍵詞:adrenoceptornorepinephrinegene knockoutgene tatgetingserotoninmu opioid receptoradra2c
相關次數:
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(正)腎上腺素受體屬於G蛋白偶合受體家族,□2(正)腎上腺素受體乃是其三類受體之一,又可進一步分成□2A、□2B、□2C三種亞型;分別由Adra2a、Adra2b、Adra2c三個不同基因所製造。□2受體三種亞型在中樞神經系統不同的分佈情形可看出三種亞型在不同的區域執行各自的功能。由於缺乏具有專一性的催動劑與拮抗劑,為了研究三種亞型中□2受體的功能,本實驗室建立了Adra2c基因剔除 / LacZ基因嵌入小鼠,其體內正常基因和標的載體進行同源互換,將Adra2c基因原位置換成lacZ報導基因。如此一來,利用X-gal染色產生的藍色反應產物以標示出應該表現Adra2c的細胞本體,利於觀察□2C受體在不同時期和區域的表現;另外製造□2C受體的Adra2c基因遭到置換破壞,可利用這種小鼠模式研究□2受體在活體動物中所負責的生理功能。
本論文主要是進一步了解□2受體在小鼠發育過程中所受的調控進而推測其可能的功能。各個腦區域的表現主要分成三種型態,一種乃是在出生後7天或14天才開始觀察到表現並持續增強到成鼠;第二種是一直到成鼠才被觀察到報導基因的表現;第三種則是在小鼠青春期,也就是第14天或是21天時,基因表現到達一個高峰值,而之後就持續下降至一個基本值直到成鼠。
由於實驗室藥物實驗的初步結果顯示此基因剔除小鼠有著低於正常小鼠的藥物刺激走動活性、對於藥物的敏感化以及偏好也較不明顯。因此,採用免疫染色的方法,藉由□亞型鴉片受體的免疫訊號和X-gal藍色小點的位置重合,可以觀察到在許多區域有著明顯的位置重合,這些區域中或是在表現□亞型鴉片受體的神經細胞所投射的軸突範圍,或是與□亞型鴉片受體表現在同一個細胞內。表示在鴉片成癮的機制中,□2C受體可能與□亞型鴉片受體受相同調控,或是在□亞型鴉片受體相鄰的位置間接地接受鴉片類藥物刺激。
實驗室更在飼養此基因剔除小鼠過程中,發現此小鼠會表現有別於一般正常小鼠的攻擊性行為,不但攻擊潛伏期較短、而且頻率也明顯高出許多;由許多文獻指出,五羥色胺對於攻擊性的行為直接參與調控,故本論文以免疫染色的方式,將五羥色胺的免疫訊號與X-gal的藍色小點做位置重合的結果,在許多腦核區都有重合的分佈,因此暗示□2C受體缺失小鼠所表現的行為性狀,其機制可能與□2C受體和五羥色胺系統的交互作用有關。
由於□2C受體的分佈區域與情緒、行為、學習記憶等諸多認知功能、甚至藥物成癮有相關,而這些行為表現型背後的機制也多互相牽連,包括不同腦區域以及不同神經傳導物質系統。由目前證據指出,進一步了解□2C受體所受之調控及在不同性狀後分子機制,不但可以幫助解釋其生物意義,也希望對有暴力傾向或是藥物成癮者可以提供更好的控制或是治療方法。
Adrenoceptors(AR) belongs to the G protein-coupled receptor superfamily. □2AR is one of the three types AR and can be divided into □2A、□2B、□2C subtypes which were encoded by Adra2a、Adra2b、Adra2c gene, respectively. The distribution of □2AR subtype in CNS reveals the specific functions of three subtypes in different regions. Since the lack of subtype-specific ligand, we established Adra2c knockout (KO)/lacZ knock-in mice to analyze □2CAR. The wild-type Adra2c gene is in-frame replaced by lacZ gene via homologous recombination between genomic DNA locus and targeted vector. Thus, we can use X-gal staining to produce the ”blue” product consequently to label the soma used to express Adra2c gene. This made monitoring the temporal and spatial expression of □2CAR easily; besides, we can study the in vivo physiological functions of □2C AR in the Adra2c gene KO mice,.
The goal of this thesis is to understand the regulation of □2CAR during development therefore to deduce the possible functions. The expression pattern of □2C AR can be divided into three types. First, expressions are seen at P7 or P14 then gradually increase to adult. Secondly, expression is observed only in adulthood. Third, gene expression culminates at puberty then diminishes to a basal level till adulthood.
The preliminary data in our laboratory reveal that the KO mice have lower drug-induced locomotor activity and blunt drug sensitization and preference. Thus, we adopt immunohistochemistry(ICC) to observe the colocalization of □□opioid receptor (MOR) and X-gal blue spot, it can be seen highly colocalization in many regions. They may express in the projection of the interneurons produce MOR or even □2CAR and MOR express in the same cell. It imply that □2CAR maybe receive the stimuli indirectly from MOR-expressing neuron or under the same regulation with MOR in the opiate addiction mechanism.
During breeding process, we found the KO mice show unusual aggression behaviors including shorter attack latency and higher frequency compared with wild type control. In literatures, 5-hydroxytryptamine (5-HT) involves directly into the regulation of aggression behaviors. Via 5-HT ICC, it can be seen colocalization of 5-HT and X-gal signal in many regions. It suggest that the mechanism of aggression phenotype in this KO mice involve the interaction of □2CAR and 5-HT system.
Because the brain regions □2CAR expresses involve most cognitive functions. The mechanisms behind these behavior phenotype are interrelated, comprise different brain structures and neurotransmitter systems. The information so far imply advanced understanding the regulation of □2CAR and the mechanism of functional-defect phenotype can help us to elucidate its biological meanings and further to provide control and cure strategies to people with violence tendency and drug addiction.
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