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研究生:王怡汎
研究生(外文):Wang, Yee-Fan
論文名稱:第五亞型蕈毒乙醯膽鹼受體在大鼠腦中之免疫細胞化學分佈
論文名稱(外文):Immunocytochemical localization of m5 muscarinic acetylcholine receptor in rat brain
指導教授:廖欽峰
指導教授(外文):Liao, Ching-Fong
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生理學研究所
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:1996
畢業學年度:84
語文別:中文
論文頁數:33
中文關鍵詞:第五亞型蕈毒乙醯膽免疫細胞
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蕈毒乙醯膽鹼受體(mAChR)已被證實顯著表現在脊椎動物腦中。從分子選殖的研究,證實目前至少有五種亞型的蕈毒乙醯膽鹼受體(m1-m5)存在。蕈毒乙醯膽鹼受體與G蛋白偶合,穿越細胞膜七次,內外環交互組成,其中細胞內第三內環特異性最大。一系列蕈毒乙醯膽鹼受體第三內環特異性抗體被製造並用來偵測蕈毒乙醯膽鹼受體在腦中分佈的情形,然而第五亞型蕈毒乙醯膽鹼受體至今仍未明確的被偵測到。
本論文之研究目的在發展第五亞型蕈毒乙醯膽鹼受體特異性抗體,應用特異性抗體定出(一)第五亞型蕈毒乙醯膽鹼受體的分子量,(二)第五亞型蕈毒乙醯膽鹼受體N端及C端在細胞胞膜內外之方向性。(三)第五亞型蕈毒乙醯膽鹼受體在何種腦細胞表現及(四)其在腦中分布情形。
運用多重性抗原胜月太的設計,合成第五亞型蕈毒乙醯膽鹼受體N端及c端胜月太(m5N-MAP,m5c-MAP)。合成的胜月太注射於雞皮下以引發抗體,純化抗體並做免疫化學分析。經西方點墨法分析,証實第五亞型蕈毒乙醯膽鹼受體會表現在腦中許多區域 小腦、海馬迴、下視丘/視丘、紋狀體及除上述區域的大腦部分)且其分子量為58-60kDa。藉由免疫細胞化學染色,atti-m5N及anti-m5c會染上LM5細胞,不染上Ltk-或LM5/p細胞;且第五亞型蕈毒乙醯膽鹼受體會表現在初級培養的神經細胞及星狀神經膠細胞。運用免疫細胞化學的技術,初步確定第五亞型蕈毒乙醯膽鹼受體會表現在嗅徑,尾核,豆狀核,黑質緻密區及胼胝體。
綜合上述結果,我們確定第五亞型蕈毒乙醯膽鹼受體分子量為58-60 kDa,第五亞型蕈毒乙醯膽鹼受體N端位在細胞外,C端位在細胞內第五亞型蕈毒乙醯膽鹼受體會表現在神經細胞及星狀神經膠細胞,及第五亞型蕈毒乙醯膽鹼受體在腦中主要分布在嗅徑、尾核、豆狀核殼區、黑質緻密部及胼胝體。
Muscarinic acetylcholine receptors (mAChR) are predominate cholinergic receptors in vertebrate brains. Molecular cloning studies have revealed that there are at least five distinct mAChR subtypes (m1-m5). The mAChRs belong to G protein-coupled receptors, consisting of seven-transmembrane domains joined by alternating extracellular and intracellular loops. Intracellular third (i3) loop is unique to each mAChR. A panel of subtype-specific antibodies against the i3 loop of ml-m5 mAChRs have been developed and used to localize mAChRs in the rat brain. However, m5 mAChRs in brain have not been detected by immunohistochemistry with certainty. The purpose of this study was to develop antibodies against peptides of the rat m5 mAChR and use these antibodies to determine (1) molecular eight of m5 mAChR, (2) orientation of N- and C-terminals of 5 mAChRs in cells, (3) types of brain cells expressing m5 mAChRs, and (4) distribution of m5 mAChRs in rat brain.
N- and C-terminal peptides of the m5 mAChR were synthesized on Fmoc MAP resins. The multiple antigen peptides, designed as m5N-MAP and m5C-MAP, were injected into chicken to produce antisera. Affinity-purified anti-m5 antibodies were used for following immunochemical analysis. Western blotting of membrane proteins prepared from mouse Ltk- cells expressing transfected m5 mAChRs (LM5) and several rat brain regions (cerebellum, cortex, hippocampus, hypothalamus, striatum, etc.) revealed that apparent molecular weight of the m5 mAChR was 58-60 kDa. The LM5 cells were detectably with both anti-m5N and anti-m5C, whereas the Ltk- cells expressing transfected chimeric m5/β2 receptors (LM5/β)were detectably with the anti-m5N, but undetectable with anti-m5C as expected. A few primary cultured cells prepared from hippocampus of embryonic rat brain co-expressed m5 mAChRs and neuron- or astroglia-markers. By immunohistochemical technique, the m5 mAChRs were detected in anterior commissure, corpus callosum, caudate putamen, medial septum, hippocampus, lateral olfactory tract, and substantia nigra compact, etc.
In summary, the apparent molecular weight of m5 mAChRs is 58-60 kDa. N- and C-terminals of m5 mAChRs in LM5 cells localize extracellularly and intracellularly, respectively. Some brain neurons and astroglial cells express m5 mAChR. The m5 mAChRs express primarily in lateral olfactory tract, caudate/putamen, substantia nigra compact, and corpus callosum, etc.



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