運用螢光方法研究RAB3A的訊息傳遞機制

中山醫學大學機構典藏 CSMUIR > 醫學科技學院 > 生物醫學科學學系暨碩士班 > 研究計劃 > Item 310902500/2717

Please use this identifier to cite or link to this item: https://ir.csmu.edu.tw:8080/ir/handle/310902500/2717

Title:	運用螢光方法研究RAB3A的訊息傳遞機制 Studies in Signal Transduction Mechanism of Rab3A by Fluorometric Methods
Authors:	林崇智;高閬仙 Lin, Chung-Chih;Kao, Lung-Sen
Contributors:	中山醫學院生命科學系
Keywords:	訊息傳遞;胞泌作用;綠螢光蛋白;Rab蛋白;胞飲體;綠螢光蛋白基因 Signal transduction；Exocytosis；Green fluorescent protein；Rab protein；Endosome；Green fluorescent protein gene
Date:	2000
Issue Date:	2010-11-05T10:49:05Z (UTC)
Abstract:	Rab3A被認為是一種參與調控式胞吐的小G蛋白。根據目前的研究認為Rab3A的作用是在囊泡的導引與膜融合，但是沒有直接證據證明其作用的位置與實際參與的生化反應。為了解決這個問題，首先一定要了解Rab3A在活體細胞中的位置與其在胞吐作用時的分佈變化。本計劃將Rab3A以綠螢光蛋白(GFP)結合，在SK-N-SH神經瘤細胞中表現，以螢光顯微鏡來觀察Rab3A在活體細胞中的位置與其在胞吐作用時的分佈變化。本年度成功地完成計劃所需的基本實驗配備，實驗材料與實驗條件，包括(1)GFP-Rab3A的真核表達載體的構築，(2)建立轉染神經胞株的條件，(3)建立觀察螢光蛋白質動態分布的顯微影像分析系統。根據這些基礎工作所得的實驗條件，進行觀察GFP-Rab3A在SK-N-SH細胞中的分布。與過去免疫螢光染色結果相似，GFP-Rab3A在細胞呈點狀分布，且集中在核周圍與神經纖維末端，證明GFP-Rab3A不因為GFP而使其分布改變。隨後分析GFP-Rab3A光點的大小與形狀，發現其大小約0.3微米到1微米之間，小光點其形狀近似圓形，大多分布在神經纖維呈靜止狀態，此顯示小光點可能為準備分泌的囊泡，顯示Rab3A可能作用於囊泡的導引與膜融合反應。大光點分布在細胞本體，呈不規則的結構，少部份大光點會移動(0.5微米/秒)與互相融合，此種現象類似內胞食體(Endosome)的行為，此顯示出Rab3A亦有可能參與突觸囊泡的回收與生成。 Rab3A is a small GTP-binding protein thought to regulate regulated exocytosis. Recent investigations indicate Rab3A plays a role in docking and fusion steps of exocytosis, but there is no direct evidence to prove where Rab3A acts. To solve this problem, tracking Rab3A localization during exocytosis in vivo need to be established. In this study, we have setup a simple system to detect Rab3A translocalization in vivo. Rab3A is fused with green fluorescence protein (GFP) and the fusion protein is expressed in neuroblastoma cell line, SK-N-SH. Transfected cells are grown in a chambered coverlass and observed by inverted fluorescence microscopy. Fluorescence indicates subcellular localization of Rab3A and is recorded by microphotography. Photograph is scanned and NIH Image and Photoshop analyze translocalization of Rab3A. Part of Rab3A is cytosolic and over the whole cell except nucleus, and this is quite different from GFP distribution. Some Rab3A is associated with small organelles, i.e. vesicles, to form small dots, and these dots are concentrated in perinuclear area and neurites. Sizes of dots in perinuclear area, several microns, are larger than those in neurites, 0.3-1 microns. Structures of dots in perinuclear area are various, but those in neurites are very unique. Small dots in neurite terminus are very steady and this seems to indicate that these dots are in ready-to-release pools. Although these results are similar to distribution of synaptic vesicle associated proteins in PC12 cells, these dots need more evidence to know their exact location. Several larger dots are very mobile among neurites and cell body, and some of them are fused each other or break down into small vesicles, and this seems to indicate that Rab3A may also play a role in vesicles recycle. In this study, we have setup a system to track Rab3A translocalization in living cells, this system will be applied to know the exact locations of Rab3A during exocytosis and find out the role of Rab3A in regulation of exocytosis.
URI:	https://ir.csmu.edu.tw:8080/handle/310902500/2717
Appears in Collections:	[生物醫學科學學系暨碩士班] 研究計劃

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