Chia Nan University of Pharmacy & Science Institutional Repository:Item 310902800/1800
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    CNU IR > Pharmacy and Science > Dept. of Pharmacy > MOST Project >  Item 310902800/1800
    Please use this identifier to cite or link to this item: http://ir.cnu.edu.tw/handle/310902800/1800


    Title: 以生物晶片技術研究陽離子性高分子基因傳送系統對巨噬細胞運作基因表現之影響
    The Effects of Cationic Polymer Gene Delivery Systems on Cellular Gene Expression Profiles in Macrophages by Using Dna Microarray Technology
    Authors: 郭榮華
    詹明修
    Contributors: 藥物科技研究所
    Keywords: 非病毒性載體
    陽離子性高分子
    基因
    巨噬細胞
    DNA 生物晶片
    Non-viral vector
    Cationic polymer
    Gene
    Macrophages
    DNA
    cDNAmicroarray
    Date: 2008
    Issue Date: 2008-07-18 15:36:07 (UTC+8)
    Publisher: 台南縣:嘉南藥理科技大學藥物科技研究所
    Abstract: 本計畫係一跨領域整合性的研究計畫,結合了高分子,生物科技,及奈米材料領域,主要工作是以DNA 生物晶片研究陽離子性高分子基因傳送系統與巨噬細胞間在分子層次上對細胞運作基因表現之影響,探討DNA/陽離子性高分子體複合體在體內(in vivo) 被網狀內皮系統(RES)吞噬之細胞運作之基因調控與陽離子性高分子結構之相關性,以應用於巨噬細胞之目標基因傳送。本計畫的目的有:(1) 了解陽離子性高分子載體結構之變化與其DNA 複合體間對巨噬細胞基因表現模式之差異性。(2)針對各種基因表現模式中去全面性分析各項細胞功能運作之應答,進而從中尋找新的傳送目標及應用。(3) 分析複合體之轉染效率與相關基因之表現型錄有無存在相關性,進而了解其與巨噬細胞之分子作用機轉。本計畫預計以DNA 生物晶片研究陽離子性奈米樹狀體(polyamidoamine (PAMAM) (第五代及第七代( PAMAM 5.0 and 7.0)) ); 分枝型高分子PEI (25K and 800K)及直鏈狀高分子幾丁胺醣(High and low viscosity)與其與DNA 結合形成之複合體對人類巨噬細胞(THP-1)之細胞運作基因表現與基因調控之全方位影響進行深入而有系統之研究。我們除了探討三種空間結構不同的陽離子性高分子對細胞基因調控的影響外,亦設計在各種高分子中又改變其分子量大小來研究對基因調控及轉染效率之相關性。研究一開始我們即將針對各種載體本身及其與DNA 形成之複合體先測試其最佳轉染效果,然後將在最佳轉染效果之下之物理化學特性(顆粒大小及表面電位)詳細測量記錄並進行細胞毒性試驗,然後將再最佳轉染條件下進行生物晶片分析實驗進而得知其全方位基因表現資料,我們將利用其中有顯著者差異之基因加以分類整理,進而獲得各項有關影響細胞功能及可能引發之途徑機轉。我們可以得知那些功能性基因被上調(up-regulation)或者下調(down-regulation),最後我們將比較各種高分子載體基因傳送系統間之基因表現差異性與其結構變化間(空間結構及分子量大小)之相關性並進而找出各項關鍵基因變化之模式,以期對巨噬細胞之各項基因應答有全方位的了解。我們將以real-time PCR (Polymerase Chain Reaction),西方墨點法(Western Blot),ELISA (enzyme-link immunosorbent assay)及ROS (Reactive Oxygen Species)產生粒腺體電位,細胞週期等各項實驗來交叉印證資料之可靠性。
    The proposed project is an interdisciplinary research consisting of polymer, biotechnology, and nanotechnology to study the effects of cationic polymer gene delivery systems on cellular gene expression profiles in macrophages by using microarray technology. The relationships and mechanisms of gene regulation between cationic polymer gene delivery systems and the responses after in vivo uptake by reticuloendothelia systems will be investigated. The results will be applied to target gene delivery to macrophages. The aims of this study are to: (1) understand the effects of the structure of cationic polymers and their DNA complexes on gene expression patterns in macrophages. (2) analyze, from the global gene expression patterns, the responses of cellular processes in macrophages induced by cationic polymer gene delivery systems in order to identify novel gene for target gene delivery. (3) analyze the relationships between transfection efficiencies and gene expression patterns in macrophages treated with cationic polymer gene delivery systems for understanding their molecular mechanisms. We will use a whole-genome analysis by cDNA microarray to explore global gene expression profiles of cellular processes in THP-1 macrophages induced by three cationic polymer gene delivery systems (dendrimer, polyethylenimine (PEI), and chitosan). In addition to the study of structure changes among these cationic polymer systems, we also investigate the effects of molecular weight of each cationic polymer on gene expression profiles in macrophages. Firstly, we will determine the optimal conditions of transfection efficiencies for each cationic polymer and its physiochemical characteristics will be analyzed. Under optimal conditions, we will then obtain global gene expression profiles of cellular process in macrophages induced by cationic polymer gene delivery systems by using microarray technology. By applying cluster analysis, we will obtain the significant differences of genes and its regulation patterns in cellular processes and pathways. Finally, we will relate these gene expression differences to the structure of cationic polymers and also identify the responsive patterns of macrophages induced by cationic polymer gene delivery systems. The validity of microarray results will be confirmed by real-time PCR,Western blot, ELISA, mitochondria membrane potential, ROS, cell cycle analysis etc..
    Relation: 計畫編號:NSC97-2221-E041-005
    Appears in Collections:[Dept. of Pharmacy] MOST Project

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