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Title: | 正價微脂體攜帶寡核苷酸複合物與穿膜促進劑在哺乳類細胞內之吸收與分佈分析 Analysis of Cellular Uptake and Distribution of Cationic Micelles Complexed with Oligonucleotides and Transmenbrane Enhancers in Mammalian Cells |
Authors: | Wei-Ju Chen 陳薇如 |
Advisor: | 張富雄(Fu-Hsiung Chang) |
Keyword: | 磁性奈米粒子,寡核苷,酸,細胞核遞送, magnetic nanoparticles,oligonucleotides,nuclear targeting, |
Publication Year : | 2009 |
Degree: | 碩士 |
Abstract: | 基因治療提供了治療疾病的前景,一般來說,利用奈米粒子來攜帶藥物或治療性的分子,例如: DNA、RNA、胜肽、蛋白質等進入標的細胞,對於核酸分子而言,有兩大標的位置-細胞質與細胞核;因此如何有效地將分子穿過細胞膜以到達每個標的位置是重要的課題。由於細胞膜是個動態的結構,傾向於親脂性的分子通過,而限制了親水性或帶電荷性的大分子物質的穿透,所以核酸類的物質比較難以自由進入到細胞內,為此,發展了以非病毒型的微脂體遞送系統,配合在奈米粒子的表面修飾物,提高進入細胞的效率;本篇研究利用微脂體-氧化鐵奈米粒子表面修飾生物素與鏈黴親和素融合SV40-Tag核位訊號 (NLS) 建構之蛋白質,階段式輔以磁力將奈米磁性粒子與螢光標定之寡核苷酸送入細胞質內後,並透過NLS與細胞內分子將奈米粒子送入細胞核內。
本論文提出利用磁力代替穿透膜胜肽的方法,觀察奈米粒子在癌細胞與老鼠骨髓間質幹細胞之細胞內分佈。實驗結果為1. 施予磁力後可有效增加細胞對奈米粒子的吸收;2. 自然處理細胞後其NLS-ST蛋白質本身並不能進入細胞質,唯有加入磁力後可顯著加強細胞質中之奈米粒子進入細胞核;3. 奈米粒子進入細胞後可能脫離溶體路徑,有助於標靶奈米粒子進入細胞核。 Gene therapy offers the promise of treating disease. In general, two target sites inside cells are cytoplasm or nucleus in case of delivering therapeutic molecules such as DNA, RNA, peptides and proteins by nanoparticles. There are complicated problems in targeting these molecules. It was difficult for the large charge molecules such as DNA to pass across plasma and nucleic membrane on their own. For these reasons, I developed a non-viral micelle delivery system using the surface-modified lipid nanoparticles. It was composed one positive charge lipid, called GEC-cholesterol with cholesterol, PEG lipid and biotinylated lipid. The surface-exposed biotin moiety could interact with the bifunctioned fusion protein containing streptavidin. Adding magnetic field sequentially could force the lipid nanoparticles complexed with oligonucleotides into the cytosol; meanwhile NLS associated with intracellular binding proteins then transported into the nucleus of cancer cells and rat bone marrow stem cells. The results indicated that manipulating the magnetic force could efficiently enhance nanoparrticles transported into cells. Besides, NLS-Streptavidin fusion proteins themselves couldn’t enter into the cytoplasm. But magnetic nanoparticles inside the lipid micelles were entered significantly into the nucleus with the assistant of magnetic fields. Thus, magnetic field might be benefit for targeting nanoparticles with cargos into nucleus. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/8840 |
Fulltext Rights: | 同意授權(全球公開) |
Appears in Collections: | 生物化學暨分子生物學科研究所 |
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