中孔洞奈米矽材於轉染基因上的應用

Non-Viral Gene Delivery by Mesoporous Silica Nanoparticles

10.6342/NTU201800011

張仁瑄

中孔洞奈米矽材 ； 基因轉染 ； 誘導型多功能幹細胞 ； 多巴胺神經 ； 睡美人轉位子 ； 門冬?胺? ； 肺腺癌細胞 ； mesoporous silica nanoparticles ； gene delivery ； induced pluripotent stem cells ； dopaminergic neurons ； Sleeping beauty transposon ； asparaginase ； human adenocarcinoma cells

國立臺灣大學化學系學位論文

2018年

博士

牟中原

繁體中文

基因轉染，指的是將外源基因插入特定細胞，藉由基因重組的方式讓細胞能夠朝著特定方向發展，或是能夠應用至基因治療。神經再生是被廣泛討論的議題，緣由是神經細胞一但受損，其自癒能力差，容易造成永久性傷害，如阿茲海默症、帕金森氏症等。因此，近幾年來研究學者於再生醫學的領域不斷在探討神經分化的相關研究，並且引入基因治療。提及基因治療，過去較廣泛使用病毒載體來進行基因轉染，進而讓細胞能表達特定基因，也進一步研究此特定基因後續影響的機制。也有不少研究，透過轉染基因至幹細胞中，促進幹細胞分化成特定功能性細胞。然而，病毒載體轉染基因雖已商業化，但仍有潛在風險，例如：基因隨機插入細胞染色體而造成基因突變、細胞癌化等，因此後來研究學者致力於開發非病毒載體，希望能將非病毒載體提升轉染基因的效率，進一步廣泛應用於臨床實驗。本論文中共分三個主題，第一、二部分則是與神經分化相關的研究。然而短暫 (transient) 表現的質粒 (plasmid) 攜帶基因進入細胞中，其效果有限，甚至必須做連續轉染 (serial transfection) 來增強基因轉殖效率。因此在本論文中第三部分我們引入轉位子 (transposon) 質粒，透過中孔洞奈米矽材將基因送入細胞，達到永久表現、甚至將細胞做成新的細胞克隆 (cell clones)。我們將此技術應用在肺癌細胞，希望能夠讓門冬酰胺酶 (asparaginase) 基因於癌細胞中表現、進一步能夠造成癌細胞凋亡，作為飢餓治療。 以下是本論文的三個主題： (1) 透過中孔洞奈米矽材作為非病毒基因載體，轉染中樞神經重要基因 Nurr1 (Nuclear receptor related-1) 於誘導性多能性幹細胞 (induced pluripotent stem cells, 簡稱為 iPSCs)，促進 iPSCs分化為多巴胺功能性神經細胞 (dopaminergic neurons) (2) 透過中孔洞奈米矽材作為非病毒基因載體，同時轉染三種不同關鍵基因 Ascl1 (Achaete-scute homolog 1), Brn2 (POU domain transcription factor) 及 Myt1l (Myelin Transcription Factor 1 Like)，以及攜帶一促進神經分化小分子 (Isoxazole 9, ISX-9) 將老鼠體細胞直接分化為多巴胺神經細胞 (3) 結合睡美人轉位子 (Sleeping Beauty transposon) 及中孔洞奈米矽材作為非病毒基因載體，應用於肺癌細胞的飢餓治療 以上三個主題分別於本論文中的第三、四及第五章。本論文第一章中分成兩部分：第一部分介紹病毒及非病毒載體作為基因療法的作用機制及相關背景，第二部分則概述中孔洞奈米矽材的歷史及合成方法。論文中第二章則分享實驗方法，包含中孔洞奈米矽材的合成鑑定、細胞實驗以及後續分析方法。最後一章則總結所有工作，以及分享本論文的成果對於未來能夠持續發展的研究。

Gene delivery is a method of introducing the foreign genomic materials into the specific cells to carry out the gene therapy. Neural regeneration is an essential issue, because the neurons are difficult to repair after injury. The nonviral-based vehicles were needed for gene delivery, to avoid the integration effect of the chromosome, gene mutagenesis and immune issues associated with the viral vehicles. In my first research topic, mesoporous silica nanoparticles (MSNs) were used in the DNA delivery and co-delivery with siRNA for differentiation of dopaminergic neurons from induced pluripotent stem cells (iPSCs). In the second research topic, the MSNs were utilized to deliver three different genes and a small molecule for direct conversion of dopaminergic neurons from mouse fibroblasts. The results showed that MSNs as an efficient, minimally cytotoxicity and effective non-viral carriers for gene delivery, and MSNs had great potential in cell-oriented therapeutic applications. In the third research topic, we first combine the Sleeping Beauty transposon plasmid and MSNs for the delivery of asparaginase (ASN) gene into the cancer cells to prolong the ASN gene expression in the lung cancer cells. In addition, the stable cell clones could be successfully generated by the treatment of puromycin, and the ASN was expressed in the cells after doxycycline was added. There are six chapters in this thesis: Chapter 1: to give an introduction of gene therapy and the background of non-viral based nanoparticles, including mesoporous silica nanoparticles Chapter 2: the experimental section of nanoparticles synthesis, in vitro studies of plasmid release and cell-associated experiments Chapter 3: dual delivery of siRNA and plasmid DNA using mesoporous silica nanoparticles to differentiate induced pluripotent stem cells into dopaminergic neurons Chapter 4: generation of functional dopaminergic neurons from reprogramming fibroblasts by nonviral-based mesoporous silica nanoparticles Chapter 5: Sleeping Beauty transposon-mediated asparaginase gene delivery by a nanoparticle platform Chapter 6: summary and the perspective of my studies.

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