探討高分子複合基材對間葉幹細胞基因遞送的影響與其在心肌修復之應用

The effect of polymer composite substrate on gene delivery into mesenchymal stem cells and its application in myocardial repair

黃念齊(Nien-Chi Huang)

指導教授 ： 徐善慧

國立臺灣大學/工學院/高分子科學與工程學研究所/博士(2020年)

https://doi.org/10.6342/NTU202002047

摘要

人類心肌細胞雖然具有再生與更新能力，但更新率卻非常低，而心肌梗塞或衰老所引起之心肌細胞流失，其數量遠大於再生的心肌細胞數量。受損或流失的心肌細胞會由收縮性差的纖維化疤痕組織所取代，導致心肌收縮功能永久性喪失。目前臨床的治療方式有很多種，例如：心臟移植、心肌形成術、藥物治療、幹細胞治療……等，卻無法有效且顯著改善患者的症狀和生活質量。近代醫學的快速發展，除了現行的療法外，基因治療為未來之趨勢。但由於體內基因遞送缺乏高效率且安全的遞送方式，以致於在臨床應用中，僅使用於治療危及性命的惡性疾病。因此本論文提出使用一系列高分子複合基材作為基因轉殖平台，在不添加基因轉殖劑的情況下，提高裸質體傳遞進入幹細胞的效率，並探討幹細胞作為基因載體用於心肌修復上的可能性。論文將分為四個部分：第一部分為使用濕式蝕刻方法大量生產二氧化矽直立奈米片，並嘗試將裸質體遞送進入幹細胞中；二氧化矽直立奈米片可有效地將裸質體DNA遞送進入幹細胞中，其轉殖效果與使用基因轉殖劑之組別相近，卻無細胞毒性，推測原因為奈米片於12小時內活化幹細胞integrin-FAK-Rho訊息傳遞路徑，並影響細胞骨架重組進一步改變幹細胞的基因遞送效率。除此之外，遞送GATA4基因至幹細胞中，可以上調另外兩個重要的心臟特徵基因：MEF2C和TBX5，且經轉殖的幹細胞培養七天後便會表達心臟特徵蛋白質。此結果證實，二氧化矽奈米片可以藉由奈米片與幹細胞間的交互作用，有效且快速的將裸質體遞送進入幹細胞中。第二部分為結合無機與有機物質製作具有不同幾何參數及化學性質之奈米片網絡，藉以探討奈米片網絡物化性質對於幹細胞行為的影響。通過於蝕刻液中添加不同的化學物質以調節奈米片網絡的幾何形狀以及物理化學性質，我們發現在各種表面特性中，奈米片的高寬比與基因轉殖效率具高度相關性，且細胞移動速率與細胞表面integrin β3基因在轉殖效率佳的組別中被高度活化。此研究顯示，轉殖效率與幹細胞於奈米片表面之移動速率和integrin活化的程度三者間具緊密關聯性。第三部分為使用水性生物可降解帶負電荷之聚胺酯製備具有不同表面特徵的轉殖平台，以了解基材表面型態對於細胞行為與基因轉殖效率的影響。幹細胞於聚胺酯薄膜表面會自動聚集形成部分球體部分貼附的形態，並促進基因轉殖效率，進一步在薄膜表面引入微溝槽特徵，幹細胞轉為貼附於溝槽並沿著溝槽移動，基因遞送效率大幅增加。同時發現，不同基材表面影響幹細胞integrin β1、α5的活化程度，而移動速率也受到表面形態影響。此結果證實，不同基材表面會改變細胞型態、調控細胞表面之integrin活化程度並影響細胞移動與轉殖效率，而遞送效率與各種基材上的細胞移動速率呈正相關，故可以藉由細胞移動速率推測基材介導的基因轉殖效率。第四部分則是為了增加幹細胞介導的基因遞送技術於臨床之應用性，使用帶負電荷的水性生物可降解溫敏性聚胺酯水膠包覆GATA4質體與幹細胞，透過微擠壓瞬時轉殖系統進行原位基因遞送，發現經轉殖的幹細胞於水膠內具有良好的增殖能力，同時也促進其他心臟特徵基因及蛋白質表現，進一步分化為類心肌細胞。而後將包覆GATA4質體及幹細胞之聚胺酯水膠注射至斑馬魚心臟受損處，可於30天內修復斑馬魚心臟功能。本論文透過四個部分探討高分子複合材料製備之基因轉殖平台的對於裸質體DNA遞送進入幹細胞效率的影響，而後利用聚胺酯水膠包覆細胞與質體增加細胞介導之原位基因遞送效率，皆證實在心肌修復上具有正面的效果，這對於組織工程、再生基因治療應用中將具有很大的潛力。

關鍵字

裸質體轉殖 ； 細胞重新編程 ； 基材介導之基因轉殖 ； 細胞介導之基因轉殖 ； 心肌修復

並列摘要

Although human cardiomyocytes are capable of regeneration and renewal, they present a markedly low renewal rate. The number of cardiomyocytes lost due to myocardial infarction or aging greatly exceeding the regenerated cardiomyocytes. Moreover, damaged or lost cardiomyocytes are replaced by fibrotic scar tissue, further leading to loss of myocardial contractility function. Currently, there are various clinical treatments available, including heart transfection, cardiomyoplasty, drug therapies, and stem cell therapies. However, these therapies fail to effectively and significantly improve the symptoms and quality of life in patients. With the rapid development of modern medicine, in addition to current therapies, gene therapies will be the future direction. However, owing to the lack of efficient and safe delivery approaches for in vivo gene delivery, gene therapies are only used in treating life-threatening malignant diseases in clinical practice. Herein, this study focused on a series of gene transfection platforms derived from polymer composite substrates to improve the efficiency of naked plasmid delivery into mesenchymal stem cells (MSCs) without adding gene transfection agents and to investigate the possibility of using MSCs as gene carriers for myocardial repair. In the first section, the fabrication of silica upright nanosheets in large quantities using the wet etching method, and their use in MSC gene delivery experiments. Silica upright nanosheets can effectively deliver naked plasmid DNA into stem MSCs. Their transfection effect was comparable with that of a group using gene transfection agents, with no cytotoxicity. This can be attributed to upright nanosheets activating the integrin-FAK-Rho signal transduction pathway present in MSCs within 12 hours and influencing cytoskeleton reorganization, which further altered the gene delivery efficiency of MSCs. Furthermore, the delivery of the GATA4 gene into MSCs upregulated another two important characteristic cardiac genes, MEF2C and TBX5, with transfected MSCs expressing specific cardiac proteins after 7-day culture. This result confirmed that silica upright nanosheets can effectively and rapidly deliver naked plasmids into MSCs by utilizing the interaction between nanosheets and MSCs. In the second section, inorganic/organic hybrid nanosheet with different geometric parameters and chemistry were used to investigate the impact of nanosheet networks on cell behavior. Following the addition of different chemicals to the etching solution to adjust the physicochemical properties of nanosheet networks, it was observed that among various surface properties, the aspect ratio of nanosheets highly correlated with gene transfection efficiency. Additionally, both the cell migration rate and integrin β3 gene expression were markedly activated in groups presenting good transfection efficiency. This study revealed the existence of a high correlation between transfection efficiency, the migration rate of MSCs on the nanosheets, and the level of integrin activation. In the third section, we used water-based biodegradable negatively charged polyurethane with various geometric features to elucidate the transfection efficiency of naked plasmid into MSCs and the cell behavior. On polyurethane film, a part of cells formed spheroids and the other cells were attached with spread morphology, which promoting the efficiency of gene transfection. Furthermore, on microgroove, cells fell into the bottom of the grooves and moved along the microgrooves, which greatly increasing the gene delivery efficiency. Simultaneously, materials with different surfaces influenced the activation levels of integrins β1 and α5 on MSCs, thus influencing cell migration rates. This result confirmed that materials with different surfaces can affect cell morphologies, regulate integrin activation levels, and influence cell migration and transfection efficiency. The delivery efficiency positively correlated with the cell migration rates on various substrates. Therefore, the substrate-mediated gene transfection efficiency of MSCs can be estimated based on the cell migration rate. In the last section, to broaden the clinical applications of MSC-mediated gene delivery technology, both MSCs and GATA4 plasmids were encapsulated into a biomimetic and negatively charged water-based biodegradable thermo-responsive polyurethane hydrogel, and in situ gene delivery was performed using a microextrusion-based transient-transfection system. We observed that the GATA4-transfected MSCs demonstrated good proliferation ability in the hydrogel and further differentiation into cardiomyocyte-like cells. Then, GATA4 plasmids and MSCs encapsulating polyurethane hydrogel were injected into the defect sites on the zebrafish heart, restoring the cardiac function of zebrafish within 30 days. Through the above findings, a series of gene transfection platforms derived from polymer composite substrates to improve the efficiency of naked plasmid DNA delivery into MSCs. MSCs and plasmids were encapsulated into polyurethane hydrogel to improve the efficiency of cell-mediated in situ gene delivery, revealing positive effects on the myocardial repair. This has great potential for application in tissue engineering and regenerative gene therapies.

並列關鍵字

naked plasmid delivery ； cell reprogramming ； substrate-mediated gene delivery ； cell-mediated gene delivery ； cardiac repair

參考文獻

第一章

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