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首页> 外文学位 >Mechanistic studies on the uptake and intracellular trafficking of DNA complexes in primary cells using lipid-modified cationic polymers as non-viral gene carrier.
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Mechanistic studies on the uptake and intracellular trafficking of DNA complexes in primary cells using lipid-modified cationic polymers as non-viral gene carrier.

机译：使用脂质修饰的阳离子聚合物作为非病毒基因载体的原代细胞中DNA复合物的吸收和细胞内运输的机理研究。

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摘要

This thesis work is aimed at addressing some of the fundamental questions surrounding the intracellular fate of plasmid DNA in clinically-relevant primary cells, when delivered with lipid-modified cationic polymers as gene carriers. We developed an optimized procedure for the transfection of primary cells, which included modifications designed to maximize the in vitro stability of the DNA-carrier complexes and enhance their transfection utility. These polymeric gene carriers bind to DNA through electrostatic interaction, which drives a self-assembly process that condenses DNA into sub-micron particles suitable for cellular uptake. This interaction is not DNA sequence or structurally specific, and thus able to package DNA molecules with different topologies and molecular weights with equal efficiency for uptake. However, circularized DNA performed better than its linearized equivalent in transfection, suggesting intracellular processing may be dependent on the physicochemical properties of the assembled complexes. Thus, we concentrated our effort on understanding the intracellular events leading to transfection. Using a linoleic acid substituted cationic polymer, we found that transfection efficiency is correlated with the amount of pDNA associated with the nucleus and that lipid-moieties is able to facilitate nuclear association of DNA to enhance transfection. Further, lipid modification altered the uptake pathways of the complexes from ones that are predominantly driven by macropinocytosis to ones that are mediated by clathrin. Endosome escape continues to be an inefficient process with these polymeric gene carriers, suggesting methods to promote cytosolic release may be the most effective approach to enhance their transfection efficiencies.
机译：本论文的目的是解决与脂质修饰的阳离子聚合物作为基因载体一起递送时，与临床相关的原代细胞中质粒DNA的细胞内命运有关的一些基本问题。我们开发了用于原代细胞转染的优化程序，其中包括旨在最大程度提高DNA-载体复合物的体外稳定性并增强其转染效用的修饰。这些聚合的基因载体通过静电相互作用与DNA结合，从而驱动自组装过程，该过程将DNA浓缩成适合细胞摄取的亚微米颗粒。这种相互作用不是DNA序列或结构特异性，因此能够以相同的吸收效率包装具有不同拓扑结构和分子量的DNA分子。但是，环化的DNA在转染中的表现要好于其线性化的等价物，表明细胞内加工可能取决于组装的复合物的理化性质。因此，我们集中精力了解导致转染的细胞内事件。使用亚油酸取代的阳离子聚合物，我们发现转染效率与与细胞核相关的pDNA量相关，并且脂质部分能够促进DNA的核缔合以增强转染。此外，脂质修饰将复合物的摄取途径从主要由巨胞饮作用驱动的复合物改变为由网格蛋白介导的复合物的摄取途径。这些聚合物基因载体的内体逃逸仍然是一个低效率的过程，这表明促进细胞溶质释放的方法可能是提高其转染效率的最有效方法。

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  • 作者

    Hsu, Charlie Yu Ming.;

  • 作者单位

    University of Alberta (Canada).;

  • 授予单位 University of Alberta (Canada).;
  • 学科 Engineering Biomedical.
  • 学位 Ph.D.
  • 年度 2012
  • 页码 277 p.
  • 总页数 277
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 老年病学;
  • 关键词

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