Sebestyén Magdolna G, Budker Vladimir G, Budker Tatiana, Subbotin Vladimir M, Zhang Guofeng, Monahan Sean D, Lewis David L, Wong So C, Hagstrom James E, Wolff Jon A
Mirus Bio Corporation, 505 S. Rosa Rd., Madison, WI 53719, USA.
J Gene Med. 2006 Jul;8(7):852-73. doi: 10.1002/jgm.921.
The hydrodynamic tail vein (HTV) injection of naked plasmid DNA is a simple yet effective in vivo gene delivery method into hepatocytes. It is increasingly being used as a research tool to elucidate mechanisms of gene expression and the role of genes and their cognate proteins in the pathogenesis of disease in animal models. A greater understanding of its mechanism will aid these efforts and has relevance to macromolecular and nucleic acid delivery in general.
In an attempt to explore how naked DNA enters hepatocytes the fate of a variety of molecules and particles was followed over a 24-h time frame using fluorescence microscopy. The uptake of some of these compounds was correlated with marker gene expression from a co-injected plasmid DNA. In addition, the uptake of the injected compounds was correlated with the histologic appearance of hepatocytes.
Out of the large number of nucleic acids, peptides, proteins, inert polymers and small molecules that we tested, most were efficiently delivered into hepatocytes independently of their size and charge. Even T7 phage and highly charged DNA/protein complexes of 60-100 nm in size were able to enter the cytoplasm. In animals co-injected with an enhanced yellow fluorescent protein (EYFP) expression vector and fluorescently labeled immunoglobulin (IgG), hepatocytes flooded with large amounts of IgG appeared permanently damaged and did not express EYFP-Nuc. Hepatocytes expressing EYFP had only slight IgG uptake. In contrast, when an EYFP expression vector was co-injected with a fluorescently labeled 200-bp linear DNA fragment, both were mostly (in 91% of the observed cells) co-localized to the same hepatocytes 24 h later.
The appearance of permanently damaged cells with increased uptake of some molecules such as endogenous IgG raised the possibility that a molecule could be present in a hepatocyte but its transport would not be indicative of the transport process that can lead to foreign gene expression. The HTV procedure enables the uptake of a variety of molecules (as previous studies also found), but the uptake process for some of these molecules may be associated with a more disruptive process to the hepatocytes that is not compatible with successful gene delivery.
裸质粒DNA经流体动力学尾静脉（HTV）注射是一种简单而有效的向肝细胞进行体内基因递送的方法。它越来越多地被用作一种研究工具，以阐明基因表达机制以及基因及其同源蛋白在动物模型疾病发病机制中的作用。对其机制有更深入的了解将有助于这些研究工作，并且总体上与大分子和核酸递送相关。
为了探究裸DNA如何进入肝细胞，使用荧光显微镜在24小时的时间范围内追踪了多种分子和颗粒的命运。其中一些化合物的摄取与共注射质粒DNA的标记基因表达相关。此外，注射化合物的摄取与肝细胞的组织学外观相关。
在我们测试的大量核酸、肽、蛋白质、惰性聚合物和小分子中，大多数都能有效地进入肝细胞，而与它们的大小和电荷无关。甚至T7噬菌体和大小为60 - 100纳米的高电荷DNA/蛋白质复合物也能够进入细胞质。在与增强型黄色荧光蛋白（EYFP）表达载体和荧光标记免疫球蛋白（IgG）共注射的动物中，充满大量IgG的肝细胞似乎受到永久性损伤，并且不表达EYFP-Nuc。表达EYFP的肝细胞仅摄取少量IgG。相比之下，当EYFP表达载体与荧光标记的200碱基对线性DNA片段共注射时，24小时后两者大多（在91%观察到的细胞中）共定位于同一肝细胞。
一些分子（如内源性IgG）摄取增加的永久性损伤细胞的出现，增加了一种可能性，即一个分子可能存在于肝细胞中，但其转运并不表明能导致外源基因表达的转运过程。HTV方法能够使多种分子被摄取（如先前研究也发现），但其中一些分子的摄取过程可能与对肝细胞更具破坏性的过程相关，而这与成功的基因递送不兼容。
