Mechanism of plasmid delivery by hydrodynamic tail vein injection.: II.: Morphological studies
Mechanism of plasmid delivery by hydrodynamic tail vein injection.: II.: Morphological studies
复制标题
DOI：
10.1002/jgm.920
复制
发表时间：
2006-07-01
影响因子：
3.5
通讯作者：
Wolff, Jon A.
中科院分区：
文献类型：
--
作者：
Budker, Vladimir G.;Subbotin, Vladimir M.;Wolff, Jon A.
背景通过流体动力尾静脉(HTV)技术将质粒DNA(PDNA)高效地输送到肝细胞，极大地普及了裸露核酸的使用。流体动力学过程使组织上的物理力增加，压力和剪切力增加，这可能导致暂时性或永久性的膜损伤。它还可以触发一系列细胞事件来密封或重组拉伸的膜。我们的目的是通过观察肝脏的形态变化来研究HTV的摄取机制，并将这些变化与注射的DNA的命运相关联。方法我们利用光镜(LM)和电子显微镜(EM)技术，在注射后的不同时间确定HTV程序对肝细胞和非实质细胞的影响。光镜研究使用石蜡包埋的肝脏，苏木精-伊红(H&E)染色。免疫-EM研究使用标记了亚纳米金颗粒的抗体，然后进行银增强，以确定注射的PDNA在亚细胞水平上的位置。根据丙氨酸氨基转移酶(ALT)的释放和清除来评估肝细胞的整体损伤程度。结果光镜和电镜结果均显示肝细胞内早在注射后5min就出现大的囊泡。囊泡数量减少20~60min。质粒DNA分子常常出现在这样的小泡中或与之相关。在Disse间隙、胞浆和胞核中也可检测到DNA。非实质细胞中也含有DNA，但未观察到HTV诱导的囊泡。结论裸露DNA除了通过膜孔直接进入肝细胞外，还可通过其他途径进入肝细胞。可能很难证明这些途径中的哪一条会导致基因表达，但仅凭膜孔假说似乎不足以解释为什么表达优先发生在肝细胞中。需要进一步的研究来描述每一条假定的通路的重要性以及它们在使能寡核苷酸(SiRNA)活性和PDNA表达方面的相互关系。版权所有(C)2006 John Wiley&Sons，Ltd.
Background The efficient delivery of plasmid DNA (pDNA) to hepatocytes by a hydrodynamic tail vein (HTV) procedure has greatly popularized the use of naked nucleic acids. The hydrodynamic process renders onto the tissue increased physical forces in terms of increased pressures and shear forces that could lead to transient or permanent membrane damage. It can also trigger a series of cellular events to seal or reorganize the stretched membrane. Our goal was to study the uptake mechanism by following the morphological changes in the liver and correlate these with the fate of the injected plasmid DNA.Methods We utilized both light microscopic (LM) and electron microscopic (EM) techniques to determine the effect of the HTV procedure on hepatocytes and non-parenchymal cells at various times after injection. The LM studies used paraffin-embedded livers with hematoxylin and eosin (H&E) staining. The immune-EM studies used antibodies labeled with sub-nanometer gold particles followed by silver enhancement to identify the location of injected pDNA at the subcellular level. The level of overall damage to liver cells was estimated based on alanine aminotransferase (ALT) release and clearance.Results Both the LM and EM results showed the appearance of large vesicles in hepatocytes as early as 5 min post-injection. The number of vesicles decreased by 20-60 min. Plasmid DNA molecules often appeared to be associated with or inside such vesicles. DNA could also be detected in the space of Disse, in the cytoplasm and in nuclei. Non-parenchymal cells also contained DNA, but HTV-induced vesicles could not be observed in them.Conclusions Our studies suggest an alternative or additional pathway for naked DNA into hepatocytes besides direct entry via membrane pores. It may be difficult to prove which of these pathways lead to gene expression, but the membrane pore hypothesis alone appears insufficient to explain why expression happens preferentially in hepatocytes. Further study is needed to delineate the importance of each of these putative pathways and their interrelationship in enabling oligonucleotide (siRNA) activity and pDNA expression. Copyright (c) 2006 John Wiley & Sons, Ltd.