Haraguchi Ayana, Fuchigami Yuki, Kawaguchi Maho, Fumoto Shintaro, Ohyama Kaname, Shimizu Kazunori, Hagimori Masayori, Kawakami Shigeru
Graduate School of Biomedical Sciences, Nagasaki University.
Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University.
Biol Pharm Bull. 2018;41(6):944-950. doi: 10.1248/bpb.b18-00094.
We developed a tissue suction-mediated transfection method (suction method) as a relatively reliable and less invasive technique for in vivo transfection. In this study, we determined hepatic transgene expression characteristics in the mouse liver, using a suction device, collecting information relevant to gene therapy and gene functional analysis by the liver suction method. To achieve high transgene expression levels, we developed a suction device with four holes (multiple hole device) and applied it to the larger portion of the left lateral lobe of the mouse liver. Hepatic transfection with physical stimuli was potentially controlled by activator protein-1 (AP-1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). We examined the spatial distribution of transgene expression in the suctioned lobe by 2-dimensional imaging with histochemical staining and 3-dimensional multicolor deep imaging with tissue clearing methods. Through monitoring spatial distribution of transgene expression, the liver suction method was used to efficiently transfect extravascular hepatocytes in the suction-deformable upper lobe of the liver. Moreover, long-term transgene expression, at least 14 d, was achieved with the liver suction method when cytosine-phosphate-guanine (CpG)-free plasmid DNA was applied.
我们开发了一种组织抽吸介导的转染方法（抽吸法），作为一种相对可靠且侵入性较小的体内转染技术。在本研究中，我们使用抽吸装置确定了小鼠肝脏中的肝转基因表达特征，通过肝脏抽吸法收集与基因治疗和基因功能分析相关的信息。为了实现高转基因表达水平，我们开发了一种具有四个孔的抽吸装置（多孔装置），并将其应用于小鼠肝脏左外侧叶的较大部分。物理刺激介导的肝转染可能受激活蛋白-1（AP-1）和活化B细胞核因子κB（NF-κB）调控。我们通过组织化学染色的二维成像和组织透明法的三维多色深度成像检查了抽吸叶中转基因表达的空间分布。通过监测转基因表达的空间分布，肝脏抽吸法被用于有效地转染肝脏抽吸可变形上叶中的血管外肝细胞。此外，当应用无胞嘧啶-磷酸-鸟嘌呤（CpG）的质粒DNA时，肝脏抽吸法可实现至少14天的长期转基因表达。
