Department of Pediatrics, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA.
Gene Ther. 2010 Sep;17(9):1098-104. doi: 10.1038/gt.2010.57. Epub 2010 Apr 29.
Our laboratory has previously demonstrated that cytoplasmic trafficking and subsequent nuclear entry of nonviral plasmid DNA can be significantly enhanced through the application of cyclic stretch after transfection in vitro. In this study, we show that cyclic stretching of the murine lung using ventilation immediately after endotracheal administration and transthoracic electroporation of plasmid DNA increases exogenous gene expression up to fourfold in mice that were not ventilated after plasmid administration and transfection by electroporation in vivo. This increase is both time and sequence specific (that is, the ventilation must occur immediately after the transfection event). The ventilation-enhanced gene transfer is also amplitude dependent, confirming similar studies completed in vitro, and is mediated, at least in part, through the cytoplasmic tubulin deacetylase, HDAC6. Using immunohistochemistry, we show that this increase in expression is due to an increase in the number of cells expressing the exogenous protein rather than an increase in the amount of protein produced per cell. These studies show the potential mechanical stimulation has in vivo in significantly increasing nonviral DNA gene expression, and may ultimately pave the way for more successful clinical trials using this type of therapy in the future.
我们的实验室之前已经证明，通过在体外转染后施加循环拉伸，可以显著增强非病毒质粒 DNA 的细胞质运输和随后的核内进入。在这项研究中，我们表明，在气管内给药后立即对小鼠进行循环拉伸，并对质粒 DNA 进行经胸电穿孔，可使未通气的小鼠的外源基因表达增加四倍，而体内电穿孔转染后则不会增加。这种增加是时间和序列特异性的（即通气必须在转染事件后立即发生）。通气增强的基因转移也与幅度有关，这与体外完成的类似研究一致，并且至少部分通过细胞质微管去乙酰化酶 HDAC6 介导。通过免疫组织化学，我们表明这种表达的增加是由于表达外源蛋白的细胞数量增加，而不是每个细胞产生的蛋白量增加。这些研究表明，机械刺激在体内具有显著增加非病毒 DNA 基因表达的潜力，并且可能最终为未来使用这种类型的治疗方法进行更成功的临床试验铺平道路。
