Gene transfer to the alveolar epithelium: effects of stretch

暴露在机械通风环境中会导致肺泡上皮细胞发生深刻变化。我们建议开发非病毒基因治疗方法来治疗急性肺损伤。因此，我们必须了解肺泡上皮细胞(AEC)在模拟肺损伤及其处理(即，换气)时的基因转移机制。机械拉伸可诱导血管内皮细胞发生多种生物学反应，包括细胞骨架的改变、细胞信号通路的激活和转录因子的上调。这些反应与基因传递过程直接相关。外源DNA必须穿过质膜，穿过细胞质，进入细胞核，并得到表达，才能使基因治疗取得成功。在原代大鼠肺泡II型细胞、人或小鼠AEC细胞系中，暴露于等轴拉伸下的基因传递和表达效率是在静态条件下生长的细胞的10倍。我们假设细胞骨架 机械拉伸诱导的重组和转录因子激活刺激外源DNA通过细胞质进入细胞核进行基因表达的能力。尽管这不是一个生理上“正常”的过程，但对于科学家日常使用的方法和形成基因治疗的基础来说，质粒与宿主细胞的相互作用是至关重要的。本方案中的实验将阐明拉伸增强基因的机制。 在肺泡上皮细胞内交付。我们还将确定一种新开发的非病毒基因转移到肺的电穿孔方法的有效性，这种方法产生高水平的表达，而没有任何与腺病毒介导的基因治疗相关的炎症反应或副作用。这些研究的具体目的是：(1)确定牵拉引起的细胞骨架组织的改变是否改变AEC中质粒DNA的胞浆迁移率；(2)确定周期性拉伸是否激活转录因子并导致AEC中DNA核输入增加；(3)确定机械通气是否增加电穿孔介导的基因转移到在体小鼠和大鼠肺泡上皮细胞。

Exposure to mechanical ventilation can cause profound changes in the alveolar epithelium. We propose to develop non-viral gene therapy approaches to treat the acutely injured lung. As such, we must understand the mechanisms of gene transfer in alveolar epithelial cells (AEC) under conditions that mimic those found during lung injury and its management (i.e., ventilation). Mechanical stretch induces numerous biological responses in AEC, including alterations in the cytoskeleton, activation of cell signaling pathways, and upregulation of transcription factors. These responses are directly related to the process of gene delivery. Exogenous DNA must cross the plasma membrane, travel through the cytoplasm, enter the nucleus, and be expressed in order for gene therapy to be successful. Gene delivery and expression in primary rat alveolar type II ceils, human, or mouse AEC cell lines exposed to equibiaxial stretch is 10-fold more efficient than in cells grown under static conditions. We hypothesize that the cytoskeletal reorganization and transcription factor activation induced by mechanical stretch stimulates the ability of exogenous DNA to travel through the cytoplasm and into the nucleus for gene expression. Although not a physiologically "normal" process, the interactions of plasmids with the host cell are vital to methods scientists use everyday and form the basis of gene therapy. The experiments in this proposal will elucidate the mechanisms of stretch-enhanced gene delivery in the alveolar epithelium. We also will establish the efficacy of a newly developed electroporation method for non-viral gene transfer to the lung that yields high level expression without any of the inflammatory response or side effects associated with adenoviral mediated gene therapy. The specific aims are (1) To determine whether stretch-induced changes in cytoskeletal organization alter the cytoplasmic mobility of plasmid DNA in AEC; (2) To determine whether cyclic stretch activates Transcription Factors and leads to increased DNA nuclear import in AEC; and (3) To determine whether mechanical ventilation increases electroporation-mediated gene transfer to the alveolar epithelium in the mouse and rat lung in vivo.