PDF(Pubmed)
Abstract：
Hypoxia-Inducible Factor-1α (HIF-1α) has presented a new direction for ischemic preconditioning of surgical flaps to promote their survival. In a previous study, we demonstrated the effectiveness of HIF-1a DNA plasmids in this application. In this study, to avoid complications associated with plasmid use, we sought to express HIF-1α through mRNA transfection and determine its biological activity by measuring the upregulation of downstream angiogenic genes. We transfected six different HIF-1a mRNAs-one predominant, three variant, and two novel mutant isoforms-into primary human dermal fibroblasts using Lipofectamine, and assessed mRNA levels using RT-qPCR. At all time points examined after transfection (3, 6, and 10 h), the levels of HIF-1α transcript were significantly higher in all HIF-1α transfected cells relative to the control (all p < 0.05, unpaired Student\'s T-test). Importantly, the expression of HIF-1α transcription response genes (VEGF, ANG-1, PGF, FLT1, and EDN1) was significantly higher in the cells transfected with all isoforms than with the control at six and/or ten hours post-transfection. All isoforms were transfected successfully into human fibroblast cells, resulting in the rapid upregulation of all five downstream angiogenic targets tested. These findings support the potential use of HIF-1α mRNA for protecting ischemic dermal flaps.
摘要：
缺氧诱导因子-1α(HIF-1α)为外科皮瓣缺血预处理促进其存活提供了新的方向。在之前的研究中,我们证明了HIF-1aDNA质粒在此应用中的有效性。在这项研究中,为了避免与质粒使用相关的并发症，我们试图通过mRNA转染表达HIF-1α，并通过测量下游血管生成基因的上调来确定其生物学活性。我们转染了六种不同的HIF-1amRNA-一种主要的，三个变体，和两个新的突变同工型-使用脂质体进入原代人真皮成纤维细胞，并使用RT-qPCR评估mRNA水平。在转染后检查的所有时间点（3、6和10小时），HIF-1α转录物的水平在所有HIF-1α转染的细胞中相对于对照显著更高(所有p<0.05,未配对的学生T检验)。重要的是,HIF-1α转录反应基因的表达(VEGF，ANG-1,PGF,在转染后6和/或10小时，用所有同种型转染的细胞中的FLT1和EDN1）明显高于对照。所有同种型均成功转染人成纤维细胞，导致所测试的所有五个下游血管生成靶标的快速上调。这些发现支持HIF-1αmRNA用于保护缺血性皮瓣的潜在用途。
