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首页> 外文期刊>Translational Stroke Research >Transgene Delivery to Endothelial Cultures Derived from Porcine Carotid Artery Ex Vivo
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Transgene Delivery to Endothelial Cultures Derived from Porcine Carotid Artery Ex Vivo

机译：转基因递送至源自猪颈动脉的内皮培养物

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摘要

Carotid artery disease is a widespread cause of morbidity and mortality. Porcine models of vascular disease are well established in vivo, but existing endothelial systems in vitro (e.g. human umbilical vein endothelial cells, rat aortic endothelial cultures) poorly reflect carotid endothelium. A reliable in vitro assay would improve design of in vivo experiments and allow reduction and refinement of animal use. This study aimed (1) to develop ex vivo endothelial cultures from porcine carotid and (2) to test whether these were suitable for lentivector-mediated transgene delivery. Surplus carotid arteries were harvested from young adult female Large White pigs within 10 min post-mortem. Small sectors of carotid artery wall (approximately 4 mm × 4 mm squares) were immobilised in a stable gel matrix. Cultures were exposed to HIV-derived lentivector (LV) encoding a reporter transgene or the equivalent integration-deficient vector (IDLV). After 7–14 days in vitro, cultures were fixed and labelled histochemically. Thread-like multicellular outgrowths were observed that were positive for endothelial cell markers (CD31, VEGFR2, von Willebrand factor). A minority of cells co-labelled for smooth muscle markers. Sensitivity to cytotoxic agents (paclitaxel, cycloheximide, staurosporine) was comparable to that in cell cultures, indicating that the gel matrix permits diffusive access of small pharmacological molecules. Transgene-expressing cells were more abundant following exposure to LV than IDLV (4.7, 0.1 % of cells, respectively). In conclusion, ex vivo adult porcine carotid artery produced endothelial cell outgrowths that were effectively transduced by LV. This system will facilitate translation of novel therapies to clinical trials, with reduction and refinement of in vivo experiments.
机译：颈动脉疾病是发病和死亡的广泛原因。在体内已经很好地建立了血管疾病的猪模型，但是体外现有的内皮系统（例如人脐静脉内皮细胞，大鼠主动脉内皮培养物）很难反映出颈动脉内皮。可靠的体外测定将改善体内实验的设计，并减少和改善动物的使用。这项研究的目的是（1）从猪的颈动脉开发离体内皮培养物，以及（2）测试它们是否适合慢载体介导的转基因递送。死后10分钟内从成年雌性大白猪中收获多余的颈动脉。将颈动脉壁的小扇区（约4mm××4mm的正方形）固定在稳定的凝胶基质中。将培养物暴露于编码报告基因转基因的HIV衍生慢病毒载体（LV）或等效的整合缺陷载体（IDLV）。体外培养7-14天后，将培养物固定并进行组织化学标记。观察到线样多细胞产物对内皮细胞标志物（CD31，VEGFR2，von Willebrand因子）呈阳性。少数细胞被共同标记为平滑肌标记。对细胞毒性剂（紫杉醇，环己酰亚胺，星形孢菌素）的敏感性与细胞培养物中的敏感性相当，这表明凝胶基质允许小药理学分子的扩散途径。转基因表达细胞暴露于LV后比IDLV更为丰富（分别为4.7％，0.1％细胞）。总之，离体成年猪颈动脉产生的内皮细胞增生物可被LV有效地转导。该系统将有助于减少和完善体内实验，从而将新疗法转化为临床试验。

著录项

  • 来源
    《Translational Stroke Research》 |2013年第5期|507-514|共8页
  • 作者

    J. Andoh; B. Sawyer; K. Szewczyk; M. Nortley; T. Rossetti; I. M. Loftus; R. J. Yáñez-Muñoz; A. H. Hainsworth;

  • 作者单位

    Stroke and Dementia Research Centre Division of Clinical Sciences St Georges University of London">(1);

    Stroke and Dementia Research Centre Division of Clinical Sciences St Georges University of London">(1);

    Microscopy Imaging Unit Division of Biomedical Sciences St George’s University of London">(2);

    Renal Transplantation Unit St George’s Healthcare NHS Trust">(3);

    Oxford Transplant Centre Churchill Hospital">(6);

    School of Biological Sciences Royal Holloway University of London">(4);

    St George’s Vascular Institute St George’s Hospital">(5);

    School of Biological Sciences Royal Holloway University of London">(4);

    Stroke and Dementia Research Centre Division of Clinical Sciences St Georges University of London">(1);

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    Endothelial cells; Carotid artery; Lentiviral vector; Porcine model;

    机译：内皮细胞颈动脉;慢病毒载体猪模型;

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