No currently available treatment is able to generate new contractile tissue or significantly improve cardiac function after myocardial infarction (MI), a leading cause of morbidity and mortality worldwide. Although gene transfer-enhanced endothelial progenitor cells (GTE-EPCs) show effectiveness in MI treatment in small animal models, no clinical trials using GTE-EPCs have been documented. Before the introduction of GTE-EPCs into human trials, gene-transfer-mediated augmentation of EPC function in animal models that reflect the human MI scenario should be tested. In this regard, a porcine model is the best choice since pigs have cardiac size, hemodynamics and coronary anatomy similar to that of humans. To examine GTE-EPC therapeutic efficacy in pig MI models, an efficient method for gene transfer into pig EPCs is required, which however, has been poorly documented. Pig bone marrow mononuclear cells were isolated and cultured in EGM-2 medium to obtain bone marrow-derived EPCs (BM-EPCs) that were characterized by immunostaining and the tube formation assay. Gene transfer was optimized in 6-well plates using a GFP and a VEGF plasmid, and scaled up in T75 flasks. Gene transfer efficiency was determined by fluorescence microscopy and flow cytometry. VEGF levels were measured by ELISA. Cell proliferation was assayed by the CCK-8 kit. (1) BM-EPCs expressed VEGFR2 and eNOS but not CD45 protein, and formed tube structures on Matrigel; (2) several chemical compounds were explored with the highest transfection efficiency of 41.4% ± 5.8% achieved using Lipofectamine 3000; (3) the VEGF level in culture medium after VEGF transfection was 378 ± 48 ng/10⁶ cells; and (4) BM-EPCs overexpressing VEGF had significantly enhanced proliferation than GFP-transfected EPCs. A simple, easy and cheap method that can be applied to produce a large number of genetically-modified BM-EPCs was established, which will facilitate the study of GTE-EPC therapeutic efficacy in pig MI model.

目前没有可用的治疗方法能够在心肌梗塞（MI）后产生新的收缩组织或显着改善心脏功能，心肌梗塞是全世界发病和死亡的主要原因。尽管基因转移增强内皮祖细胞 (GTE-EPC) 在小动物模型中显示出对 MI 治疗的有效性，但尚未记录使用 GTE-EPC 的临床试验。在将 GTE-EPC 引入人体试验之前，应在反映人类 MI 情况的动物模型中测试基因转移介导的 EPC 功能增强。在这方面，猪模型是最好的选择，因为猪的心脏大小、血流动力学和冠状动脉解剖结构与人类相似。为了检查 GTE-EPC 在猪 MI 模型中的治疗效果，需要一种将基因转移到猪 EPC 中的有效方法，然而，这种方法的记录很少。分离猪骨髓单核细胞并在EGM-2培养基中培养，获得骨髓源性EPCs（BM-EPCs），并通过免疫染色和成管实验进行表征。使用 GFP 和 VEGF 质粒在 6 孔板中优化基因转移，并在 T75 烧瓶中放大。通过荧光显微镜和流式细胞术测定基因转移效率。通过ELISA测量VEGF水平。通过CCK-8试剂盒检测细胞增殖。(1) BM-EPCs表达VEGFR2和eNOS但不表达CD45蛋白，并在Matrigel上形成管状结构；(2) 探索了几种化合物，使用 Lipofectamine 3000 达到最高转染效率 41.4% ± 5.8%；(3)转染VEGF后培养基中VEGF水平为378±48ng/10 ⁶个细胞；(4)过表达VEGF的BM-EPCs比转染GFP的EPCs增殖显着增强。建立了一种简单、容易、廉价的方法，可用于大量生产转基因BM-EPC，这将有助于研究GTE-EPC在猪MI模型中的治疗效果。