Division of Cardiology, Keenan Research Center for Biomedical Science, St. Michael's Hospital, Unity Health Toronto, University of Toronto, Toronto, ON, Canada.
Department of Cardiovascular Surgery, Renji Hospital, Shanghai Jiaotong University, Shanghai, China.
Sci Prog. 2021 Jul-Sep;104(3):368504211024113. doi: 10.1177/00368504211024113.
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/10 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.
目前尚无治疗方法能够在心肌梗死（MI）后产生新的收缩组织或显著改善心脏功能，MI 是全球发病率和死亡率的主要原因。尽管基因转导增强的内皮祖细胞（GTE-EPC）在小动物模型中显示出对 MI 治疗的有效性，但尚未有使用 GTE-EPC 的临床试验记录。在将 GTE-EPC 引入人体试验之前，应该在反映人类 MI 情况的动物模型中测试基因转导介导的 EPC 功能增强。在这方面，猪模型是最佳选择，因为猪的心脏大小、血液动力学和冠状动脉解剖结构与人类相似。为了在猪 MI 模型中检验 GTE-EPC 的治疗效果，需要一种有效的方法将基因转入猪 EPC，然而，这方面的研究还很少。从猪骨髓中分离出单核细胞，并在 EGM-2 培养基中培养，获得骨髓来源的内皮祖细胞（BM-EPC），通过免疫染色和管形成试验对其进行鉴定。在 6 孔板中使用 GFP 和 VEGF 质粒优化基因转导，并在 T75 培养瓶中放大。通过荧光显微镜和流式细胞术确定基因转导效率。通过 ELISA 测量 VEGF 水平。通过 CCK-8 试剂盒测定细胞增殖。（1）BM-EPC 表达 VEGFR2 和 eNOS，但不表达 CD45 蛋白，并在 Matrigel 上形成管结构；（2）探索了几种化学化合物，其中使用 Lipofectamine 3000 获得的最高转染效率为 41.4%±5.8%；（3）VEGF 转染后培养基中的 VEGF 水平为 378±48ng/10 细胞；（4）过表达 VEGF 的 BM-EPC 比 GFP 转染的 EPC 具有明显增强的增殖能力。建立了一种简单、易用、廉价的方法，可以大量生产基因修饰的 BM-EPC，这将有助于研究 GTE-EPC 在猪 MI 模型中的治疗效果。
