Department of Physiology, Favaloro University, Buenos Aires, Argentina.
Cytotherapy. 2013 Feb;15(2):163-70. doi: 10.1016/j.jcyt.2012.11.004.
Given the close similarity between ovine and human cardiomyocytes, sheep models of myocardial infarction and heart failure are increasingly used in studies of stem cell-mediated heart regeneration. In these studies, mesenchymal stromal cells (MSCs) are frequently employed. To enhance the paracrine effects of these MSCs, ex vivo transfection with genes encoding growth factors has been proposed. Although viral vectors exhibit higher transfection efficiency than plasmids, they entail the risks of uncontrolled transgene expression and immune reactions that preclude repeated administration. Our aim was to optimize the efficiency of plasmid-mediated transfection of ovine MSCs, while preserving cell viability.
Varying amounts of diverse cationic lipids were used to obtain the reagent-to-DNA mass ratio showing highest luciferase activity. Transfection efficiency (flow cytometry) was tested on plasmid-green fluorescent protein-transfected MSCs at increasing DNA mass.
Lipofectamine LTX 5 μL and Plus reagent 4 μL with 2 μg of DNA yielded 42.3 ± 4.7% transfection efficiency, while preserving cell viability. Using these transfection conditions, we transfected MSCs with a plasmid encoding human vascular endothelial growth factor (VEGF) and found high VEGF protein concentrations in the culture supernatant from day 2 (1968 ± 324 pg/mL per μg DNA) through at least day 12 (888 ± 386 pg/mL per μg DNA) after transfection.
Plasmid-mediated transfection of ovine MSCs to over-express paracrine heart-regenerative growth factors is feasible and efficient and overcomes the risks and limitations associated with the use of viral vectors.
由于绵羊和人类的心肌细胞非常相似，因此绵羊心肌梗死和心力衰竭模型越来越多地被用于研究干细胞介导的心脏再生。在这些研究中，经常使用间充质基质细胞（MSCs）。为了增强这些 MSC 的旁分泌作用，已经提出了用编码生长因子的基因对其进行体外转染。尽管病毒载体的转染效率高于质粒，但它们存在着不可控的转基因表达和免疫反应的风险，从而排除了重复给药的可能。我们的目的是优化绵羊 MSC 的质粒介导转染效率，同时保持细胞活力。
使用不同量的阳离子脂质来获得显示最高荧光素酶活性的试剂与 DNA 的质量比。在转染质粒-绿色荧光蛋白的 MSC 时，通过增加 DNA 的质量来测试转染效率（流式细胞术）。
Lipofectamine LTX 5 μL 和 Plus 试剂 4 μL 与 2 μg 的 DNA 产生了 42.3 ± 4.7%的转染效率，同时保持了细胞活力。使用这些转染条件，我们将编码人血管内皮生长因子（VEGF）的质粒转染到 MSCs 中，发现转染后第 2 天（每 μg DNA 为 1968 ± 324 pg/mL）和至少第 12 天（每 μg DNA 为 888 ± 386 pg/mL）的培养上清液中 VEGF 蛋白浓度很高。
绵羊 MSC 的质粒介导转染以过表达旁分泌心脏再生的生长因子是可行且高效的，并且克服了与使用病毒载体相关的风险和限制。
