Division of Physiology, Department of Basic Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.
Stem Cell Biology and Regenerative Medicine Research Group, Research Institute of Biotechnology, Ferdowsi University of Mashhad, Azadi Square, Mashhad, 91779-48974, Iran.
Arch Razi Inst. 2021 Nov 30;76(5):1315-1325. doi: 10.22092/ARI.2021.354659.1645. eCollection 2021 Nov.
Conventional cancer therapies, including surgery, radiotherapy, and chemotherapy, are not tumor site-specific and have cytotoxic and harmful side effects for normal cells. Mesenchymal stem cells (MSCs), due to their tumor-tropism migration property, are a promising alternative to deliver and produce antitumor agents. However, MSCs are difficult-to-transfect cells, and introducing the exogenous therapeutic gene into MSCs is challenging yet needs improvement. Transfection using chemical reagents, including Lipofectamine, is more convenient and less cytotoxic compared with different methods of introducing exogenous DNA into MSCs. Nonetheless, the major limitation of Lipofectamine is low transfection efficiency in MSCs. Therefore, the purpose of this study was to evaluate and suggest the optimum quantities of lipoplex components to enhance the transfection efficiency of human adipose tissue-derived MSCs (hASCs). Finding the best transgene expression time point and the optimum concentration of G-418 for antibiotic-based selection was another goal of this study. hASCs were transfected in a series of experiments with altering the quantities of Lipofectamine LTX® (Lip-LTX), the related "PLUS" reagent, and a plasmid DNA (pDNA) expressing the enhanced green fluorescent protein (eGFP). After transfection, the percentage of eGFP-expressing cells was evaluated using fluorescence microscopy and ImageJ software in 12-hour intervals for 48 hours. Also, the viability of hASCs exposed to different concentrations of G-418 was measured using an MTT assay. The results demonstrated that a combination of 2 µL Lip-LTX, 0.75 µL of its "PLUS" reagent, and 0.75 g pDNA (6484 bp) improve the transfection efficiency of hASCs (23.75%), and the best period for evaluation of fluorescence for these cells is 12 to 24h post-transfection. Also, the optimum concentration of G-418 for antibiotic-based selection of hASCs was 0.25mg/mL. In conclusion, this study indicates that the setting up of optimized quantities of lipoplex components and the golden time of evaluation for transgene expression could increase the possibility of transgene expression in hASCs before beginning research and clinical application. Also, the definition of optimal dose of selection antibiotic for purification of transfected hASCs seems to be necessary for maximum transgene expression effects in the cell population.
传统的癌症治疗方法，包括手术、放疗和化疗，对肿瘤部位并不具有特异性，并且对正常细胞具有细胞毒性和有害的副作用。间充质干细胞（MSCs）由于其具有肿瘤趋向性迁移的特性，是一种很有前途的替代方法，可以用于递送和产生抗肿瘤药物。然而，MSCs 是难以转染的细胞，将外源性治疗基因引入 MSCs 具有挑战性，需要改进。与将外源性 DNA 引入 MSCs 的不同方法相比，使用化学试剂（包括 Lipofectamine）进行转染更加方便，细胞毒性也更小。然而，Lipofectamine 的主要局限性是转染效率低。因此，本研究的目的是评估并建议优化复脂质体成分的数量，以提高人脂肪组织源性间充质干细胞（hASCs）的转染效率。寻找最佳的转基因表达时间点和最佳的 G-418 浓度以进行基于抗生素的选择也是本研究的另一个目标。通过改变 Lipofectamine LTX®（Lip-LTX）、相关“PLUS”试剂和表达增强型绿色荧光蛋白（eGFP）的质粒 DNA（pDNA）的数量，在一系列实验中转染 hASCs。转染后，在 48 小时内每隔 12 小时使用荧光显微镜和 ImageJ 软件评估表达 eGFP 的细胞的百分比。还使用 MTT 测定法测量 hASCs 暴露于不同浓度的 G-418 时的活力。结果表明，组合使用 2 µL Lip-LTX、0.75 µL 的“PLUS”试剂和 0.75 g pDNA（6484 bp）可提高 hASCs 的转染效率（23.75%），这些细胞的最佳荧光评估期是转染后 12 至 24 小时。此外，用于 hASCs 基于抗生素选择的最佳 G-418 浓度为 0.25mg/mL。总之，本研究表明，优化复脂质体成分的用量和转基因表达的最佳评价时间，可以增加在开始研究和临床应用之前 hASCs 中转基因表达的可能性。此外，定义用于纯化转染 hASCs 的最佳选择抗生素剂量似乎对于细胞群体中转基因表达效果的最大化是必要的。
