Markmee Runchana, Aungsuchawan Sirinda, Pothacharoen Peraphan, Tancharoen Waleephan, Narakornsak Suteera, Laowanitwattana Tanongsak, Bumroongkit Kanokkan, Puaninta Chaniporn, Pangjaidee Nathaporn
Department of Anatomy, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand.
Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand.
Heliyon. 2019 Jul 17;5(7):e02018. doi: 10.1016/j.heliyon.2019.e02018. eCollection 2019 Jul.
The aim of this study was to evaluate the efficiency of ascorbic acid (AA) on cell viability, cytotoxicity and the effects on cardiomyogenic differentiation of the human amniotic fluid mesenchymal stem cells (hAF-MSCs). The results of methylthiazole tetrazolium (MTT) assay and cell apoptosis assay indicated that after 24, 48 and 72 h of treatment, AA had no effect on cells viability and cytotoxicity. After treating the hAF-MSCs with 5-azacytidine (5-aza) and a combination of AA and 5-aza, the alamar blue cells proliferation assay showed the normal growth characteristic similar to control group. Especially, the morphological changes were observed between day 0 and day 21, and it was revealed that the hAF-MSCs exhibited myotube-like morphology after 7 days of cell culturing. Moreover, the treatment with a combination of AA and 5-aza was able to up-regulate the cardiomyogenic specific gene levels, which are known to play an important role in cardiomyogenesis. This was specifically notable with the results of immunofluorescence and immunoenzymatic staining in the AA combined with 5-aza treatment group, the highest expression of cardiomyogenic specific proteins was revealed including for GATA4, cTnT, Cx43 and Nkx2.5. It could be concluded that AA may be a good alternative cardiomyogenic inducing factor for hAF-MSCs and may open new insights into future biomedical applications for a clinically treatment.
本研究的目的是评估抗坏血酸（AA）对人羊水间充质干细胞（hAF-MSCs）的细胞活力、细胞毒性以及心肌分化的影响。噻唑蓝（MTT）法和细胞凋亡检测结果表明，处理24、48和72小时后，AA对细胞活力和细胞毒性没有影响。在用5-氮杂胞苷（5-aza）以及AA与5-aza的组合处理hAF-MSCs后，alamar蓝细胞增殖检测显示其生长特征与对照组相似。特别是，在第0天和第21天之间观察到形态变化，结果显示hAF-MSCs在细胞培养7天后呈现出肌管样形态。此外，AA与5-aza联合处理能够上调心肌特异性基因水平，这些基因在心肌生成中起重要作用。在AA联合5-aza处理组的免疫荧光和免疫酶染色结果中这一点尤为显著，显示出心肌特异性蛋白的最高表达，包括GATA4、肌钙蛋白T（cTnT）、连接蛋白43（Cx43）和Nkx2.5。可以得出结论，AA可能是hAF-MSCs良好的替代性心肌诱导因子，可能为未来临床治疗的生物医学应用开辟新的思路。
