Experimental Induction of Myogenic Differentiation in Rabbit Bone Marrow Mesenchymal Stem Cells Using Different Concentrations of 5-Azacytidine
Received Date: Feb 24, 2019 / Accepted Date: Mar 23, 2019 / Published Date: Mar 30, 2019
Purpose: To investigate the proliferation and myogenic differentiation abilities of rabbit bone marrow mesenchymal stem cells (BMSCS) in the presence of different concentrations of 5-azacytidine (5-Aza).
Method: BMSCS from adult New Zealand male white rabbits were expanded and cultured in vitro. Cells were induced with 0, 5, 10, 20, and 40 μmol/L 5-Aza for 24 hours, and these groups were denoted groups A,B,C,D, and E. Group A served as the control group, and groups B, C, D, and E were the experimental groups. The absorbance of the cells at 570 nm was measured using the MTT method, which is commonly used for assessing cell viability. On days 1, 3, 5, 7 and 9 after induction, the proliferation and morphological changes of BMSCS were observed using an inverted light microscope. On day 9 of cultivation, we digested and collected the cells and prepared slides of the cells. The expression of actin was assessed by immunocytochemistry, and the percentage of actin expression was determined through a semi quantitative analysis of fluorescence. Quantitative RT-PCR was used to detect the mRNA expression of troponin and myosin.
Results: The flow cytometry results showed that the surface markers CD34, CD44 and CD90 were expressed at percentages of 2.52%, 94.6% and 92.8%, respectively. The 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-tetrazolium bromide (MTT) assay revealed that cell viability was significantly lower in group E than in group A (p <0.001). On day 9, the cell morphology changes observed in group C were the most obvious. Actin expression was detected in groups A, B and C by immunohistochemistry, and semi quantitative fluorescence showed that group C exhibited significantly higher myogenic differentiation in BMSCS than group A (p=0.034). On day 9 after induction, troponin and myosin mRNA expression levels were higher in group C than in group A (p=0.04 and p <0.001, respectively), as demonstrated by quantitative real-time PCR.
Conclusion: Different concentrations of 5-Aza exert different effects on the proliferation and myogenic differentiation of fifth-generation BMSCS. In addition, 10 μmol/L, 5-Aza significantly promoted myogenic differentiation, whereas 40 μmol/L 5-Aza exerted a certain toxic effect on BMSC proliferation.
Keywords: 5-Azacytidine; Bone marrow mesenchymal stem cells; Proliferation; Myogenic differentiation; Actin; Troponin; Myosin
Citation: Fei W, Zhang Y, Cao S, Liu M, Tan J, et al. (2019) Experimental Induction of Myogenic Differentiation in Rabbit Bone Marrow Mesenchymal Stem Cells Using Different Concentrations of 5-Azacytidine. J Clin Toxicol 9: 413.
Copyright: © 2019 Fei W, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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