Effects of Excessive Retinoic Acid on C2C12 Myogenesis
Effects of Excessive Retinoic Acid on C2C12 Myogenesis
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DOI：
10.2485/jhtb.25.97
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发表时间：
2016
影响因子：
0.4
通讯作者：
Bo Liu;Nan Li;Yuling Jiang;Chao Liu;Le-Ping Ma;W. Cong;Jing Xiao
中科院分区：
文献类型：
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作者：
Bo Liu;Nan Li;Yuling Jiang;Chao Liu;Le-Ping Ma;W. Cong;Jing Xiao
维甲酸（RA）是一种广泛应用的细胞分化诱导剂。为了研究过量RA对成肌的影响，将C2 C12细胞培养在不同浓度的RA中。本研究中，当RA浓度大于10 μM时，细胞周期阻滞于G 0 /G1期。超过40 µM的RA可抑制细胞增殖。因此，选择1和10 μM RA分别处理C2 C12细胞，然后通过实时PCR检测肌源性调节因子（MRF）。结果发现，RA处理后，Myf 5转录维持在较低水平。MyoD和Myogenin转录的变化曲线与溶剂对照一致，但MRF 4转录在第6天高于溶剂对照。此外，与10 μM RA处理的细胞相比，MyoD和Myogenin的转录升高持续时间更长，并且1 μM RA处理的细胞中第6天的MRF 4转录更高。用蛋白质印迹法测定肌球蛋白重链（MyHC），分析肌源性终末分化。我们的研究结果表明，10 μM RA处理的细胞中MyHC蛋白水平明显下降。最后，免疫染色显示，RA处理的肌球蛋白阳性细胞中越来越多的分叉伴随着更长和更薄的细胞体，特别是在10µM RA处理的细胞中。结论：过量RA仍能诱导成肌细胞肌化分化。然而，较高剂量的RA可能会阻碍肌管的成熟。
: Retinoic acid (RA) is a widely-used agent inducing cell differentiation. In order to study the effect of excessive RA on myogenesis, C2C12 cells were cultured in gradient concentrations of RA. In this study, the cell cycle arrested in G 0 /G 1 phase when the concentration of RA was higher than 10 µM. Cell proliferation was inhibited by RA over 40 µM. Therefore, 1 and 10 µM RA were chosen to treat C2C12 cells under serum withdrawal, respectively, and then, myogenic regulatory factors (MRFs) were measured by real-time PCR. It was found that after RA treatment, Myf5 transcription was maintained in a lower level. The change curves of MyoD and Myogenin transcription were consistent with the vehicle control, but MRF4 transcription was higher than that of the vehicle control at day 6. Additionally, compared with 10 µM RA-treated cells, the elevated transcription of MyoD and Myogenin insisted longer and the transcription of MRF4 at 6 day was higher in 1 µM RA-treated cells. Myosin heavy chain (MyHC) was also calibrated by Western blot to analysis myogenic terminal differentiation. Our findings illustrated that MyHC protein level was obviously declined in 10 µM RA-treated cells. Finally, immunostaining revealed that a growing number of bifurcations in RA-treated myosin-positive cells were accompanied by longer and thinner cell bodies, especially in 10µM RA-treated cells. In conclusion, excessive RA could still induce myoblast myogenic differentiation. However, the maturation of myotubes might be discouraged by a higher-dose of RA.