Trichostatin A Promotes Cardiomyocyte Differentiation of Rat Mesenchymal Stem Cells After 5-Azacytidine Induction or During Coculture With Neonatal Cardiomyocytes Via a Mechanism Independent of Histone Deacetylase Inhibition
Trichostatin A Promotes Cardiomyocyte Differentiation of Rat Mesenchymal Stem Cells After 5-Azacytidine Induction or During Coculture With Neonatal Cardiomyocytes Via a Mechanism Independent of Histone Deacetylase Inhibition
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曲古抑菌素 a 通过独立于组蛋白脱乙酰酶抑制的机制，在 5-氮杂胞苷诱导后或与新生心肌细胞共培养期间促进大鼠间充质干细胞的心肌细胞分化
DOI：
10.3727/096368911x593145
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发表时间：
2012-01-01
影响因子：
3.3
通讯作者：
Zhu, Jing
中科院分区：
文献类型：
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作者：
Yang, Ge;Tian, Jie;Zhu, Jing
本研究旨在探讨组蛋白去乙酰化酶（HDAC）抑制剂甲氨蝶呤（TSA）对骨髓间充质干细胞（MSCs）体外心肌分化的影响。分离大鼠MSC并分为六组：1）对照; 2）5-氮杂胞苷处理（5-aza，10 μ M）; 3）用TSA处理（100、3（10和500 nM）; 4）用5-aza处理，然后与TSA孵育; 5）与新生心肌细胞（CM）共培养;和6）用TSA处理，然后与CM共培养。在TSA处理的细胞中HDAC活性被显著抑制，在暴露于300 nM的TSA 24 h后具有最大抑制。在对照组、5-氮杂处理组或共培养组中未观察到HDAC活性的变化。在分化7天后，使用实时PCR、免疫荧光染色和蛋白质印迹法测定，5-氮杂处理、共培养或TSA处理组中的细胞中早期心脏转录因子加塔-4、NKx 2.5、MEF 2c和心肌肌钙蛋白T（cTnT）的表达比对照组增加6-8倍。然而，cTnT阳性细胞百分比显著不同，对照组为0.7%，5-氮杂处理组为10%，共培养组为25%，TSA处理组为4%（500 nM）。TSA处理用5-aza预处理或与CM共培养的细胞显著增加加塔-4、NKx 2.5和MEF 2c的表达，比对照增加35-50倍。TSA处理（500 nM）后，5-aza处理组和共培养组的cTnT蛋白表达均显著高于对照组（P < 0. 05）。TSA诱导分化1周后，5-aza预处理组和共培养组的cTnT阳性细胞百分比分别显著增加至92.6%（从10.3%增加至19.8%）和23.9%（从24.5%增加至30.2%）。这些数据表明，TSA通过抑制HDAC活性以外的机制增强5-aza诱导或诱导与CM共培养后的MSC的心脏分化。
This study was to investigate the effect of trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, on cardiac differentiation of bone marrow mesenchymal stem cells (MSCs) in vitro. Rat MSCs were isolated and divided into six groups: 1) control; 2) 5-azacytidine treatment (5-aza, 10 mu M); 3) treatment with TSA (100, 3(10, and 500 nM); 4) treatment with 5-aza followed by incubation with TSA; 5) coculture with neonatal cardiomyocytes (CMs); and 6) treatment with TSA then coculture with CMs. HDAC activity was significantly inhibited in TSA-treated cells with the maximal inhibition after 24 h of exposure to TSA at 300 nM. No changes in HDAC activity were observed in control, 5-aza-treated, or coculture groups. Following 7 days of differentiation, the expression of early cardiac transcription factors GATA-4, NKx2.5, MEF2c, and cardiac troponin T (cTnT) was increased by 6-8 times in the cells in 5-aza-treated, coculture, or TSA-treated groups over control as determined using real-time PCR, immunofluorescence staining, and Western blotting. However, the percent cTnT-positive cells were dramatically different with 0.7% for control, 10% for 5-aza-treated, 25% for coculture, and 4% for TSA-treated group (500 nM). TSA treatment of the cells pretreated with 5-aza or cocultured with CMs dramatically increased the expression of GATA-4, NKx2.5, and MEF2c by 35-50 times over control. The cTnT protein expression was also significantly increased by over threefold by TSA treatment (500 nM) in both 5-aza-treated and coculture group over control. The percent cTnT-positive cells in both 5-aza-pre-treated and coculture groups were significantly increased by TSA treatment after 1 week of differentiation by up to 92.6% (from 10.3% to 19.8%) and 23.9% (from 24.5% to 30.2%), respectively. These data suggested that TSA enhanced the cardiac differentiation of MSCs after 5-aza induction or (luring coculture with CMs through a mechanism beyond the inhibition of HDAC activity.