脉冲电磁场诱导大鼠骨髓间充质干细胞体外心肌样细胞分化的研究

The Effects of Pulsed Electromagnetic Fields on the Induction of Rat Bone Marrow Mesenchymal Stem Cells to Differentiate into Cardiomyocytes-like Cells in Vitro

观察脉冲电磁场(PEMFs)诱导大鼠骨髓间充质干细胞(rBMSCs)在体外向心肌样细胞分化的影响。选用第3代rBMSCs用于实验。实验分为PEMFs诱导组、5-氮胞苷(5-Aza)诱导组和对照组:PEMFs诱导组给予rBM-SCs 50 Hz1、mT低频脉冲电磁场刺激,30 min/d,分别刺激10 d、15 d和20 d;5-Aza诱导组给予rBMSCs 10μmol/L 5-Aza诱导1d,对照组不予处理;后两组与PEMFs诱导组同步培养10、15和20 d。通过倒置相差显微镜逐日连续观察细胞的生长状况和形态特征,采用实时荧光定量PCR检测肌钙蛋白T(TNNT2)mRNA和α-辅肌动蛋白(ACTN2)mRNA表达量的变化。结果显示:对照组rBMSCs呈纺锤形、多角形或梭形,随时间延长,细胞形态无明显变化;5-Aza或PEMFs诱导后,细胞形状逐渐变长,大量细胞聚集生长,且随着诱导时间的延长,聚集趋势不断增加,刺激20 d,细胞聚集呈长链状,高倍镜下可见细胞伸长明显,且部分细胞与邻近细胞融合。与对照组相比,5-Aza诱导组TNNT2 mRNA在3个时间点的表达量分别为相应对照组的19.40倍(P<0.01)、21.02倍(P<0.01)和2.38倍;ACTN2 mRNA在3个时间点的表达量分别为相应对照组的6.64倍(P<0.01)、6.67倍(P<0.01)和0.76倍。PEMFs诱导组中TNNT2 mRNA在3个时间点的表达量分别为相应对照组的15.78倍、6.73倍和2.73倍(均有P<0.05);ACTN2 mRNA在3个时间点的表达量分别为相应对照组的4.93倍(P<0.01)、1.89倍和0.64倍。而与5-Aza诱导组比较,PEMFs诱导组中TNNT2 mRNA在3个时间点的表达量分别为相应5-Aza诱导组的0.81倍、0.32倍(P<0.01)和1.15倍;ACTN2 mRNA在3个时间点的表达量分别为相应5-Aza诱导组的0.74倍、0.28倍(P<0.01)和0.83倍。本研究结果表明,在我们所选用的PEMFs条件下,PEMFs可以诱导rBMSCs在体外向心肌样细胞分化,最佳作用时间可能为第10 d。

The aim of this study is to investigate the effects of pulsed electromagnetic fields（PEMFs） on the induction of rat bone marrow mesenchymal stem cells（rBMSCs） to differentiate into cardiomyocytes-like cells in vitro.rBMSCs were randomly divided into PEMFs groups,5-Azacytidine（5-Aza） groups and control groups.PEMFs groups were exposed to 50 Hz,1 mT PEMFs for 30 min every day,lasting for 10 d,15 d and 20 d,respectively.5-Aza groups were induced by 10 μmol/L 5-Aza for 1 day,then the medium was changed to complete medium without 5-Aza.And control groups were only cultured with complete medium.rBMSCs＇ growth status and morphological features were observed by inverted phase microscope every day.The mRNA expressions of cardiac troponin T（TNNT2）and α-actinin（ACTN2） were determined by Real-Time PCR.The results showed that rBMSCs were spindle,polygon or fusiform in control groups.The cells gradually got longer and grew close together after being stimulated by PEMFs and 5-Aza,and with the extension of induction time,the tendency became obvious.At 20th day after PEMFs or 5-Aza treatment,rBMSCs gathered like a long chain,got much longer obviously at the high magnification,and some of them even fused with their neighbors.Compared with control groups,the levels of TNNT2 mRNA expression in 5-Aza groups were 19.40 fold（P0.01）,21.02 fold（P0.01） and 2.38 fold at 10 d,15 d,20 d and the levels of ACTN2 mRNA expression in 5-Aza groups were 6.64 fold（P0.01）,6.67 fold（P0.01） and 0.76 fold at 10 d,15 d,20 d.However,the levels of TNNT2 mRNA expression in PEMFs groups were 15.78 fold（P0.01）,6.73 fold（P0.05） and 2.73 fold（P0.01） of control groups at 10 d,15 d,20 d and the levels of ACTN2 mRNA expression in PEMFs groups were 4.93 fold（P0.01）,1.89 fold and 0.64 fold,respectively.Compared with 5-Aza groups,the levels of TNNT2 mRNA expression in PEMFs groups were 0.81 fold,0.32 fold（P0.01） and 1.15 fold at 10 d,15 d,20 d and the levels of ACTN2 mRNA expression in PEMFs groups were 0.74 fold,0.28 fold（P0.01） and 0.83 fold at 10 d,15 d,20 d.PEMFs could contribute to the induction of rat marrow rBMSCs to differentiate into cardiomyocytes-like cells in vitro,and the best exposure time might be 10 days,but further investigation is still needed.