低频脉冲电磁场诱导大鼠骨髓间充质干细胞ACTN2、α-actin和TNNT2表达

Expression of ACTN2,α-actin and TNNT2 in Rat Bone Marrow-derived Mesenchymal Stem Cells Induced by Low Frequency Pulsed Electromagnetic Fields

目的探讨低频脉冲电磁场(PEMFs)对大鼠骨髓间充质干细胞(rBMSCs)α-辅肌动蛋白-2(ACTN2)、α-肌动蛋白(α-actin)和肌钙蛋白T-2(TNNT2)表达的影响。方法采用差速贴壁分离方法获取rBMSCs,选用第3代rBMSCs用于实验。实验分为4组:①PEMFs诱导组,给予50Hz、1mT低频PEMFs刺激,30min/d,分别刺激10d、15d和20d;②5-氮胞苷(5-Aza)诱导组,给予10μmol/L 5-Aza诱导1d,然后采用完全培养液培养10d、15d和20d;③PEMFs+5-Aza诱导组:采用10μmol/L 5-Aza诱导1d后,再给予PEMFs诱导组相同的处理;④对照组,采用完全培养液培养10d、15d和20d。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方法检测ACTN2和TNNT2的mRNA表达,采用Western blot方法检测ACTN2、α-actin和TNNT2的蛋白表达。结果在各时间点,PEMFs诱导组、5-Aza诱导组和PEMFs+5-Aza诱导组的ACTN2、α-actin和TNNT2mRNA表达与蛋白表达明显高于对照组。结论 PEMFs可促进体外培养的rBMSCs向心肌样细胞分化。

Objective To study the expression of Actinin, alpha 2 （ACTN2）, α-actin and Troponin T type 2 （TNNT2） in rat bone marrow-derived mesenchymal stem cells （rBMSCs） induced by low frequency pulsed electromagnetic fields （PEMFs）. Methods The rBMSCs were isolated by adherence method and the third generation of the rBMSCs were randomly divided into PEMFs groups, 5-Azacytidine groups （5 Aza）, PEMFs ＋5 Aza group and control groups. PEMFs groups with complete medium 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. PEMFs ＋5-Aza group were firstly induced by 10μmol/L 5-Aza for 1 day, and then were exposed to PEMFs according to the way of PEMFs group. And control groups were only cultured with complete medium, rBMSCs＇ growth status and morphological features were observed by inverted phase microscope. The mRNA expressions of ACTN2 and TNNT2 were determined by real- time fluorescent quantitation PCR. And the protein expression of the ACTN2, α-actin and TNNT2 were detected with Western blot method. Results The result showed that the mRNA expression level of the ACTN2 and TNNT2 and the protein expression level of the ACTN2, α-actin and TNNT2 were significantly higher in the PEMFs group, 5-Aza group and PEMFs＋ 5-Aza group when compared with the control group. Conclusion PEMFs may induce rBMSCs differentiation into cardiomyoeyte-like cells in vitro.