环磷酸腺苷葡甲胺诱导骨髓间充质干细胞向心肌细胞分化的实验研究

Meglumine cyclic adenylate induces differentiation of bone marrow mesenchymal stem cells into cardiomyocytes in vitro

目的:探讨环磷酸腺苷葡甲胺(MCA)对骨髓间充质干细胞(BMSCs)向心肌细胞分化的影响。方法:采用全骨髓贴壁培养法培养BMSCs,流式细胞术鉴定细胞表面抗原CD71、CD45和CD44的表达情况。用含各浓度MCA的细胞培养液干预后:(1)MTT法测定MCA对细胞活性的影响;(2)ELISA法测定细胞内cAMP水平;(3)荧光定量RT-PCR测定BMSCs心肌特异性基因表达情况;(4)利用流式细胞术比较MCA和5-氮杂胞苷(5-Aza)的诱导分化率。结果:采用全骨髓贴壁培养法获得BMSCs,流式细胞术测定细胞表面抗原结果符合BMSCs标准。MCA抑制BMSCs细胞活性,表现为时间和剂量依赖性,其中10-2mol/L对细胞活性的抑制作用尤为显著。随着MCA浓度的升高细胞内cAMP浓度也升高。BMSCs经10-3 mol/L、10-4 mol/L和10-5 mol/L MCA干预后均有GATA-4、β-MHC和Cx43 mRNA的表达,其中10-3 mol/L MCA诱导干预3 d,能发挥最佳的诱导分化作用。给予PKA抑制剂H89干预后,GATA-4、β-MHC和Cx43 mRNA的表达量显著减少(P<0.05)。流式细胞术测定MCA组诱导分化率略高于5-Aza组的诱导分化率(20.24%±1.02%vs 18.39%±0.58%,P<0.05)。结论:MCA可以进入BMSCs细胞内,升高cAMP水平,抑制BMSCs活性,通过cAMP/PKA信号通路促进BMSCs表达心肌特异性基因。

AIM：To study the effect of meglumine cyclic adenylate（MCA） on the differentiation of bone marrow mesenchymal stem cells（BMSCs） into cardiomyocytes in vitro.METHODS： The whole bone marrow adherent culture method was used to isolate,culture and amplify the BMSCs.The surface markers of BMSCs were determined by flow cytometry analysis.MCA at concentrations of 10-2 mol/L,10-3 mol/L,10-4 mol/L,10-5 mol/L,10-6 mol/L and 10-7 mol/L was added to the culture medium containing the second generation of BMSCs.5-Azacytidine（5-Aza） was used as a positive control.The cell viability was measured by MTT method.The cAMP content in BMSCs was detected by ELISA.The mRNA expression of GATA-4,Cx43 and β-MHC in MCA group and MCA＋H89（a PKA inhibitor） group was measured by SYBR-RT-PCR.The differentiation effects of MCA and 5-Aza were compared by flow cytometry.RESULTS： Most of the BMSCs expressed CD44 and CD71,and did not express CD45.MCA inhibited the viability of BMSCs in a time-and dose-dependent manner,and MCA at the concentration of 10-2 mol/L showed particularly remarkable effect.MCA significantly increased intracellular cAMP level in BMSCs in a concentration-dependent manner.The mRNA expression of GATA-4,β-MHC and Cx43 in MCA group were significantly higher than that in blank group（P〈0.05）,and the highest effect was under the condition of MCA induction at the concentration of 10-3 mol/L for 3 days.The mRNA expression of GATA-4,β-MHC and Cx43 in MCA group was higher than that in 5-Aza group and H89＋MCA group（both P〈0.05）.Differentiation rate in MCA group was slightly higher than that in 5-Aza group（20.24%±1.02% vs 18.39%±0.58%,P〈0.05）.CONCLUSION： MCA stimulates BMSCs to increase intracellular cAMP production and inhibits the viability of BMSCs,thus promoting the mRNA expression of GATA-4,β-MHC and Cx43 through the cAMP/PKA signaling pathway.