Objectives We blocked the canonical wnt signalling pathway through knocking down β-catenin in Mesenchymal stem cells (MSCs), and investigated the role of 5-Azacytidine, salvianolic acid B and cardiomyocytes lysis in inducing non-β-catenin expression MSCs differentiate into cardiomyocytes.
Methods Culturing MSCs with adherence screening method, cell surface markers CD29, CD90, CD34, CD45 were detected by flow cytometer. Separating cardiomyocytes from rat heart tissue and detected by Immunofluorescence. We built the non-β-catenin expression MSCs by infected the MSCs with Lentivirus containing β-catenin SiRNA. The well-cultured MSCs were divided into eight groups: the control group, 5-Azacytidine group (5-aza group), salvianolic acid B group (salB group), cardiomyocytes lysis group (CM group) , 5-Azacytidine combined with salvianolic acid B group (5-aza + salB group), 5-Azacytidine combined with cardiomyocytes lysis group (5-aza + CM group), salvianolic acid B combined with cardiomyocytes lysis group (salB + CM group), 5-Azacytidine combined with salvianolic acid B and cardiomyocytes lysis group (5-aza + salB + CM group). After induced for two weeks, we use QPCR and wextern blotting to detect the expression of cTnT, β-myosin, α-actin.
Results MSCs were positive for CD29, CD90 and negative for CD34, CD45. Immunofluorescence showed that cardiomyocytes were positive for cTnT. QPCR and western boltting results showed that cTnT, β-myocin, α-actin expression in 5-aza + SalB + CM group is significant higher than 5-aza + CM group and SalB + CM group; 5-aza + SalB group is significant higher than 5-aza group and SalB group.
Conclusions Blocking canonical wnt signaling pathway is a necessary condition for cardiomyocytes differentiation from MSCs.5-aza combined with salB can induce Nβ-MSCs differentiate into cardiomyocytes effectively under cardiomyocytes microenvironment.
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