| Background and Objectives Cardiomyocytes do not regenerate after birth, and they respond to mitotic signals by cell hypertrophy rather than by cell hyperplasia. Loss of cardiomyocytes leads to regional contractile dysfunction, and necrotized cardiomyocytes in infarcted ventricular tissues are progressively replaced by fibroblasts to form scar tissues. The regenerative medicine which repaires damage tissue with autologous cell transplantation has been developed recent years. Studies revealed that transplantation of cultured cardiomyocytes into the damaged myocardium could prevent postinfarction heart failure. This new way which was called cellular cardiomyoplasty has been proposed as a future method for the treatment of heart failure. Many achievements have been gained in the feild of inducing the differentiation of embryonic stem cells into cardiomyocytes. But there are still problems we didn't solved, such as immunologic rejection and ethnics. Recent published reports have revealed that mesenchymal stem cells (MSCs) from mesoderm are multipotential stem cells. MSCs induced with 5-Aza can differentiate into cardiac-like muscle cells in culture and in vivo in ventricular scar tissue and improve myocardial function. In addition, the cells are easy to be collected and cultured, and have the potential ability to expansion. Unfortunately, only about 30% MSCs induced with 5-Aza can differentiate into cardiomyocytes, and little is known about the molecular mechanism.PPARs (peroxisome proliferator activated receptor) are a family of ligand-activated nuclear hormone receptors which include three sub-types, PPARa, PPARP and PPARr. It was reported that activation of PPARy is involved in cell proliferation, differentiation and apoptosis. But its certain role and regular mechanism in the differentiation of MSCs into cardiomyocytes is to be determined. The current study is initiated to investigate the regulate mechanism of PPARr and its ligand indomethacin in the differentiation of mouse MSCs into cardiomyocytes in vitro.Materials and Methods We examined the isolation, purification, expansion of mouse MSCs and capacity to differentiate into cardiomyocytes in vitro. First, bone marrowmononuclear cells were harvested from male Balb/c mouses femur and tibia with Percoll separating medium at the density of 1.082g/mL. Then, cells were cultured with DMEM/ Low glucose medium and 10% foetus cattle blood serum. Mesenchymal stem cells were obtained by removing the non-adherent cells 24h after seeding and purified and expanded through passaging in time. Primary cells were passgaed at a ratio of one to three plates when they reached 70-80% confluence. After 2-3 passages, cells were induced to differentiate into three different directions, such as osteoblasts, lipoblast and cardiomyogenesis, with three different inducing methods. Different concentrations of 5-Aza were selected including 3, 5 and 10umol/L. Cell cycles were measured with flow cytometry.To examine the effects of PPARy on the differentiation of mouse MSCs into cardiomyocytes, we constructed the mammalian expression plasmid pEGFP-N1-PPARr2 for mouse PPARy2. Indomethacin was choosed as ligand to activate PPARy. Cells were randomly divided into groups as follows: Control group; 5-Aza group (induced with 5-Aza); indomethacin group (induced with indomethacin); 5-Aza/indomethacin group(induced with 5-Aza/indomethacin); PPARr2 transfection group (transfected with expression plasmid pEGFP-N1- PPARr2 and activated with indomethacin). Cells were transfected using the cationic liposome-mediated transfection method and incubated under conditions permissive for differentiation in the presence of indomethacin. Cardiomyogenesis was monitored by immunostaining with an antibody, Troponin T, directed against the troponin. Adipogenesis was measured as lipid droplet by staining with oil red O. Osteogenesis was measured as calcium nod and by fluorescence staining with tetracyn. The changes of PPARy,GATA4, MEF-2C and Nkx2.5 mRNA expression were observed with RT-PCR at the time of lh,3h,5h,1...
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