Cardiomyocyte-like Cells Differentiation Of BMMSCs Induced By PFT-α And BMP-2 In Rat

Objective:Nowadays,the incidence ofischemic heart disease is higher in our country even in the whole word. It has influence the health of people. For this kind disease, the traditional methods were just cause the remission of disease, while not repair the dead or lost cells. The treatment of ischemic heart disease with the method of separating the stem cells into cardiomyocytes may promote cell repair and angiogenesis. In recent years, using cell transplantation in the treatment of cardiovascular disease has been highly valued experts and scholars both at home and abroad.Mesenchymal stem cells(BMSCs) have the ability to self-renew and differentiate into varieties of cells, including cardiomyocytes. Besides, it has the advantage of no-ethical controversy, easy-derived isolated and cultured, low immunogenicity, etc. So it became the "seed cells" of cell transplantation in the treatment of ischemic heart disease. While, the number of this cell is rare, and it gradually reduces with the increasing age, In order to ensure the treatment effect, it is essential to amplificate, purificate and improve the conversion rate of BMSCs.Since now, large numbers of experimenthave been studiedon usinginducer alone. This experiment will explore the effecton by the PFT-α(P53 gene inhibitor) and BMP2(Bone Morphogenetic Protein 2, bone morphogenetic protein-2) inducing BMSCs different into cardiomyocytes. The aim of this experiment is to provide reference for the treatment of ischemic heart disease by cell transplantation.Methods:1 isolate and purified BMSCs by centrifuging from the long bone marrow which from healthy SD rats, cultured in IMDM medium which contains 10% fetal bovine serum, changed the medium every 3d. Separately four groups to inducte BMSCs from well-grown second generation by IMDM culture medium containing PFT-α20μmol / L, BMP-2 0.2ng / ml and added PFT-α20μmol / L and BMP-2 0.2ng / ml. Meanwhile,take the liquid with no induction medium ascomparison group.After that, 4 days later, removed the liquid containing induction medium, added new media(excluding induction medium) and cultured 4w.2 Immunocytochemical staining: chose the 4 weeks cultured cells from the induce experimental of the 4 groups and noinduction medium liquid, observed cell α-actin(α-striated muscle actin), desmin(desmin), c Tn T(cardiac troponin) and the expression of c Tn I(troponin-I) positive cells in light microscopy.3 Immunofluorescence double staining technique: the first step is to take cells cultured 4 weeks of PFT-α with BMP-2 induced group; then use fluorescence to label cell α-actin, c Tn T protein and observe α-actin, c Tn T in co-expression.4 TEM: This technical is to observe the internal structure of the 4-week induced cell of PFT-α with BMP-2 by TEM, which were titled into slice by the methods of centrifugation, dehydration and other treatments.Results:1 BMSCs cultureThe cells were round and gradually increased after been inoculating for 24 h, which were isolated and cultured from centrifuging and separation screening adherent BMSCs. It also showed that the number of adherent fusiform cells significantly increased after 3d and proliferated 6d later.2 Immunochemical detection of cell:It showed that the expression of cells from desmin, α-actin, c Tn T and c Tn I were seen in induced group. Through observation, cells of PFT-α with BMP-2 in induced group were significantly higher than the other groups(P<0.01 or P<0.05).3 Immunofluorescence double staining:It confirmed that there was red positive expression cells in PFT-α combined with BMP-2 andgree positive expression cells in c Tn T in induced differentiation group. When these two were overlapped, it was observed yellow, so we can indicate that there were circumstances that α-actin and c Tn T express same in co-inducing group.4 TEM:The TEM showed us the ship of differentiated cells was light and dark strip with fine muscle parallel wire-like structure, and some of them appeared projection surface, the nucleus located center. The cytoplasm not only contained Golgi complex, glycogen granules, rough endoplasmic reticulum, but also other rich organelles.Conclusions:1 BMSCs can be differentiated into cardiomyocytes successfully by the lonely induction of BMP-2, PFT-α and combined induction of PFT-αBMP-2.2 With the comparison of expression of α-actin, c Tn T and c Tn I of differentiated cells in each experimental group and comparison group, as well as morphology comparison, there was significantly higher positive differentiation rate of PFT-α with BMP-2 than that of PFT-α and BMP-2 alone, which were used to be induced bone marrow mesenchymal stem cells into cardiomyocytes differentiated. The way of combining PFT-α with BMP-2 was more efficient.