The Differentiation Of MSCs Into Cardiomyocyte-like Cells Induced By Astragaloside Ⅳ Combined With5-aza In A Cardiac-like Microenvironment In Vitro

Myocardial infarction (MI) is a kind of cardiovascular diseases with high morbidity and mortality, significantly threatens peoples' health. The decrease of cardiac muscle and ventricle reconstructions after myocardial infraction is the important reason that leads to heart failure, arrhythmia and even death. Now days, in clinic, most of the treatment methods including the drug treatment, interventional therapy and operative therapy could not replace the necrotic cardiac muscle because of the impossible regeneration of cardiomyocytes. Thus to find out some implant to replace the dead cardiomyocytes is one of the important topic in cardiovascular research field. Therefore, CCT(Cardiac Cell Therapy)to regenerate functional myocardial cells becomes research focus in recent years. Many research show that differentiation of bone marrow mesenchymal stem cells (MSCs) into cardiomyocytes has good application prospect. At present, there are mainly two methods about differentiation of bone marrow mesenchymal stem cells into cardiomyocytes, one is induced by differentiation inducer(such as5-aza), another is induced by myocardial microenvironment. But both of them have the lower inducing differentiation rate. It's still an important subject needed to resolve as soon as possible that how to improve the differentiation rate of MSCs into cardiomyocytes.In recent years, with the rapid development of traditional Chinese medicine (TCM) in the field of stem cells, the researchers found that Traditional Chinese medicine in promoting MSCs proliferation and differentiation into myocardial cells could play a positive role. Traditional Chinese medicine can not only promote MSCs proliferation, differentiation in vivo, but also have effect on cardiac microenvironment, promote the survival and function of MSCs, so have a good prospect of application. Astragalus and its effective monomer composition of astragaloside IV is one of them. Earlier period research results show that astragaloside IV can successfully induce MSCs differentiation into cardiomyocytes in vitro, AST and5-aza combined induction can reduce5-aza induced concentration, strengthen cells activity.In view of the present situation that the lower differentiation rate of MSCs into cardiomyocytes, based on the early research foundation, this study used AST and5-aza for combined induction in cardiac-like microenvironment. We observed the differentiation rate of combined induction and compared the functional differences in induced cells to show the advantages of combined induction by AST and5-aza in cardiac-like microenvironment. At the same time, we preliminary discuss the possible mechanism of AST during the process. Aims to explore the feasible and effective methods of enhancing differentiation rate, and provide a theoretical basis and technical support for its clinical application.ObjectiveTo observe whether the combined induction by AST,5-aza in cardiac-like microenvironment can improve the differentiation rate of bone marrow mesenchymal stem cells into cardiomyocyte-like cells or not, and evaluate whether the induced cardiomyocyte-like cells can be with the function of cardiomyocytes. At the same time, we preliminary discuss the possible mechanism of AST during the process.MethodsMale/female SD Rats, SPF, weighted50g and aged3-4weeks, were purchased from Laboratory Animal Center of Guangzhou University of Traditional Chinese Medicine. MSCs were isolated from SD rat's leg by density gradient centrifugation and adherent culture. MSCs were purified by culture-expanded, labeled with mouse anti-rat anti-bodies for CD34(FITC-conjugated) and CD44(FITC-conjugated). The labeled cells were analyzed by FACScan flow cytometry, and expanded as undifferentiated cells in culture for8passages. A certain amount of MSCs were inoculated into a culture plate with6holes with1×104/mL density,24h later, replaced the culture medium. Thereafter, the cells were assigned into5group:group I (replaced basic culture medium); groupⅡ (cells were incubated in10umol/L5-aza,24h later, replaced basic culture medium); groupⅢ (replaced basic culture medium containing myocardial cell lysate, as the simulated cardiac microenvironment in vitro); groupⅣ (cells were incubated in100mg/l AST and10umol/L5-aza,24h later, replaced basic culture medium containing myocardial cell lysate); group Ⅴ (cells were incubated in10umol/L5-aza,24h later, replaced basic culture medium containing myocardial cell lysate).2weeks after induction respectively, Cells were identified by the method of Immunohistochemistry to observe the expression of cardiac-specific structural proteins cTnT, Desmin, Cx43; calculated the differentiation rate; We also observed the levels of ANP mRNA expression and intracellular second messenger cyclic adenosine monophosphate levels (isoproterenol stimulation); at the same time, tested intracellular HGF and IGF-1content in reduced cells.Results1.72-96hours after culturing, anchorage-dependent cell appear and become fusiformis gradually.14days later, cells begin to merge and connect with each other which manifest as lamellar structure. The expression rates of superficial symbol antigen CD34on MSCs is0.4%while that of CD44is98.5%which conform to the feature of expressions of the surface markers of MSCs. Above all, we think the cultured cells are undifferentiated cells with high purity.2. After2w induction, the cells in all of the induction groups could be detected the expression of cTnT, Desmin, Cx43. The induction effect of cTnT, Desmin, Cx43is higher in group Ⅳ than that in group Ⅰ/Ⅱ/Ⅲ/Ⅴ(P<0.01). The differentiation rate of group Ⅳ respectively reached31.22%,36.65%,32.42%.3. After2w induction, the expression of ANP mRNA in group Ⅳ was significantly higher than that in group Ⅱ, group Ⅲ and group Ⅴ(P<0.01). Isoproterenol can significantly improve the level of cAMP in induced cells, most obviously in gruop Ⅳ.4. After2w induction, intracellular HGF, IGF-1content in group Ⅲ, Ⅳ and V was significantly higher in that in group Ⅰ and group Ⅱ (P<0.01或0.05). There were no statistically difference between group Ⅰ and group Ⅱ (P>0.05). Among all of the induction groups, the level of Intracellular HGF, IGF-1in group Ⅳ was the highest. Conclusion1. The combined induction by AST and5-aza in cardiac-like microenvironment can be efficient expression of cardiac-specific proteins cTnT, Desmin and Cx43, with the higher differentiation rate.2. In cardiac-like microenvironment, AST combined with5-aza induces MSCs into cardiomyocyte-like cells, the induced cells not only have the similar forms with cardiomyocytes but also have the good functions.3. During the course of differentiation, the effect of AST may be related to promote MSCs paracrine growth factor HGF, IGF-1in cardiac-like microenvironment, indirectly contributed to improve the efficiency of MSCs induced differentiation.4. It's proved that the method of combined induction by AST and5-aza in cardiac-like microenvironment is a kind of feasible, effective induction method with certain advantages, and worth further study, popularization and application.