Cardiomyocyte-Likecells Differentiation Of BMSCs Induced By 5-AZA And Tanshinoneâ…¡A In Rat

With improving living standard and changing of diet structure the incidence of cardiovascular system disease is an upward trend. The cardiovascular system diseases such as myocardial infarction rely on traditional thrombolysis, anti-heart failure and resisting arrhythmia treatment, which can not repair the dead or lost cells. There are no specific efficient drugs or treatment for dilated cardiomyopathy, but there is symptomatic treatment aiming at heart failure symptoms. The existing researches also confirmed that Chinese medicine treatment of ischemic heart disease such as myocardial infarction(mi), to a certain extent, alleviated the patient’s clinical symptoms with less side effect and high safety. However, simple traditional Chinese medicine treatment was unable to supply new cells for damaged heart muscle cells in a short period of time. Heart transplantation is the only effective treatment for advanced cases, so the researchers and clinical doctors are trying to find new treatment of repairing or regenerating necrotic myocardial tissue and cells. The treatment of ischemic heart disease by separating the stem cells into cardiomyocytes may promote cell regeneration and angiogenesis. In recent years, Stem cell transplantation has become the hot topics in the study of diseases of the cardiovascular system and has been highly valued by experts and scholars both at home and abroad.Mesenchymal stem cells(BMSCs) have stronger ability of differentiation with the potential of multi-directional differentiation and strong plasticity, so they become the "seed cells" of cell transplantation in the treatment of ischemic heart disease. Under a specific tissue microenvironment BMSCs can differentiate into mature organizational needs cells, such as endothelial cells, nerve muscle cells, liver cells and cardiomyocytes. In order to ensure the treatment effect, it is essential to amplificate, purificate and improve the conversion rate of BMSCs. At present 5- nitrogen cytidine(5- azacytidine, 5- aza) is inducer used by most researchers for directional differentiation of BMSCs into myocardial cells. Inducers from traditional Chinese medicine in the clinical treatment of cardiovascular diseases also gradually become the object of attention. Tanshinone Ⅱ A is an effective component extracted from traditional Chinese medicine salvia miltiorrhiza. Studies have shown that tanshinone Ⅱ A has great potential in the process of inducing stem cell proliferation and differentiation.This study explores the joint of 5- nitrogen cytidine and tanshinone Ⅱ affect directional differentiation of BMSCs into myocardial cells. The experiment is aiming at providing reference for the treatment of ischemic heart disease by cell transplantation.First separate and collect BMSCs by centrifuging from the long bone marrow which from healthy SD rats, take the second generation of cell and cultivate 48 h, divide the cells into the following four groups:(1) add the culture medium containing tanshinone Ⅱ A(final concentration of 500 ng/ml); 3 days later, remove the liquid containing induction medium, add new media(excluding induction medium) and culture 4w.(2) add the culture medium containing 5- cytidine(final concentration of 250 mu g/L); 3 days later remove induction medium; add medium without containing nitrogen cytidine 5 and continue to develop 4 w.(3) add the culture medium with both tanshinone Ⅱ A and 5- nitrogen cytidine(final concentration respectively 500 ng/ml and 250 mu g/L); 3 days later remove induction medium; add medium without containing tanshinone Ⅱ A and cytidine 5- nitrogen to develop 4 w.(4) cultivate control groups containing no inducers medium; change the liquid after three days; exchange liquid at a regular intervals(1/3 days); observe cells daily; when the density of cell fusion of adherent cells reach 90%, 0.04% EDTA and 0.5% pancreatin(1:1) should be used to digest; then inoculation and passage with the density of 2: 1.. Test the expression of protein, striated muscle actin(a- SCA- actin) and myocardial specificity of troponin T(cTnT) with Uimmunocytochemistry techniques. detect the expression of joint induced group—SCA- actin and cTnT by Immunofluorescence double standard dyeing technology. Observe ultrastructural changes of combined induced group by transmission electron microscope. Immunocytochemical staining: chose the 4 weeks culture cells from the induce experimental of the 4 groups, observe cell α-actin(α- striated muscle actin), desmin(desmin), cTnT(cardiac troponin) positive cells in light microscopy. Immunofluorescence double staining technique: the first step is to take cells of TanshinoneⅡA with 5-aza induced group cultured 4 weeks; then use fluorescence to label cell α-actin, cTnT protein and observe α-actin, cTnT in co-expression. This technology is to observe the internal structure of induced cell of TanshinoneⅡA with 5-aza by TEM, which are titled into slice by the methods of centrifugation, dehydration and other treatments. Results: BMSCs culture: The cells are round and gradually increase after been inoculating for 24 h, which are isolated and cultured from centrifuging and separation screening adherent BMSCs. It also shows that the number of adherent fusiform cells significantly increase after 3d and proliferated 6d later. Immunochemical detection of cell: It shows that the expression of cells from desmin, α-actin, cTn T is seen in induced group. Through observation, cells of TanshinoneⅡA with 5-aza in induced group are significantly higher than the other groups(P <0.01 or P <0.05). Immunofluorescence double staining: It confirms that there are red and green positive expression cells in TanshinoneⅡA combined with 5-aza and in cTnT from induced differentiation group. When these two are overlapped, it is observed yellow. TEM:The TEM shows us the ship of differentiated cells was light and dark strip with fine muscle parallel wire-like structure, and some of them appear projection surface, the nucleus located center. The cytoplasm not only contain Golgi complex, glycogen granules, rough endoplasmic reticulum, but also other rich organelles.The Conclusion shows that BMSCs can be differentiated into cardiomyocytes successfully by the individual induction of TanshinoneⅡA, 5-aza and combined induction of TanshinoneⅡA and 5-aza. With the comparison of expression of α-actin, cTnT of differentiated cells in each experimental group, as well as morphology comparison, there was significantly higher positive differentiation rate of TanshinoneⅡA with 5-aza than that of TanshinoneⅡA and 5-aza alone, which were used to be induced bone marrow mesenchymal stem cells into cardiomyocytes differentiated. The way of combining TanshinoneⅡA with 5-aza was more efficient.