Down Regulation Of Sox2 Gene Expresion Affecting P19 Stem Cell Differentiation Into Cardiomyocytes

The main factors of exasperate heart function are myocardial necrosis, cicatrization and ventricular remodeling, which are both the primary pathological bases of chronic congestive heart failure. Cellular transplantation treatment is a new method with treating heart disease. Stem cell is the most perfect seeded cell for cellular transplantation treatment. For that reasons, the researches focused on the mechanism of stem cell differentiation into cardiomyocytes, which have important significance in theory and clinic. P19 cell is a embryonic stem cell line which possesses the capability of differentiation into cardiomyocyte. The cellular transplantation treatment that is injection or transplantation the cardiac stem cells by coronary reflow which are cultured in vitro to the position of myocardial infarction could make myocardial remodeling in the area of lesion and improve the function of failing heart. The research studies the change in morphology and the cardio-specific gene expression, which provide a perfect cellular model for the fundamental researches of cellular transplantation treatment for myocardial infaction. The normal P19 cells and the treated P19 cells whose Sox2 gene was interfered by RNAi were separately induced and differentiated by dimethyl sulfoxide (DMSO) into cardiomyocytes. Then, the process of cell aggregation and differentiation into cardiomyocytes was tested. Detect the change of cells shape, the cardio-specific marker expression and expression of mRNA of cardio-specific genes during the differentiation into cardiomyocytes period by morphocytology, immunohistochemistry and RT-PCR tests. It was shown that cell aggregations were cohering-cultured with growth culture medium after DMSO induced for 2 days, spontaneous and rhythmically beating cells were present within the aggregation outgrowths on 10th day. On 4th day cells appeared fusiform shape and on 11th day cells were arrayed parallelly connecting with each other. The cells showedα-actin-positive and on 5th day expressed cardiomyocyte specific mRNA. The cardio-special genes were expressed by RT-PCR, and the expression increases with the inducing and differentiation period. The treated P19 could not form the aggregations and differentiate into cardiomyocyte with co-hering cultured. This experiment choose the fragment with the best interfering efficiency and use siRNA interfering Sox2 gene . It showed the influence of stem cells differentiation into cardiomyocytes with deficiency the pluripotency factor Sox2 gene. We choose the best concentration of DMSO for differentiation and use a new method on aggregating cells, which could obtain numerous and uniformitarian embryonic bodies easily for a large scale differentiation experiments with a short term.