Experimantal Study Of Sodium Tanshinone â…¡A Silate On The Proliferation Of The BMSCs And The Differentiation Into Cardiomyocyte-like Cells

BackgroundBone mesenchymal stem cells(BMSCs), besides hematopoietic stem cells, another stem cells existing in bone marrow, could self-replicate, self-renewal and have multilineage differentiation potential. It has been considered as a promising candidate for their some characteristics such as obtained easily, the less immunogenicity, easiness to separation and amplification in vitro, the power of differentiation into the myocardial cells, endotheliocytes and vascular smooth muscle cells. Therefore, BMSCs are thought to be a suitable source of cell transplantation for the therapy of infarcted myocardium. But its applied into clinic is restricted because of its less source. And its differentiation into the other cells needs help from exogenous factors. Therefor, this experiment use the sodium tanshinonⅡA silate(DS-201) to intervent the BMSCs, aiming to study the effects of the DS-201 on the proliferation of the BMSCs and the process of differentiating into cardiomyocyte-like cells.Objectives1 To study the separation, purification and amplification of the BMSCs of Sprague Dawley(SD) rat in vitro, to establish the mature and stable culture system of BMSCs, to provide sufficient cell support for posterior experimental research.2 To research the effects of DS-201 on the proliferation of BMSCs. Find the optimum concentration of DS-201 which promots the proliferation of BMSCs. Use this concentration to intervent the BMSCs continuously and detect the effects of this concentration on the growth trand of the BMSCs.3 To detect the effects of the DS-201 in the process of differentiating into cardiomyocyte-like cells.Methods 1 The methods of the separation, purification, amplification and identification of the BMSCs of SD rat in vitro. To culture the rat BMSCs using the adherence screening method, to detect the antigenic expression of CD29,CD34,CD44 using the immunofluorescence staining method. With the MTT method, determine the absorbance of the BMSCs of passage P1-P5 in different time at 24h,48h,72h,96h,120h, to describe its growth process, and draw the growth curve.2 The effects of the sodium tanshinon IIA silate on the proliferation of BMSCs. Choose eight different concentration of sodium tanshinon IIA silate to intervent the P3 BMSCs. Every team has five repeat hole. After 24h, use the MTT method to determine the absorbance of every team. Choose the optimum concentration promoting the proliferation of BMSCs. And use this concentration intervent continuously for 3 days. Statistic the absorbance data at different time points of 24h,48h,72h,96h., and 120h. According to the date draw the curve of the growth of BMSCs.3 The effects of the sodium tanshinon IIA silate on the differentiation of BMSCs to the cardiomyocyte-like cells. Use different medicines to intervent the process of the BMSCs differentiation to the cardiomyocyte-like cells. With the Rever transcriptase-polymerase chain Reaction method, determine the expresss of the gene of SDF-1,αMHC,βMHC to research which is the best intervention way of the BMSCs to the cardiomyocyte-like cells. Results1 BMSCs were isolated by the whole bone marrow adherent cultivation. We operate the first medium change after 48 hours. The cells begin to enter the exponential phase of growth phase after 3 days, cells begin rapid proliferation, as well as the majority of cells displayed spindle-like shape. The cell would spread out fully at the ninth or tenth day for original generation. They have radiation or swirl shape. After the cell carried out passage, they adhere the culture dish quickly, no eclipse period, entering the growth period, at the fifth day cell might enter the next passage. The cell have been depurated at passage 3. Cells of passage 3 were used identification. The results using the immunofluorescence staining are positive expression of CD29 (expressed BMSCs) andCD44(expressed BMSCs), negative expression of CD34(positive expression on the hemopoietic stem cells and endothelia cells).2 DS-201 has effects on the proliferation of BMSCs which is concentration-dependent:thick concentration of DS-201 inhibit the proliferation and optimum concentration is helpful to the proliferation. The optimum concentration of DS-201 to the proliferation of BMSCs is 1.5×10-2umol/L.3 With the Rever transcriptase-polymerase chain Reaction method, we determined the expresss of the gene of SDF-1,αMHC,βMHC and according the data we found that the team of DS-201 together with 5-aza is the best to the differentiation of the BMSCs to the cardiomyocyte-like cells. Conclusions1 We have established a successful culture system for BMSCs of SD rat in vitro. The cell were rapid proliferation, good condition and stable biological character, more purification. The cells identified by cell appearance and cell-surface marker was BMSCs.2 DS-201 has double direction to the proliferation of BMSCs:thick concentration of DS-201 inhibit the proliferation and optimum concentration is helpful to it.3 The team of DS-201 together with 5-aza is best to the differentiationg of the BMSCs to the cardiomyocyte-like cells.