Abstract:

Objective To explore the effect of the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into cardiomyocyte-like cells in vitro using different concentrations of TGF-β1(transforming growth factor beta1). Methods BMSCs of the SD rat were isolated and cultured from rat bone marrow, then the 2nd-generation BMSCs were induced by 2, 5, 10, 15μg/L TGF-β1 for 72 hours. The cultured cells were observed under a phase-contrast microscope. The immunohistochemical technique was used to examine the expression of desmin,α-sarcomeric actin, cTnT and Cx43. The laser scanning confocal microscope (LSCM) was used for the expression of α-sarcomeric actin and cTnT. GATA-4 and α-myosin heavy chain (α-MHC) expression were detected by relative quantitative RT-PCR after 7, 21, 28 days of induction respectively. Results 1.The morphous of BMSCs cell was different in each treatment group. BMSCs after being induced by 2, 15μg/L TGF-β1 on 7days were the same as the control group. BMSCs induced by 5μg/L TGF-β1 on 28 days showed a fusiform shape, orientating with one accord, forming myotubule-like structure. The morphous of BMSCs after being induced by 10μg/L TGF-β1 was the same as the 5μg/L group, but the number of cells was relatively small. 2. BMSCs induced by different concentrations of TGF-β1 on 28 days were identified by the positive staining for desmin, α-sarcomeric actin and Cx43. The positive rates were higher than that of the control group. 3. The rate of cTnT-positive cells in the 5μg/L treatment group was greater than that of the 15μg/L treatment group (P =0.000) and the 2μg/L treatment group(P =0.028). The number in the control group was lower than that of the treatment groups(P <0.01). 4. Under LSCM after immunoflurescence labeling, we found that bright red fluorescence in the cytoplasm was observed in the α-sarcomeric actinpositive cells, green fluorescence in the cytoplasm was in the cTnT-positive cells while the part that overlaps was yellow. 5.RT-PCR assessment showed that the differentiated cells began to express GATA-4 from day 7 to day 28 of differentiation and began to express α-MHC from day 21 to day 28 of differentiation. Conclusion TGF-β1 may induce BMSCs to acquire cardiogenic phenotype and 5μg/L is a suitable induced concentration.

Key words: Bone marrow mesenchymal stem cells, Transforming growth factor beta 1, Cardiomyocytes, Differentiation, Immunohistochemistry, Rat