关键词: 骨髓源性心肌干细胞, 衰老, 骨形态发生蛋白-2, 细胞分化, β-半乳糖苷酶, 活性氧, 流式细胞术

Abstract: Objective To investigate the changes in morphology and senescence-associated markers of the marrow-derived cardiac stem cells (MCSCs) from rats at different ages and to explore the impacts of age on proliferation, survival and differentiation of MCSCs. Methods With single-cell cloning culture, MCSCs were selected from the bone marrow of young, adult and aged male SD rats respectively. Ultrastructural changes of the cells were viewed under a transmission electron microscope. The senescence-associated changes were examined with SA-β-galactosidase staining and reactive oxygen species(ROS) staining. Distribution of cell cycle of MCSCs from different age groups was evaluated with flow cytometric analysis. Rates of the survived and apoptotic cells were determined by Annexin V/PI double-labeled flow cytometric analysis and Hochest33342 staining. Differentiation of the MCSCs toward cardiomyocytes was induced with BMP-2. Expression of cardiac transcription factors and cardiac specific genes of the cells after induction were examined with RT-PCR. CTnT expression of the cells also be examined with immunocytochemistry. Results The nucleus/plasma ratio of the cells from aged rats decrease and there are some myelin bodies in the cells of aged group. With increasing of age, the MCSCs in S+GSUB>2/SUB>/M phase reduce, while β-galactosidase-positive cells and ROS-positive cells increase. Survival rate of the cells from aged rats is lower than that of the cells from young rats. At four week after induction with BMP-2, expression of Nkx2-5, GATA-4, cTnT mRNA and Cx-43 mRNA of the cells of young group increase significantly. In adult and aged group, expression of the cardiac transcription factors and cardiac specific genes is lower than that of the cells in young group. In immunocytochemical staining, cTnT expression of the cells in young group is stronger after induction with BMP-2. As compared with that of the cells in young group, cTnT expression of the cells in aged group is weak after induction. Conclusion With increasing of age, MCSCs show senescent changes, including their abilities of proliferation, survival and differentiation toward cardiomyocytes decrease.

Key words: Marrow-derived cardiac stem cells, Aging, BMP-2, Cell differentiation, β-galactosidase, Reactive oxygen species, Flow cytometry