关键词: 骨髓间充质干细胞, 1,25-维生素-D₃, 心肌样细胞, 细胞分化, 免疫印迹法, 大鼠

Abstract:

Objective To explore the optimal concentration of 1,25-vitamin-D₃ for inducing the differentiation of bone marrow mesenchymal stem cells (BMSCs) into cardiomyocyte-like cells in vitro. Methods BMSCs of SD rats were isolated and cultured by whole bone marrow adherent method combined with density gradient centrifugations. Depending on the final concentration of 1,25-vitamin-D3, the 2nd-generation BMSCs were divided into five groups: 3 nmol/L group, 6 nmol/L group, 12 nmol/L group, 24 nmol/L group and the control group. The adherent cells were observed dynamically under the inverted phase contrast microscope, including their morphology and growth status. The surface antigens, morphological characteristics, protein expression and mRNA expression of the cells in each group were assessed. Results 1. Under the inverted phase contrast microscope, most of the primary cells showed a short spindle shape after 72 hours of culture. After 1 week of culture, the cells showed diversified morphology. The adjacent cells of BMSCs, induced after 4 weeks, were closely connected with each other, and the arrangement had obvious directivity. There were differences in the number and morphology of BMSCs, induced by 1,25-vitamin-D₃ at different concentrations. 2. The result of flow cytometry showed that the positive expression rates of CD29, CD45 and CD90 were 97.4%, 3.3% and 91.4%, respectively, 3. The results of immunofluorescence, immunocytochemistry and Western blotting revealed that the expressions of tropomyosin(TPM), connexin43(Cx43) and cardiac troponin T(cTnT) in 6 nmol/L group were significantly higher than that of the other groups, while that in control group were weak or negative (P<0.05). 4. Transmission electron microscope(TEM) observation showed that the induced cells had a cardiomyocyte-like ultra structure: there were many parallel arranged myofilaments, mitochondria, ribosomes, rough endoplasmic reticulum and other organelles in the cytoplasm. 5. The result of Real-time PCR showed that the induced cells could express GATA binding protein 4(GATA 4) and Nkx2.5 at the 1st week, and then the expression of them decreased at the 2nd week, but then increased at the 4th week. The 6 nmol/L group was superior to the other three groups in gene expression (P<0.05). Conclusion 1,25-vitamin-D₃ can induce BMSCs to obtain myocardial differentiation phenotype, and the optimum concentration of inducing differentiation is 6 nmol/L.

Key words: Bone marrow mesenchymal stem cell, 1,25-vitamin-D₃, Cardiomyocyte-like cell, Cell differentiation, Western blotting, Rat