OBJECTIVE: To investigate the effect of salidroside on rat bone marrow mesenchymal stem cells (BMSCs) differentiation into the cholinergic nerve cells, so as to provide the theory basis of the combination of salidroside and stem cells for clinical therapy of nervous system diseases.METHODS: BMSCs were isolated from 2 Wistar rats (aged 4-6 weeks,weighing 120 g), which were identified by CD34, CD45, CD90, and CD106 with flow cytometry. According to inducing method, BMSCs at passage 2 were divided into 3 groups: In groups A and B, BMSCs were induced by salidroside (20 microg/mL) and retinoic acid (5 micromol/mL) respectively for 1, 3, 6, and 9 days, in group C, BMSCs were cultured with serum-free DMEM/F12 medium as control. MTT assay was used to detect the cellular proliferation activity. The immunofluorescence chemical technology was used to detect the expressions of nerve growth factor (NGF) and relevant marker molecule of nerve cells, including neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP2), beta-Tubulin III, glial fibrillary acidic protein (GFAP), and the marker of cholinergic neuron, such as Acetylcholine (Ach) and NGF. RT-PCR was used to detect mRNA expressions of NSE, beta-Tubulin III, GFAP,brain derived neurotrophic factor (BDNF),and gamma-aminobutyric acid (GABA). ELISA was used to detect the levels of BDNF and NGF, and the expression level of NGF protein was analyzed by Western blot.RESULTS: The results of the flow cytometry showed that the cultured cells were CD90 and CD106 positive, and CD34 and CD45 negative,which indicated that the cells were BMSCs. The cellular proliferation activity in groups A and B were significantly higher than that in group C at 6 days and 9 days (P < 0.05). RT-PCR results showed that the expression level of NSE,BDNF, beta-Tubulin III,GFAPmRNA were increased in group A at 6 days; In group B, that expression level of NSE mRNA was up-regulated at 6 days, that expression level of BDNF mRNA increased at 1 days and reached the peak at 6 days, and that expression level of beta-Tubulin III mRNA was up-regulated at 3 days, which was significantly higher than that at the other time points, and than that in group C (P < 0.01). But no GABA mRNA expression was detected in each group. Immunofluorescence chemical technology staining showed that the positive rates of NSE, MAP2, beta-Tubulin III, and GFAP were significantly higher in group A than those in group C at 3 days; the positive rates of Ach were significantly higher at 3, 6, and 9 days than those at 1 day in groups A and B, and in groups A and B than in group C (P < 0.01); the positive rates of NGF in groups A and B were significantly higher than those in group C (P < 0.01). The levels of BDNF and NGF in groups A and B were significantly higher than those in group C at 1, 3, 6, and 9 days (P < 0.01), but no significant difference of BDNF was found between groups A and B (P > 0.05). The expression level of NGF protein in groups A and B were significantly higher than that in group C (P < 0.01). The NGF expression reached the peak at 6 days in group A and at 3 days in group B.CONCLUSION: Salidroside could induce rat BMSCs differentiate into cholinergic nerve cells in vitro.
Animals Bone Marrow Cells