Study On The Relationship Between Rac1 And The Neurogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells

Objective: To study the relationship between Rac1 and the neurogenic differentiation of mesenchymal stem cells, broadening our understanding of molecular mechanism to the neurogenic differtiation of BMSCs.Methods: BMSCs were isolated by Percoll gradient centrifugation and the purified cells were induced into neuron-like cells by using two treatments: BHA in combination with bFGF, RA in combination withβ-ME. Morphological changes of cells during the induction by two treatments were observed under microscopes. The expression of neural markers nestin,β-tubulin and NSE, of induced cells were showed by immunofluorescence. We used BHA in combination with bFGF to induce BMSCs into neuron-like cells after comparing the effect of two methods. BMSCs markers CD90 and neural markers nestin,β-tubulin, NSE, NCAM were detected by immunofluorescence during the neurogenic differentiation of BMSCs. Rac1 was verified by immunofluorescence staining as well. Rac1, Cdc42 and RhoA expression was analyzed by western blot. The active proteins of Rac1, Cdc42, RhoA were pulled-down and analyzed by western blot.Results: Treatment of BMSCs with BHA for 5 hours led to a dramatic change in morphology, such as cell body contraction and neural-like axis production, and the expression of nestin andβ-tubulin, characteristic markers for neural precursors and neurons. NSE positive cells were detected by 24 hours. In contrast, cells induced by RA andβ-ME didn't show typical neural appearance. However, neural markers could be detected 7 days later, indicating a neurogenic differentiation phenotype. During the differentiation of BMSCs induced by BHA and bFGF, Rac1 expression was persistently positive. BMSCs lose their typical markers after induing 5 hours, while the neural markers were showed positive at the same time.Rac1,Cdc42 and RhoA are molecular switches that use a simple biochemical strategy to control complex cellular processes. They cycle between two conformational states: one bound to GTP active state, the other bound to GDP inactive state.The expression of Rac1 was decreased obviously after inducing by BHA and bFGF for 5 hours by western blot. Then we maintained the induced cells with serum-free medium, the expression of Rac1 keeped in a low level. Maintaining the induced cells with BMSCs medium containing 10% serum resulted in a change in morphology, which remind me the appearance of BMSCs, and an increased Rac1 expression. It's worth noting that these cells can be induced for the second time, and the Rac1 expression decreased again. Cdc42 and RhoA expression was in accordance with Rac1.The expression level of Rac1,Cdc42 and RhoA protein was decreased during the neurogenic differentiation of bone marrow mesenchymal stem cells. The activity of Rac1 and Cdc42 was upregulated when BMSCs differentiating into neuron-like cells while the RhoA activity was downregulated.Conclusion: Rac1 is closely related to the neurogenic differentiation of BMSCs.