Effect Of Rac1 Inhibition On Neurogenic Differentiation Of Bone Marrow Mensenchymal Stem Cells

Objective: To study the role of Rac1 during the neurogenic differentiation of mesenchymal stem cells by inhibiting the expresstion of Rac1 through RNAi.Methods: BMSCs were obtained by Percoll gradient centrifugation technique, and phenotypically identified by immunofluoresce staining, and further confirmed by both osteogenic and neurogenic differentiation. BMSCs were induced to neural-like cells by the following steps: cells were treated with 10 ng/mL basic fibroblastic growth factor (bFGF) for 24 h, followed by 200μMβ-hydroxyanisole (BHA) plus 2% dimethylsulfoxide (DMSO) for 5 h. Cells were maintained with DMEM (H) plus N2 for 48 h before immunostaining to detect the expression of neuronal markers like Nestin,β-Ⅲ-Tubulin and NSE. We designed three interference microRNA sequences targeting specifically Rac1, and clone them into pcDNA?6.2-GW/EmGFP-miR plasmid. Transfected the recombination plasmids into BMSCs and subjected to Western blotting analysis. With Rac1 inhibition, BMSCs morphology changes and expression of Nestin and NSE is studied during the neurogenic differentiation.Results: Immunofluoresce analysis showed that BMSCs of 5 and 20 passages were positive for CD29, CD90, CD106, CD71 and negative CD45. After neurogenic differentiation for 5 h, BMSCs displayed long bipolar branching with small, spherical, and refractive cell bodies. The number of cells with morphology changed increased progressively by 48 h, some of them formed secondary, tertiary, and long branches. Nestin expression was significantly increased after induced for 5 h and the percentage of Nestin positive cells was 66.52%±0.8%. Nearly all treated cells expressedβ-tubulin at the same time and the percentage increased over 95%. After maintaining for 48 h, NSE expression was significantly elevated while Nestin decreased. Transfected the recombination plasmids into BMSCs and Western blotting analysis demonstrate that miRac1 constructs efficiently specifically downregulate Rac1 protein levels. In neurogenic differentiation, BMSCs with the inhibition of Rac1, can not display the morphology changes and can not express the neuronal marker.Conclusion: Inhibition of expression of Rac1 in BMSCs will inhibit the neuronal differentiation. Written by Song Lin...