Fasudil hydrochloride differentiates bone marrow mesenchymal stem cells into neurons via notch signaling

摘要

BACKGROUND: Notch signaling regulates bone marrow mesenchymal stem cell (MSC) proliferation, differentiation, and apoptosis, Notch signaling and Rho kinase signaling exhibit a crosstalk phenomenon with JAK/STAT, and both participate in the neuronal dendritic spine development. Inhibition of RhoA/Rho kinase signaling may regulate MSC differentiation into neuronal-like cells. OBJECTIVE: To investigate the effect of Notch1 signaling on the differentiation of rat MSCs into neurons induced by fasudil hydrochloride (C14H17N3O2S·HCl), a Rho kinase inhibitor, through a siRNA approach. DESIGN, TIME AND SETTING: An in vitro cytological experiment was performed in the Cell Laboratory of Henan Academy of Medical and Pharmaceutical Sciences between December 2007 and May 2009. MATERIALS: MSCs were obtained from Wistar rat femoral bone, fasudil hydrochloride was provided by Tianjin Chase Sun Pharmaceutical Co., Ltd. Rn-notch1-siRNa, negative control siRNA (Cy3 label) and Rn-MAPK1 control siRNA were provided by QIAGEN, Coloqne, German. METHODS: The cultured MSCs were divided into non-transfected, transfected group (transfected with Rn-Notch1-siRNA), positive control (transfected with Rn-MAPK-1 control siRNA), and negative control (transfected with negative control siRNA) groups. Fasudil hydrochloride was applied to induce MSCs to differentiate into neurons. MAIN OUTCOME MEASURES: The fluorescence expression by the transfected MSCs was observed under an inverted fluorescence microscope; the expression of Notch1 mRNA, Hes1 mRNA, and MAPK1 mRNA in MSCs was detected by reverse transcription polymerase chain reaction; the expression of Notch1 protein, nestin, neurofilament M, and glial fibrillary acidic protein was detected by immunocytochemistry. The viability of MSCs was detected by tetrazolium bromide assay. RESULTS: MSC fluorescence increased following a 72-hour siRNA transfection, with transfection efficiencies of up to (0.91 ± 0.04); the Notch1 mRNA and Hes1 mRNA expressed by transfected MSCs was significantly decreased (P < 0.05) compared with non-transfected cells. Fasudil hydrochloride induced MSCs to differentiate into neurons with greater efficiency in the transfected group (P < 0.05). CONCLUSION: Fasudil hydrochloride induces rat MSCs to differentiate into neurons; inhibition of Notch1 signaling and Hes1 expression may jointly promote the differentiation of MSCs into neurons.