Effect Of Notch1 Signaling On Differentiation Of Rat Bone Marrow Stromal Cells Into Neuron-like Cells Induced By Fasudil Hydrochloride

Background & ObjectiveMarrow mesenchymal stem cells which are isolated from mature bone marrow, adipose tissue, umbilical cord blood and so on have a strong capacity of proliferation, self-renewal and differentiation. It can not only differentiate into the mesodermal cells, such as skeletal muscle cells, cadiocytes and adipocytes, but also differentiate into the ectodermal cells, such as neurocytes, epithelial cells.The finding of MSCs has brought a new strategy of therapy for a variety of neurodegenerative diseases, central nervous system injury and stroke.It has brought new hope for patients and may have extensive prospects of clinical application. Thus the research about biological characteristics, isolation and culture, induced differentiation, the mechanism of neurocyte differentiation of MSCs and transplantation to treat neurological diseases has become an important domain of neuroscience research. So far, There is low differentiation and differentiated mechanism is not clear when bone marrow mesenchymal stem cells differentiate into neural cells in vitro. Therefore, The methods of inducing bone marrow-derived mesenchymal stem cells differentiation into neural cells and the differentiated mechanism needs further study.Notch signaling pathway is an important cellular signaling pathway in biological body.Notch signaling involves in embryonic nervous system development, regulation of stem cell proliferation, differentiation and apoptosis and other biological activity. Some studies have shown that the expression of Notch signaling molecules in bone marrow-derived mesenchymal stem cells decreases during differentiation into neural cells.It is favorable for their differentiation into neurons when the notch signaling is inhibited while the role of Hesl protein is not very clear in this process. Recent studies have found that CoC12 combines with Rho kinase inhibitors can induce bone marrow mesenchymal stem cells differentiate into nerve cells. This result prompt us that the RhoA/Rho kinase signaling pathway may be involved in regulating the process of bone marrow mesenchymal stem cells differentiating into neuron-like cells.Therefore, this study is to explore the role of fasudil hydrochloride in the course of bone marrow-derived mesenchymal stem cells differentiating into neuron-like cells, and whether the Notch signaling inhibition is conducive to promot the efficiency of differentiation on bone marrow-derived mesenchymal stem cells into neuron-like cell induced by fasudil hydrochloride. There is an important significance in studing deeply the differentiated mechanisms of bone marrow-derived mesenchymal stem cells differentiating into neuron-like cells. This is also important for clinical application of bone marrow-derived mesenchymal stem cell transplantation combined with fasudil hydrochloride to treat some related nervous system diseases in the future.Materials and MethodologyThe experimental animals are Wistar rats with aged from February to March, weight from 150 to 200g and either sex. The cells are subcultured from the MSCs more than 10 generations for experiments. The MSCs for experiments were divided into non-transfected group, transfected group (transfected with Rn-Notch1-siRNA), positive control group (transfected with Rn-MAPK-1 Control siRNA) and negative control group (transfected with Negative Control siRNA). Fasudil hydrochloride induces MSCs differentiating into neurons. The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope. The expression of notchl mRNA, Hesl mRNA and MAPKl mRNA in MSCs was detected by RT-PCR. The expression of Notchl protein, Nestin, neurofilament M (NF-M) and glial fibrillary acidic protein (GFAP) was detected by immunocytochemical method. The viability of MSCs was detected by MTT assay.Results1. The fluorescence of MSCs was mostly displayed after transfection of 72 hour and the efficiency of transfection was up to 0.91±0.04. Meanwhile, the notchl mRNA and Hesl mRNA expressed by MSCs of transfected group was significantly decreased (P<0.05) and MTT displayed that the viability of MSCs was also significantly reduced (P<0.05). This difference between control group has statistic significance2. Fasudil hydrochloride can induce MSCs differentiating into neuron-like cells and the best efficiency of induction was observed in the transfected group. There was higher expression of Nestin and neurofilament-M (NF-M) than the other group (P<0.05).This difference has statistic significanceConclusion1. Fasudil hydrochloride can induce MSCs differentiating into neuron-like cells.2. The Notch signaling inhibition is conducive to promot the efficiency of differentiation on bone marrow-derived mesenchymal stem cells into neuron-like cell induced by fasudil hydrochloride.3. There may be notchl signaling and Rho/Rho GTPase signaling synergy on differentiation of rat bone marrow stromal cell into neurons induced by fasudil hydrochloride and they jointly promoted the differentiation of MSCs into neurons.