| Objective Establishing training system of SD rat bone marrow mesenchymal stem cells(BMSCs) and screening out a better method for inducing BMSCs differentiate into neuron-like cells in vitro,so as to lay the foundation for the next experiments of inner ear transplantation.Method First, cultured BMSCs:First, BMSCs were cultured and subcultured by the main method of whole bone marrow directly adherent in flasks, and BMSCs of the third generation used for flow cytometric detection about the surface markers CD29,CD90of BMSCs and the surface antigen CD45of hematopoietic cell. Second,BMSCs of the fourth generation were pre-induced24h by basic fibroblast growth factor(bFGF),and then divided into2groups:group A was added the inducers of neurotrophin-3(NT-3) and retinoic acid (RA),and group B was added the inducer of beta-mercaptoethanol(BME). After induction of1h,5h,24h,7d,14d and21d,the2groups cells were identified by immunofluorescence whether express neuron-specific enolase(NSE) as markers of mature neurons,and were observed whether positive staining of neuron-like cells and compare the level of the positive rate.Result First we can harvest many cells,which can proliferation and differentiation quickly,by the method of whole bone marrow directly adherent in flasks. Second,BMSCs can espress CD29,CD90and can not express CD45by flow cytometric detection.And combined with cell’s morphology,the cells is comply with BMSCs characteristic.Third, the result of cell immunofluorescence show NSE expression and the positive rate of induced to differentiate into neuron-like cells in group A is significantly higer than group B.Conclusion First BMSCs cultured by the method of whole bone marrow directly adherent in flasks can harvest many high purity cells, which can proliferation and differentiation quickly. Second, the method of step-by-step guidance and the combined NT-3with RA is a better method of inducing,which induced by bFGF in the pre-24h,and then switch to NT-3+RA induced to differentiate into neuron-like cells which can express NSE was significantly higher than BME. |
