Study On The Effect Of Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells Into Neuron-like Cells Inducing By Liuwei Dihuang Wan And Basic Fibrolast Growth Factor In Vitro

Objective:This study is to observe the separation, cultivation and purification of BMSCs, explore the effect of Chinese medicine Liuwei dihuang wan which nourishing liver and kidney and basic fibrolast growth factor(bFGF) on the differentiation of rat bone marrow mesenchvmal stem cells (BMSCs) into neuron-like cells by cell biology techniques, we try to find a method to revecie a higher proportion of neuron-like cells.Methods:We cultured the flushing fluid of Sprague-Dawley rat bone marrow,, purified BMSCs by removing the unadherent cells to observe changes in morphology and proliferation. Detection and identification of cell surface markers CD34and CD44by immunocytochemical staining method to the3rd generation of cells to know whether the purified cells were BMSCs. To divided the BMSCs into control group, bFGF group, Liuwei dihuang wan group and bFGF+Liuwei dihuang wan group,they were induced by adding the corresponding induction medium after pre-induction for24hours by bFGF.Observing morphological changes of the cells in every group, identifying the induced cells by immunocytochemical staining of nestin,neuron-specific enolase(NSE), glial fibrillary acidic protein (GFAP) to judge whether the differentiation was occurrence in7days later.Results:1.We got purified BMSCs by removing the unadherent cells and other adherent cells after isolated the primary cells. BMSCs had good vitality which were cultured in L-DMEM containing10%fetal bovine serum in the3rd generation,it was good for the next induction. The immunocytochemical staining show that the cells expressed CD44while not expressed CD34.2. The constract between the3rd generation BMSCs and the control group after pre-induction for24hours showed cell number was increased and stereoscopic was enhanced. After induced by Liuwei dihuang wan for7days, BMSCs formed the more typical form of neural cells, bFGF+Liuwei dihuang wan group was more obvious.The cell morphology of control group did not have significantly changes in1to3days, but grow speed had slowed significa-ntly, and later they turnd to aging even death.3.The immunocytochemical staining results show that the neural cell makers were not expressed in the control group, but expressed in the other groups.Positive cells rate of Nestin、NSE and GFAP in the bFGF+Liuwei dihuang wan group were higher than the bFGF group and Liuwei dihuang wan group.Conclusion:1.The BMSCs had high activity and stability biological traits which were separated、cultured and purified by the method of bone marrow adherent seperation.2.The BMSCs could be differentiated into neuron-like cells in vitro with the induction of Liuwei dihuang wan, the combined induction of Liuwei dihuang wan and bFGF revecied a higher proportion of neuron-like cells.