| Objective: To establish an optimal protocol for the isolation and cultivation of and the possibility of nerve growth factor(NGF) inducing differentiation of human bone marrow stromal cells(hMSCs) into neuron-like cells into vitro.Methods:①hMSCs from adult normal volunteer flank bone were isolated by imm- unohistochemistry staining by means of gradient centrifugation and anchoring culture.②After 3 passages were obtained,and then digested by DMEM medium containing fetal calf serum, The growth curve of P2,P4,P6 were drawn with MTT.③After the 3-5 passages of MSCs were induced, when it was near to 70%-80% fusion, experimental group:hMSCs were maintained in the preinduction medium consisting of 10ng/ml basic fibroblast growth factor for 24 hour,then the preinduction medium was removed, The hMSCs were affected by the concentration of 50ng/ml,100ng/ml,150ng/ml and 200ng/ml NGF respectively, and the morphological changes of these cells were observed after induction, immunohistochemistry staining was used to explore the rate about positive cells of NGF on MSCs'differentiation;negative control group:they were cultured in the serum free medium.④Nestion and neuronspecific enolase(NSE) the special mark of neural stem cells,the tag of motor neurons were detected on the 0.5th,1th,3th,5thday after seeding with immunocytochemical method.On the other hand, reasonable sight was selected to count positive cells and compared the expression of each mard among five group.Results:①The proliferation curve of MSCs:the first 1-2 days after cultivation was latent period ,and then MSCs proliferated rapidly and reached the exponential phase of growth .The number of MSCs reached the peak time at the 6-7th of culture since the proliferation decreased and entered flat form phase.②The morphologies of MSCs were observed after induction:12 hours after adding inductive agent, the form of MSCs changed,large cell body and similar thin long processes of axon and dendrite, and obviously at hour 24, partial cells were with net-shaped arrangement, but death cells were few. The negative control induction scheme; no markedly morphological changes, still were long spindle cells.③The immunohistochemical assay result of neuron-like cells: The differentiated neuron-like cells in different concentration of NGF were NSE and Nestion staining positive. showing buffy cell body and process; No positive result appeared in the negative control group.④nestin and neurons specific enolase(NSE) were evaluated by indirect immunocytochemisty staining. NGF in different concentration have different influence on NSC's differentiation. The differentiation rates of NSE positive cells in the counts in NGF groups were (35.6±4.4)%,(61.9±3.4)%,(57.3±5.7)% and(57.2±4.8)%, there was significantly different among them by one-way ANOVA(F=338.71,P<0.05), it was also found the rate positive cells of 100ng/ml ,150ng/ml and 200ng/ml groups were much higher than that of 50ng/ml group(all P<0.01), but the difference in first three groups was not different.⑤NGF in different time had different influence on NSC's differentiation. The differentiation rates of NSE positive cells in the counts in 0.5th,1th ,3th ,5thday were(27.8±5.6)%,(60.5±6.7)%,(61.9±3.4)%and(30.7±3.5)%, respectively. There was significantly different among them(F=136.03,P<0.01), it was also found the rate positive cells of 1th ,3th were much higher than those of 0.5th and 5th(all P<0.01), but the difference between 1th and 3th was not different(P>0.05).Conclusion:①A simple new method which is used for the isolation , purification and cultivation in vitro of MSC form hMSCs by total marrow culture associating with adhering to flash has been established .This method is easy to manipulate ,hard to co- ntaminate and with high purified of isolation.②NGF may induced MSC into neuro- like cells and live time of cells is prolonged. 100ng/ml NGF can induce differenttiati- on of hMSCs into neuron-like cells in higher rate. |
