The Effect Of BMP-4 On Neural Stem Cells Co-cultured With Rat Bone Marrow Mesenchymal Stem Cells

Objective To explore the effect of bone morphogenetic protein-4(BMP-4) on the survival and differentiation of neural stem cells(NSCs) co-cultured with bone marrow mesenchymal stem cells(BMSCs) and to provide further theoretical basis for cell transplantation in the treatment of spinal cord injury.Methods The brain tissue was isolated from neonatal SD rats in 24 hours and neural stem cells were cultured in serum free medium for 7 days. The bone marrow mesenchymal stem cells were extracted from the bone marrow of SD rats and cultured by adherent bone marrow gradient.Then the co-cultured model of bone marrow mesenchymal stem cells and neural stem cells was established. Then the BMP-4 was put into this co-cultured system, after that, the survival rate of NSCs which were attended by Annexin-V apoptosis Kit were treated by the flow cytometry(FCM). The immunofluorescence staining was used to detect and statistic the differentiation of NSCs included MBP of the oligodendrocyte-specific protein, MAP-2 of the neuron-specific protein and GFAP of the astrocytes protein. And the total protein were extracted from the differentiated cells, after that, MBP of the oligodendrocyte-specific protein, MAP-2 of the neuron-specific protein and GFAP of the astrocytes protein were detected by Western-blot method. By comparing the rates of cells,survival and differentiation, the effect of BMP-4 on the survival and differentiation of NSCs was analyzed.Result(1) The cellular morphology of bone marrow mesenchymal stem cells after cultured by three generations tended to identical like fusiform or spiral. The neural stem cell balls were suspended growth. Those neural stem cells could differentiate into astrocytes cells, neurons and oligodendrocytes after common cultured.(2)The apoptosis model was produced by H2O2, the neural stem cells of common cultured were divided into 4 groups: the control group, the H2O2 group, the H2O2+ common cultured group and the H2O2+ common cultured +BMP-4 group. The survival of neural stem cells which were treated by Annexin V-PI cell apoptosis kit was detected by the flow cytometry(FCM). The result of the flow cytometry analysis showed that the survival rates of common cultured cells improved,but the survival rates declined after adding BMP-4.This data gap was significant after analysis of variance(F=29.373,P<0.05).(3) The co-cultured model of BMSCs and NSCs was divided into two groups: the control group and the BMP-4 group, there was a significant differentiation of NSCs after cultured by 7 days, those cell balls had surrounding radial growth, the cells arranged very closely and intercellular narrow showed by micriscope. of MBP of the oligodendrocyte-specific protein, MAP-2 of the neuron-specific protein and GFAP of the astrocytes protein were found by immunofluorescence staining.(4) After extracting the total protein of cells, MBP of the oligodendrocyte-specific protein, MAP-2 of the neuron-specific protein and GFAP of the astrocytes protein were detected by Western-blot method.The statistical data showed the expression of GFAP in the experimental group was higher than the control group(t=9.638,P<0.05), and the expression of MBP was lower than the control group(t=4.902,P<0.05), the expression of MAP-2 has no significant difference(t=0.71,P>0.05).Conclusion BMP-4 hinders the differentiation effect of BMSCs,S promotion on NSCs.