Effect Mechanism Of EphB4 On Proliferation, Differentiation Of Human Embryonic Neural Stem Cells And The Treatment Effect By Morroniside

Repair and neurogenesis in the injured late stage of ischemic stroke is a key for rehabilitation therapy of stroke. At present, the focus research of neuroscience is stem cell therapies. Stroke can stimulate the proliferation of transition on endogenous neural stem cell in short time, the underlying mechanisms are regulated by multiple signaling pathways. Based on the neurovasular unit, our team proposed the hypothesis of ―neurovascular homeostasis rehabilitation‖ for the treatment of stroke at 2007, hope to regulate specific molecular mechanisms through giving exogenous drugs. Then, proliferation and differentiation of stem cells induced by brain injured will be enhanced. Finally, promote the neurovascular homeostasis rehabilitation. Our previous studies suggested Eph/ephrin signal pathway may be involved in the mechanisms of promoting neurogenesis of morroniside, component extracted from Cornus Officinalis which is a traditional Chinese herb. Several members of the tyrosine kinase family play an important role in cell proliferation and angiogenesis. The aim of the present study is to inverstigate the effects and mechanisms of EphB4 receptor on proliferation, differentiation of human embryonic neural stem cells(NSCs), and observe the treatment effect by morronside, to explore the possible mechanism of drug morronside to treat a variety of neurological diseases.Take neurospheres cultured for 7 days, the formed neurospheres were planted into the petri dishes. Immunofluorescent staining for the NSCs marker nestin was performed after 3 days culture in basal DMEM/F12. Immunofluorescent staining for the neuron β-Ⅲ tublin(Tuj1) and for the mature astrocyte marker glial fibrillary acidic protein(GFAP) was performed after 10 days culture in specific differention medium. Immunofluorescent staining for the oligodendrocytes was performed after 16 days culture in specific differention medium, NSCs were identified by confocal laser scanning image. We have developed three groups of recombinant lentiviral short hairpin RNA(sh RNA) vector targeting the human EphB4 gene, the real-time PCR and Western blot were used to select the best one, real-time PCR and Western blot results showed that the EPHB4-RNAi(28947-1-1 shRNA) has the best interference effection. Also we have developed only one group of overexpression(OE) lentiviral vector according to the human EphB4 gene. NSCs were transfected with recombinant lentiviral shRNA vector and recombinant lentiviral OE vector targeting the human EphB4 gene, which carrying green fluorescent protein(GFP). Real time-PCR and Western blot were used to detected the transfecion efficiency. Cell proliferation were detected by Ki67 staining, cell differentiation were detected by nestin, β- III tublin and GFAP staining, cell apoptosis were observed by Hoechest 33342/PI staining.The results showed compared with the shRNA control group, after NSCs were transfected with LV-shRNA-EphB4, the expression of Eph B4 mRNA and EphB4 protein were decreased significantly(p<0.001, p<0.001); in contrast, NSCs were transfected with LV-OE-EphB4, the expression of EphB4 mRNA and protein were increased significantly(p<0.05, p<0.05). Compared with the shRNA control group, the number of Ki67+/GFP+ 、nestin+/GFP+、β-III tublin+/GFP+ cells in EphB4-shRNA groups were decreased significantly(p<0.001, p<0.001, p<0.001), GFAP+/GFP+ cells were increased significantly(p<0.001), the apoptotic cells of EphB4-sh RNA groups increased significantly(p<0.05). Compared with the Eph B4-OE control group, the number of Ki67+/GFP+ 、nestin+/GFP+、β-III tublin+/GFP+ cells cells in EphB4-OE groups were increased significantly(p<0.001, p<0.01, p<0.01), GFAP+/GFP+ cells were decreased significantly(p<0.001), the apoptotic cells of EphB4-OE groups decreased significantly(p<0.01). Meanwhile, we observed changes of proliferation, differentiation and apoptosis on NSCs the next day after given morronside(100 μmol·L-1) worked 48 hours, the results showed that after given morronside, compared with the shRNA control group, the number of Ki67+/GFP+、nestin+/GFP+、β-Ⅲ tublin+/GFP+ cells were increased significantly(p<0.001, p<0.05, p<0.001), cell apoptosis were decreased(p<0.05).So the conclusion is: the expression of EphB4 can promote the proliferation and differentiation into neuron of NSCs, meanwhile decreased the apopotosis of NSCs, it is suggested that EphB4 plays an important role on proliferation and differentiation of NSCs. Morronside can promote proliferation and differentiation into neuron of NSCs, the mechanism is not limited to EphB4 signaling pathways.