| ObjectiveCerebral ischemia is a kind of common and frequently encountered disease which seriously threatens people's health. Recently, Stem cell transplantation brings perspective for cerebral ischemic treatment. Some studies indicated: MSCs transplantation post-cerebral ischemia can improve the neurologic impairment not only through amendmenting neonatal angiogenesis in ischemic penumbra, but also through promoting endogenous NSCs proliferation. However, the mechanism of endogenous NSCs proliferation and differentiation remain unknown. This study was designed to investigate the effects of MSCs on the notch signaling pathway, which plays an important role in NSCs proliferation and differentiation, and the possible effects of ECs in MSCs.Methods1.The mouse NSCs were isolated and cultured by neurosphere suspension method; and they were identified by detecting nestin using immunocytofluorescence staining.2.The mouse MSCs were isolatated by adherence-screening method, MSCs were cultured routinely and the MSCs were identified by immunocytofluorescence staining of CD34 and CD29.3.MSCs and NSCs were cocultured in a transwell system; and the co-culture systems of MSCs with NSCs, in which the ECs function was inhibited, of HMVECs and 3T3 fibroblasts with NSCs respectively were set as controls.4.The gene expressions of notch1, hes1 and mash1 (notch signaling pathway relative genes) and nestin and GFAP (NSCs proliferation and differentiation relative genes) were dected by semiquantitative RT-PCR assay in different co-culture groups and at different time points.Results1. Mouse NSCs cultured by neurosphere suspension method proliferated prosperously and positively expressed nestin.2.Mouse MSCs proliferated prosperously; the immunocytofluorescence staining showed that CD29 was positively expressed and CD34 was negatively expressed in the cells which was the characteristics of MSCs.3.The gene expression involved in notch signaling pathway and proliferation and differentiation of NSCs in different co-culture groups and at different time points was detected:(1). In MSCs and HMVECs co-culture groups, notch1 and hes1 gene expressions in NSCs increased and semiquantitative analysis showed that they all reached the peak at 6h after co-culture. However, in the ECs function blocked MSCs groups and the ECs function blocked HMVECs groups, the gene expressions of notch1 and hes1 in NSCs were significantly lower than those of MSCs and HMVECs co-culture groups at 6h. All these suggested that the ECs from MSCs could play an important role in activating the notch1 and hes1 of NSCs gene expressions.(2).In MSCs and HMVECs co-culture groups, the mash1 gene expression of NSCs down-regulated in 24h, however, in the ECs function blocked MSCs group, the ECs function blocked HMVECs group and 3T3 fibroblasts group, the mash1 gene expressions of NSCs were all up-regulated gradually. These indicated that ECs could inhibit the mash1 gene expression in NSCs which was one of the negative-regulated genes of the notch signaling pathway activation.(3). The nestin and GFAP gene expressions of NSCs were nearly constant in the MSCs and HMVECs co-culture groups, however, in the ECs function blocked MSCs group, the ECs function blocked HMVECs group and 3T3 fibroblasts group, the nestin gene expression level in NSCs was all down-regulated but the GFAP gene expression level in NSCs was up-regulated rapidly. These indicated that ECs played a vital role in maintaining the NSCs in an undifferentiatial proliferation condition.Conclusion1.MSCs can maintain NSCs in an undifferentiatial proliferation condition, and ECs from MSCs might play an important role.2.NSCs notch signaling pathway can be activated by MSCs, in which, positive relative gene expressions, such as notch1 and hes1, were up-regulated; and the negative relative gene, such as mash1 were down-regulated. ECs from MSCs potentially play an important role in keeping NSCs in an undifferentiatial proliferation condition.
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