Hcmv Infection Hippocampal Nscs Differentiation Astrocytes And Induced Cell Apoptosis

Objective To study the susceptibility of astrocytes that differentiated from human embryonic hippocampus neural stem cells(NSCs) to HCMV AD169 infection and the effect of HCMV infection on apoptosis of astrocytes in vitro.Methods Human embryonic hippocampus NSCs were isolated and cultured in vitro and identified by detecting the expression of Nestin protein with indirect immunofluorescence method,then cells were induced to differentiate into astrocytes.Astrocytes cultured in vitro were identified by observing their morphologic characteristics under inverted microscope and detecting the glial fibrillary acidic protein(GFAP) by indirect immunofluorescence. Then astrocytes were infected with HCMV strain AD169 at 1 multiplicity of infection(MOI=1),Viral-associated cytopathic effects were observed under inverted microscope,and indirect immunofluorescence was used to examine the expression of HCMV immediate early gene product—IE proteins,PI staining by Flow cytometry and slide staining with Annexin V-FITC/PI were both used to detect the apoptosis of astrocytes. The medium from infected cells was collected to infect fibroblasts(MRC-5),which are fully permissive for HCMV infection.Results Most of NSCs cultured in vitro expressed protein—Nestin,which is the recognized NSCs marker,then were induced to differentiate,7 days later,cells strongly expressed GFAP—markers of astrocytes.7 days after HCMV infected astrocytes, cytopathic effects(Swelling,bigger and so on) were observed and HCMV gene product-IE protein were detected in most cells.The results of flow cytometry assay showed that the proportion of apoptotic cells in the infection group(14.72%±5.78%) was higher than that in the normal group(7.59%±2.33%),and there was statistical significance between the two groups(t=3.18,P<0.05).At the same time,staining on the slide also showed the same results:much more cells in the infection group than that in the normal group were positive of apoptotic stain.Fibroblasts exposed the medium from infected cells were also observed typically Virus-induced cytopathic.Conclusion(1) HCMV can infect human astrocytes that differentiated from human embryonic hippocampus NSCs in vitro;(2) HCMV infection can induce astrocytes apoptosis in vitro.This effect may provide part of the explanation for the abnormalities in brain development associated with congenital HCMV infection.