[Inhibitory effect of murine cytomegalovirus infection on neural stem cells' differentiation and its mechanisms].
OBJECTIVE
Cytomegalovirus (CMV) is the leading infectious cause of congenital anomalies of the central nervous system caused by intrauterine infection. However, the exact pathogenesis of these brain abnormalities has not been fully elucidated. It has been reported that periependymitis, periventricular necrosis and calcification are the most frequent findings in the brains of congenital CMV infection. Because a number of multipotential neural stem cells (NSCs) have been identified from ventricular zone, it is possible that NSCs in this area are primary targets for viral infection, which seems to be primarily responsible for the generation of the brain abnormalities. Therefore, the objective of the present study was to investigate the effect and mechanism of murine cytomegalovirus (MCMV) infection on neural stem cells' differentiation in vitro and its role in the mechanisms of brain abnormalities caused by congenital cytomegalovirus infection.


METHODS
NSCs were prepared from fetal BALB/c mouse and were infected with recombinant MCMV RM461 inserted with a report gene LacZ at 1 multiplicity of infection (MOI = 1). The effect of MCMV infection on neural stem cells' differentiation was observed by detecting the ratio of nestin, GFAP and NSE positive cells with immunohistochemistry and flow cytometry on day 2 postinfection. The effects of MCMV infection on gene expression of Wnt-1 and neurogenin 1 (Ngn1) related to neural differentiation were detected by RT-PCR.


RESULTS
NSCs isolated from embryonic mouse brains strongly expressed nestin, a specific marker of NSCs and had the capacity to differentiate into NF-200 and NSE positive neurons or GFAP positive astrocytes. At MOI = 1, the results of flow cytometry assay showed that nestin positive cells' proportion in the infection group [(62.2 +/- 1.8)%] was higher than that in the normal group [(37.2 +/- 2.4)%] (t = 4.62, P < 0.01). At the same time, the rates of GFAP and NSE positive cells' in the infection group were significantly lower than those in the normal group (P < 0.01). The scanning densities of Wnt-1 was 0.14 +/- 0.03 in the infection group while 0.32 +/- 0.04 in the control group (t = 7.21, P < 0.01). The scanning densities of Ngn1 were 0.09 +/- 0.01 and 0.21 +/- 0.02 in the two groups (t = 10.7, P < 0.01).


CONCLUSIONS
These results suggest that MCMV infection could inhibit neuronal differentiation, which may be primary causes of disorders of brain development in congenital CMV infection. The decreased expression of Wnt-1 and Ngn1 may be involved in the inhibitory effect of murine cytomegalovirus infection on neural stem cells' differentiation, which may lead to a new strategy for preventing and treating brain abnormalities caused by CMV infection through regulating these two signal pathways.
Zhou
Yu-feng
YF
Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Fang
Feng
F
Dong
Yong-sui
YS
Zhou
Hua
H
Zhen
Hong
H
Liu
Jin
J
Li
Ge
G
D000818:Animals; D001253:Astrocytes; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D001921:Brain / Q000166:cytology; D002352:Carrier Proteins / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D003586:Cytomegalovirus Infections / Q000151:congenital*; D004622:Embryo, Mammalian / Q000166:cytology*; D005260:Female; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D007150:Immunohistochemistry; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D039902:Multipotent Stem Cells / Q000378:metabolism / Q000821:virology*; D018146:Muromegalovirus; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D064231:Nestin; D009474:Neurons; D011247:Pregnancy; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051155:Wnt1 Protein / Q000235:genetics / Q000378:metabolism
D051792:Basic Helix-Loop-Helix Proteins; D002352:Carrier Proteins; D005904:Glial Fibrillary Acidic Protein; D007381:Intermediate Filament Proteins; D009419:Nerve Tissue Proteins; D064231:Nestin; D051155:Wnt1 Protein; C577484:NES protein, human; C105389:NEUROG1 protein, human; C533270:NSMCE1 protein, human; C577485:Nes protein, mouse