人NSCs分化的Astrocyte系中HCMV感染的建立

HCMV infects astrocytes differentiated from human embryonic hippocampus neural stem cell

目的研究HCMV能否感染体外培养的由人海马神经干细胞（NSCs）分化成的星形胶质细胞（astrocyte）。方法体外分离、培养人NSCs，诱导其定向分化为星形胶质细胞，观察细胞的形态学特点并分别进行免疫荧光鉴定。然后用HCMVAD169株感染分化细胞，观察细胞形态变化，检测胞内病毒蛋白表达，并收集感染细胞的培养液，添加到正常人胚肺细胞中，观察有无典型细胞病变出现。结果体外原代培养的人NSCs绝大多数都表达巢蛋白（nestin），诱导其分化为星形胶质细胞后，细胞高表达神经胶质纤维酸性蛋白（GFAP）。HCMVAD169感染细胞7d后，细胞出现肿胀、变大变圆等典型病变，胞核内检测到HCMV即刻早期基因表达产物-IE蛋白，添加感染细胞的培养液到人胚肺细胞中，细胞也出现典型病变。结论上述结果说明：在体外培养的由人NSCs分化成的星形胶质细胞中，HCMVAD169能够建立增殖性感染，这可能与先天性感染导致脑发育异常有关。

Objective To determine the susceptibility of astrocytes that differentiated from human embryonic hippocampus neural stem cells （NSCs） to HCMV AD169 infection. Methods Human embryonic hippocampus NSCs were isolated and cultured in vitro and validated by indirect immunofluorescence, then cells were directed to differentiate into astrocytes. Astrocytes cultured in vitro were also confirmed by indirect immunofluorescence and then infected with HCMV strain AD169 at an MOI of 1. Viral-associated cytopathic effects were observed under inverted microscope. Indirect immunofluorescence was used to examine the expression of immediate early （IE） HCMV proteins 7 days later, and the media from infected cells were 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 symbol NSCs specific antigen. After differentiation, cells expressed glial fibriUary acidic protein （GFAP） -a marker of astrocytes. 7 days after HCMV infection of astrocytes, cytopathic effects were observed and HCMV immediate early gene product-IE was detectable in most cells. Typically Virus-induced cytopathology was also observed in Fibroblasts exposed to the media from infected cells. Conclusions Astrocvtes cultured in vitro are permissive for HCMV infection. This effect may provide part of the explanation for the abnormalities in brain development associated with congenital HCMV infection.