人巨细胞病毒感染新生乳鼠原代培养脑神经细胞研究

INFECTION OF PRIMARY CULTURE OF NEWBORN MICE CEREBRAL NEURONS BY HUMAN CYTOMEGALOVIRUS

为了研究确定人巨细胞病毒（ＨＣＭＶ）体外感染Ｂａｌｂ／ｃ新生乳鼠原代培养脑神经细胞及其感染特征，制备和培养了新生乳鼠脑神经细胞并进行病毒感染。感染后每隔２～３天在倒置显微镜下观察活细胞培养物并摄影记录。在感染后第１～４周，取样进行苏木素－伊红（ＨＥ）染色及尼氏（Ｎｉｓｌ）染色。同时，用已知抗ＨＣＭＶ外膜蛋白的单克隆抗体进行免疫细胞化学染色，和地高辛标记的ＨＣＭＶ寡核苷酸特异性探针进行原位杂交，检测相应病毒核酸及胞内定位。结果在病毒感染后的第２周，受染细胞出现明显病变，表现为颗粒增多，折光性减弱。ＨＥ染色可见，受染神经细胞明显肿胀，胞浆及胞核均为嗜酸性，并可见到嗜酸性包涵体。Ｎｉｓｓｌ氏染色发现，胞核旁的尼氏小体变小，成粉末状，甚至消失。免疫细胞化学染色显示，受染神经细胞呈阳性反应。原位杂交结果证实，病毒核酸存在于受染细胞的胞浆及核内。这表明，ＨＣＭＶ能感染体外原代培养的新生乳鼠脑神经细胞，且病理特征与在人胚脑神经细胞观察到的基本模式一致。这种体外培养的细胞模型，可用于ＨＣＭＶ致中枢神经系统感染的研究。

This subject is to study the infection of primary culture of cerebral neurons from the newborn alb/c mice in vitro by human cytomegalovirus (HCMV) and the feature of infection. The cerebral neurons were prepared, cultured and HCMV infection model was made. 1～4 weeks after infection, the cell culture was observed and photographed under microscope, and the cell samples were taken for HE and Nissl's stainning, meanwhile the virus specific antigen, the virus DNA and its intracellular location were analyzed with immunocytochemical staining and in situ hybridization using digoxigenin labelled HCMV DNA probes, respectively at 1、2、3 and 4 weeks of postinfection. The neurons exhibited cytopathic effects at 2 weeks of HCMV postinfection, showing the disappearance of halo around cell body, granular degeneration in cytoplasm, unclear and coarse cell membrane, broken and thickened axon, and besides,the cells containing large, bizarre nuclei and intranuclear inclusion bodies were found. Nissl's body was blurred or dispersed. Meanwhile the viral antigen positive neurons were detected by immunocytochemical staining, and the presence of virus sequence was confirmed by in situ hybridization, however, nothing was found in negative controls. These observations are consistent with the properties of human neural cell aggregate cultures with HCMV infection. We suggest that the primary culture of cerebral neurons of newborn Balb/c mice may be used as a model in vitro for investigation of the HCMV replication and HCMV-induced pathogenesis in central nervous system.