小鼠巨细胞病毒感染神经干细胞的体外研究

The effect of MCMV infection on neural stem cells in vitro

目的研究小鼠巨细胞病毒(MCMV)对体外培养的神经干细胞(NSC)增殖和分化的影响,为研究先天性巨细胞病毒感染致脑发育异常的机制奠定理论基础。方法体外分离、培养和鉴定BABL/c胎鼠NSC,用含LacZ报告基因的重组小鼠巨细胞病毒RM461感染NSC,X-gal染色监测感染过程,透射电镜观察超微结构改变,PCR检测受染细胞及培养上清中病毒DNA及观察培养上清接种到小鼠胚胎成纤维细胞(MEF)的细胞病变效应,了解RM461对NSC的感染情况。通过绘制生长曲线、流式细胞术检测分化细胞比率,研究RM461对NSC增殖和分化的影响。结果RM461感染NSC48h后细胞出现肿胀、体积增大、神经球边缘的细胞发生脱落和死亡,细胞形态的改变随感染复数(MOI)的增加而更加明显;X-gal染色,24h受染细胞即可出现阳性改变,48h达高峰;RM461感染72h时,受染细胞及培养上清中病毒DNA呈阳性,培养上清接种到MEF后可出现明显的细胞病变效应,电镜下受染细胞胞浆中存在病毒颗粒,细胞器肿胀明显;RM461可明显抑制NSC的增殖和分化,当MOI为1时,分化培养2d,感染组胶质原纤维酸性蛋白(GFAP)和神经元特异性烯醇酶(NSE)阳性细胞的比例分别为(37.4±1.6)%和(8.9±0.8)%,远较未感染组[分别为(50.3±1.8)%和(23.4±1.3)%]低(P<0.01),且GFAP和NSE阳性细胞比率随MOI的增加降低更加明显。结论RM461可感染体外培养的NSC,并能形成产毒性感染;RM461可明显抑制NSC细胞的增殖和分化并导致干细胞死亡,这可能与先天性巨细胞病毒感染致脑发育异常有关。

Objective To study the effect of murine cytomegalovirus（MCMV） infection on proliferation and differentiation of neural stem cells（NSC） in vitro and its role involved in mechanisms of encephaledysplasia caused by congenital cytomegalovirus infection. Methods NSCs were prepared from fetal BALB/c mice and were infected with recombinant MCMV RM461 which was inserted by report gene/acZ at different multiplicity of infection（MOI）. X-gal immunohistochemistry staining was used to monitor MCMV infection. The NSC morphology changes were observed in inverted microscope and transmission electron microscopo（TEM）. The productive infection was verified by TEM, detection of MCMV DNA in infected NSC and supernatant was done by PCR and cytopathic effect（CPE） in mouse embryonic fibroblasts（MEF） caused by the supernatant from the infection group was carried out. The effects of MCMV infection on proliferation and differentiation of NSC were shown by growth curve and the ratio of nestin, GFAP and NSE positives cells were checked with flow cytometry. Results NSCs infected by RM461 were swollen and the cell volume was enlarged after 48 h of infection. The cells at the merge of the neurospheres were deciduous and dead. The cell morphology was obviously changed as the MOI increased. LacZ in NSC could be detected within 24 h of infection and peaked at 48 h as shown by X-gal staining. At 72 h of infection MCMV DNA was positive in infected NSC and supomatant as shown by PCR, viral particles and the swollen organelle were seen in the plasma in TEM. When the supomatant was inoculated on MEF, typical CPE was observed in 3 days. RM461 infection significantly inhibited proliferation and differentiation of NSC. The differentiation cells were analyzed by flow cytormetry 2 days after infection. The results of flow cytometry assay showed that GFAP and NSE positive cells＇ ratio was （37.4± 1.6）% and （8.9±0.8）% in the infection group while （50.3±1.8）% and （23.4 ± 1.3）% in the control group（ P 〈0.01）. The proportion of GFAP and NSE positive cells was signifi- cantly decreased as the MCMV MOI increased. Condusim~ NSCs were permissive for MCMV infection /n vitro and the virus infection inhibited their proliferation, differentiation and caused cell death. These results may explain at least part of pahngenesis of encephalodysplasia caused by congenital cytoms infection.