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Study on the neurons of fetal mice from the cerebral cortex
with the congenital human cytomegalovirus infection
WANG Ming-liTANG Jiu-laiShi Bai-fenet al.
Department of Microbiology,Anhui Medical University,Hefei 230032 China
AbstractObjective:To investigate is to determine the infection of neurons that human cytomegalovirus AD169 strain(HCMV AD169)could cross the placenta of the Balb/cmice and initiate congenital infection of the developing fetus cerebral cortex invivo and that the neurons of the primary cultures from the cerebral cortex of Balb/c fetal mice could be infected by HCMV AD169 in vitro.Methods:HCMV(5.0 log TCID50 ml/mouse)was injected into the intraperitoneum of mice (half of mice are female)when they were about 8～ 12 weeks old.Then,these of mice were used for mating.Pregnancies were dated as day 0 when a copulation plug was found.Fetuses on day about to give birth of gestation were removed from the uteri and its cerebral cortex was removed from the skull and fixed with 4％ paraformaldehyde,then cut into serial coronal sections 5 μ m thick,applied to slides coated with Histostik,and then stained with hematoxylin and eosin (HE).The supernatant of minced a fetal mouse cerebral cortex was used for virus isolates by HF cells.And viral DNA positive cells were examined by in situ hybridization using digoxigenin labelled HCMV DNA oligonucleotide probe in the acetone fixed imprint of the cerebral cortex.Meanwhile, The cerebral cortex neurons were prepared and cultured and HCMV infection model was made. 1～4 weeks after infection,the cultures was recorded and observed by photography under microscope, and the samples were taken for HE stainning.At the same time, the virus DNA and its intracellular location were analyzed in situhybridization～using digoxingenin labelled HCMV DNA oligonucleotide probes.Results: HCMV had been isolafed from the supernatant of fetal mice cerebral cortex.The pathological changes consisting of destructive meningoencephalitis and large、basophilic、intranuclear neuronal inclusions of viral type occurred in the fetal mouse cerebral cortex.The presence of virus sequences was confirmed by in situ hybridization,however, nothing was found in the normal controls.The neurons exhibited cytopathic effects at 2 weeks of postinfection,and the viral sequences was found when they were infected by HCMV AD169 in vitro.Conclusion:Development of the fetal mouse model of HCMV infection that mimics human congenital central nervous system(CNS)infection in vivo and in vitro will allow the study of the pathogenesis of HCMV congenital CNS infection and the development of therapeutic agents and vaccines.
KeyWordshuman cytomegalovirus(HCMV);neuron of the cerebral cortex;a fetal mouse;congenital infection
人巨细胞病毒(Human Cytomegalovirus,HCMV)是最常见的宫内传播病毒之一。人类先天性HCMV感染可使多系统、多脏器受累，而中枢神经系统(CNS)受损后果最为严重，常见为脑积水，脊柱裂，小头畸形，脑内钙化(特别是脑室周围)，非遗传性感觉神经性耳聋和四肢瘫痪等。每年仅就HCMV先天感染导致胎儿新生儿患神经系统疾病人数，Plotkin等[1]估计美国约有6000个婴儿为感染患者；按此估算，我国至少有3万多个婴儿成为患者。流行病学调查亦提示，先天畸形胎儿血清中HCMVIgM阳性率(20％)明显高于一般活产儿(1．65％～3．5％)[2]；而在脑组织中HCMVDNA阳性率高达44％[3]。证实先天性HCMV感染中，CNS感染率较高，严重危害了胎儿的生长发育，导致的智力障碍给家庭和社会带来沉重负担。
为开展研究HCMV先天性CNS感染的病理过程，亟需建立合适的体内外实验模型。长期以来，一直沿用鼠巨细胞病毒(Murine Cytomegalovirus,MCMV)感染小鼠作为研究CMV感染与疾病的模型，但MCMV经自然途径感染小鼠后，不能像HCMV一样经胎盘致发育中的胚胎发生先天性感染[4]，因此MCMV不适用于HCMV先天性感染的研究。本研究首次采用预先腹腔接种HCMV至生育龄Balb/c雌雄小鼠，然后配对喂养，在雌鼠临产时剖腹取出胎鼠及其母鼠双侧大脑皮质进行观察研究，建立了模拟人类HCMV先天性感染CNS的体内模型；同时在待产胎鼠双侧大脑皮质原代培养物上接种HCMV，连续4～5周细致地观察检测了HCMV致神经元感染的细胞病理学改变，建立了模拟人类HCMV先天性感染CNS的体外模型。现将结果报告如下。
1材料和方法
1．1病毒 HCMVAD169株由上海医科大学微生物学教研室提供，用人胚成纤维细胞(HF)培养增殖。使用前，将病毒在HF细胞上传代，以增加病毒毒力；按Gibson[5]的病毒致细胞变异作用(CPE)终点稀释法以在24～48h内出现达＋＋＋～＋＋＋＋的病毒悬液在HF细胞上滴定TCID50；以6．0logTCID50/ml的病毒作为实验用毒种。
1．2实验动物纯系Balb/c小鼠，雌雄兼用，8～12周龄，体重(30±5)g，由本校实验动物中心提供(皖实验动物号：皖准01号)。感染病毒后饲养于本室专用感染动物柜内。
1．3细胞人成纤维细胞株(HF)及原代兔肾细胞均由本室自制。本研究采用的HF株为6～8代。
1．4主要试剂所用主要化学及分子生物学试剂分别购自Sigma公司、Promega公司和华美生物工程公司；地高辛DNA标记和检测试剂盒购自Boehringer Mannheim公司。
1．5HCMV MIE和LA寡核苷酸探针序列合成与标记HCMV DNA MIE(即刻早期抗原)和LA(晚期抗原)寡核苷酸探针片段序列位置参考文献[6]设计，MIE寡核苷酸探针序列:5′GAGGCTATTGTAG、CCTACACTT-TGG3′，序列位点为第900～925核苷酸；LA寡核苷酸针片段序列为5′GTCGCC、TGCACTGCCAGGTG、CTTCG3′，序列位点第2301～2325核苷酸。用DNA合成仪固相亚磷酸三酯法合成并纯化(中国科学院上海植物生理所植物分子遗传国家重点实验室)。上述两种Digoxigenin标记的探针制备及检测按Boehringer Mannheim公司说明书进行。
1．6动物体内感染模型的建立将HCMV AD169株悬液按0．5ml/只(5．0log TCID50)注射至雌雄小鼠腹腔内，然后按1∶1比例同笼饲养，此设定为试验组；与此同时，在设定为对照组的小鼠腹腔内分别注射正常HF细胞悬液05ml/只。逐日晨检查阴栓以确证其妊娠开始时间。待雌鼠临产(约为妊娠的第20d)剖腹取出胎鼠和相应母鼠大脑皮质，分别进行病毒分离、病理学检测及其病毒核酸原位杂交检测。
1．7体外神经元感染模型的建立实验用Balb/c临产胎鼠由本校实验动物中心提供。原代培养物的制备基本参照吴希如等[7]介绍的方法进行。将细胞接种至盛有已预先覆盖鼠尾胶原薄膜盖玻片的塑料培养皿内，最终细胞浓度1×106/ml。置37℃，50％CO2饱和湿度的CO2培养箱内培养，再经48h后，分别于相应孔内加入细胞分裂抑制剂5－氟－2′脱氧尿苷15μg/ml和尿苷35μg/ml以抑制非神经细胞的过度增殖。48h后，于每个试验孔内分别加入100TCID50病毒悬液0．1ml，同时以相同病毒量感染HF细胞作为阳性对照。观察和鉴定方法，每隔2～3d于例置显微镜下观察、摄影记录、换液；并在病毒感染后的第24h、48h、72h和第1～4周分别连续取样，用于病毒TCID50滴定，细胞病理学染色及原位杂交检测等。
1．8病毒分离基本方法参照文献[8]。常规无菌操作下取出胎鼠双侧大脑皮层，用细胞维持液制备10－1/ml脑细胞悬液，低速离心后，取上清分为两份，一份直接接种至HF细胞和原代兔肾细胞；另一份经56°30′灭活后再分别接种至上述两种细胞，同时设置正常细胞对照。对出现HCMV特征性CPE者，取培养上清做PCR，检测HCMV DNA。
