小鼠MCMV先天感染模型的构建及神经系统感染状况的观察

发布时间：2018-05-27 10:28

本文选题：巨细胞病毒 + 动物模型　；参考：《华中科技大学》2011年硕士论文

【摘要】：目的建立小鼠巨细胞病毒(MCMV)先天感染模型,并观察其脑组织的病理变化及感染状况。方法 1)MCMV毒株制备及感染性滴度的测定:剖宫取出孕中晚期BALB/c胎鼠,制备鼠胚成纤维细胞(MEF);扩增MCMV RM461病毒株,用蚀斑法测定病毒滴度;稀释至造模所需浓度(1×103 PFU/μL)备用; 2)MCMV先天感染模型的构建:取孕11～13.5d(E11～13.5d)的BALB/c鼠,麻醉后逐个孕囊行羊膜腔内病毒接种(RM461病毒悬液1μL,病毒量1×103PFU),小心复位后关腹;对照组羊膜腔内注射DMEM培养液1μL。单独饲养5d后处死孕鼠,剖宫取出胎鼠,麻醉处死,取胎鼠脑组织制作冰冻切片; 3)胎脑组织病理改变及病毒抗原检测:脑组织切片常规HE染色,光镜下观察病理学变化;采用免疫酶组化及免疫荧光法检测脑组织中的MCMV早期抗原。结果 1)成功扩增MCMV RM461毒株,测得感染性病毒滴度为3.27×107PFU/mL; 2)成功构建MCMV先天感染模型。感染组和对照组的胎鼠存活率分别为71.9%和76.7%。与对照组相比,羊膜腔内接种MCMV病毒对胎鼠存活率、吸收胎率、死胎率及胎鼠头部重量无明显影响,但可致胎鼠体重降低; 3)脑组织病理学变化:对照组脑组织未见有结构异常;感染组出现脑室、脑沟回发育不良,神经细胞水肿,脑室区、脑室下区和大脑皮质区主要有淋巴细胞、单核细胞等炎症细胞浸润; 4)病毒抗原检测:在宫内感染组,免疫酶学组化法发现脑室区、脑室管膜下区、大脑皮质和海马区可见病毒感染细胞;免疫荧光观察到的主要感染部位与免疫酶组化一致。结论通过羊膜腔内注射MCMV RM461病毒悬液成功建立MCMV先天感染模型,为研究MCMV先天感染致脑发育异常机制提供合适的整体研究模型。
[Abstract]:Purpose The congenital infection model of mouse cytomegalovirus (MCMV) was established and the pathological changes and infection status of its brain tissue were observed. Method The preparation of 1)MCMV virus strain and the determination of infective titer: take out the fetal mouse of BALB/c in the middle and late pregnancy, prepare the mouse embryo fibroblast, amplify the MCMV RM461 virus strain, determine the virus titer by plaque method, dilute to 1 脳 10 ~ 3 PFU/ 渭 L to make the model. Construction of 2)MCMV congenital infection model: pregnant BALB/c mice with 1113.5 d (E1113.5 d) were given intraamniotic virus inoculation with RM461 virus suspension 1 渭 L, virus volume 1 脳 103 PFUU, careful reduction and abdominal closure, and control group was injected with DMEM culture fluid 1 渭 L. After feeding alone for 5 days, the pregnant rats were killed, the fetal mice were removed from the uterus, and the fetal brain tissue was taken out to make frozen sections. 3) pathological changes of fetal brain tissue and detection of virus antigen: routine HE staining and light microscopy were used to observe the pathological changes in brain tissue, and MCMV early antigen in brain tissue was detected by immunohistochemistry and immunofluorescence method. Result 1) the MCMV RM461 strain was successfully amplified, and the titer of infectious virus was 3.27 脳 10 ~ (7) PFU / mL; 2) successful construction of MCMV congenital infection model. The survival rates of infected group and control group were 71.9% and 76.7%, respectively. Compared with the control group, intraamniotic inoculation with MCMV virus had no significant effect on fetal survival rate, fetal absorption rate, fetal death rate and fetal head weight, but could result in fetal mice weight loss. 3) histopathological changes of brain: there were no structural abnormalities in brain tissue in control group, and there were mainly lymphocytes in cerebral ventricle, hypoplasia of sulcus gyrus, neuronal edema, ventricular area, subventricular area and cerebral cortex in infected group. Infiltration of inflammatory cells such as monocytes; 4) virus antigen detection: in the intrauterine infection group, the virus infected cells were found in the ventricular area, the subependymal area, the cerebral cortex and the hippocampal area, and the main sites of infection observed by immunofluorescence were the same as those observed by immunohistochemistry. Conclusion The model of congenital MCMV infection was successfully established by intraamniotic injection of MCMV RM461 virus suspension, which provided a suitable overall model for studying the mechanism of cerebral dysplasia caused by MCMV congenital infection.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R-332

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