小鼠原代神经元细胞狂犬病病毒滴度测定  

Titrating rabies virus in primary cerebral cortex neurons of mice

作　　者：单晶辉[1,2] 王心蕊[1] 关振宏[1] 李宁[1] 宋艳[1,3] 张茂林[1] 

机构地区：[1]吉林大学人兽共患病研究所人兽共患病研究教育部重点实验室,吉林长春130062 [2]吉林大学畜牧兽医学院 [3]北华大学护理学院

出　　处：《中国公共卫生》2011年第5期572-574,共3页Chinese Journal of Public Health

基　　金：国家科技支撑计划(2008BAB96B11-3);国家自然科学基金(31072147);吉林省科技发展计划重点项目(20080930)

摘　　要：目的探索利用小鼠原代神经元细胞测定狂犬病病毒(rabies virus,RV)滴度的可行性。方法分离培养小鼠大脑皮质原代神经元细胞,接种100小鼠脑内接种半数致死量(MICLD50)的标准攻击强毒(CVS)毒株,通过免疫荧光和反转录聚合酶链反应(RT-PCR)检测其感染性,并在此基础上在原代神经元细胞上进行了该毒株的细胞半数感染量(CCID50)测定。结果成功制备了小鼠大脑皮质原代神经元细胞;原代神经细胞培养物接种CVS 96h后,用抗微管相关蛋白质(MAP2)抗体和抗RV阳性血清作为一抗并经荧光抗体染色后,在胞体和突起上均有神经元特异性和RV特异性着染;在共聚焦显微镜下,当相同的细胞部位上出现红色和绿色交迭时呈现粉色斑点,免疫荧光证实原代神经细胞可被CVS感染,同时RT-PCR得到1 770 bp左右的目的条带,说明其被CVS感染;该原代细胞上所测定的CVS病毒滴度为104.5CCID50/0.1 mL,而该病毒的原始滴度为104.5MICLD50/0.03 mL。结论小鼠脑原代神经元细胞上测定的CVS株的病毒滴度与通过脑内接种法测定的滴度相同。Objective To explore the feasibility to titrate rabies viruses in primary cerebral cortex neurons of mice.Methods The primary cerebral cortex neurons of mice were prepared and inoculated by median mouse intracerebral lethal dose（100MICLD50） of CVS strain and the infections were confirmed by immunofluorescent and RT-PCR detection.Then the median cell culture infectious dose（CCID50）was determined on the cultured neurons.Results The results showed that primary cerebral cortex neurons were successfully prepared and could be infected by rabies viruses efficiently.The titer of CVS strain was 104.5CCID50/0.1mL,and the original inuculum was 104.5 MICLD50/0.03mL.Conclusion The titer titrated in the cultured neurons was equal to the original titer of MICLD50.It implies that primary cerebral cortex neurons might be served as a potent technique for titrating the rabies viruses in vitro.

关 键 词：狂犬病病毒 大脑皮质原代神经元 小鼠脑内接种半数致死量 细胞半数感染量 

分 类 号：R186[医药卫生—流行病学]