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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 of measuring the rabies virus (RV) titer by using mouse primary neuronal cells. Methods Primary cultured mouse cerebral cortical neurons were isolated and inoculated in 100 mice to inoculate a standard challenge virulent (CVS) strain with a median lethal dose (MICLD50). Immunofluorescence and reverse transcriptase polymerase chain reaction -PCR) was used to detect the infectivity. Based on this, CCID50 of this strain was determined on primary neuronal cells. Results Primary neuronal cells of mouse cerebral cortex were successfully prepared. Primary culture of CVCs was inoculated with CVS for 96 hours, and then primary anti-microtubule-associated protein (MAP2) and anti-RV sera were used as primary antibodies and stained with fluorescent antibody. Neuronal-specific and RV-specific staining was observed on both the soma and on the protuberances; under confocal microscopy, pink spots appeared when red and green overlapped on the same cell site, and immunofluorescence confirmed that primary neurons were CVS infection, RT-PCR to obtain a target band of about 1770 bp, indicating that it was CVS infection; CVS virus titer measured on the primary cell 104.5CCID50 / 0.1 mL, and the virulence of the virus titers 104.5MICLD50 / 0.03 mL. Conclusions The virus titer of the CVS strain determined on mouse primary brain neurons is the same as that measured by intracerebral inoculation.