痘苗病毒天坛株感染AG6小鼠脑病模型的建立与初步分析

doi:10.12300/j.issn.1674-5817.2025.070

实验动物与比较医学 ›› 2026, Vol. 46 ›› Issue (1): 3-10.DOI: 10.12300/j.issn.1674-5817.2025.070

痘苗病毒天坛株感染AG6小鼠脑病模型的建立与初步分析

杨琳¹^,², 金梦², 吴汗青¹^,², 李顺²(), 周晓辉²()

^1.上海师范大学生命科学学院, 上海 200233
^2.上海市公共卫生临床中心实验动物部, 上海 201508

收稿日期:2025-05-13 修回日期:2025-08-19 出版日期:2026-02-25 发布日期:2026-02-14
通讯作者:
周晓辉（1973—），男，博士，研究员，研究方向：新发传染病动物模型构建与免疫机制研究。E-mail： zhouxiaohui@shaphc.org。ORCID：0000-0002-7491-628X；

李顺（1986—），男，博士，副研究员，研究方向：人类疾病动物模型构建及应用研究。E-mail： lishun86@126.com。ORCID：0000-0002-9887-8879
作者简介:杨琳（1996—），女，硕士研究生，研究方向：感染性动物模型。E-mail：1016358073@qq.com
基金资助:
国家重点研发计划-基础科研条件与重大科学仪器设备研发专项“单/多病因传染病动物模型的研制及应用”(2023YFF0724800);国家重点研发计划-基础科研条件与重大科学仪器设备研发专项“单/多病因传染病动物模型的研制及应用”(2023YFF0724801);上海市公共卫生临床中心项目“流感相关脑病发生机制研究”(KY-GW-2025-22)

Establishment and Preliminary Analysis of an AG6 Mouse Encephalopathy Model Induced by Vaccinia Virus Tiantan Strain Infection

YANG Lin¹^,², JIN Meng², WU Hanqing¹^,², LI Shun²(), ZHOU Xiaohui²()

^1.College of Life Sciences, Shanghai Normal University, Shanghai 200233, China
^2.Experimental Animal Department, Shanghai Public Health Clinical Center, Shanghai 201508, China

Received:2025-05-13 Revised:2025-08-19 Published:2026-02-25 Online:2026-02-14
Contact: LI Shun (ORCID: 0000-0002-9887-8879), E-mail: lishun86@126.com
ZHOU Xiaohui (ORCID: 0000-0002-7491-628X), E-mail: zhouxiaohui@shaphc.org;

摘要/Abstract

摘要：

目的构建痘苗病毒天坛株（vaccinia virus Tiantan strain，VTT）感染AG6小鼠脑病动物模型。方法通过以0.01的感染复数（multiplicity of infection，MOI）感染Vero细胞，扩增VTT并进行浓缩和滴定，孵育72 h后，收集含病毒的细胞进行浓缩；将浓缩的病毒悬浮液连续稀释（10倍梯度）并加入到含有形成致密单层Vero细胞的6孔板中进行噬斑实验，计数每孔形成的空斑数目，并根据样品的稀释倍数计算病毒滴度。将14只5~6周龄的AG6小鼠（雌雄各半，并按性别分笼饲养）随机分为对照组（n=3，PBS）、低接种量组（n=6，1×10? PFU）和高接种量组（n=5，5×10? PFU）。将小鼠经异氟烷吸入麻醉后，进行滴鼻感染。每日观察各组小鼠精神状态，记录其体重及死亡情况，并在感染后第13天经尾静脉注射2%伊文思蓝（Evans Blue）（4 mL/kg体重）以评估各组小鼠血脑屏障（blood-brain barrier，BBB）的破损情况，然后收集小鼠脑组织样本进行星形胶质细胞和小胶质细胞活化的免疫荧光分析。结果纯化后的VTT滴度为1×10⁷PFU/mL。与对照组相比，低接种量组小鼠的体重无明显变化，且未出现死亡；高接种量组小鼠在感染后第5天开始体重显著减轻（P＜0.05），并伴有致死结果。感染后第13天，低接种量组小鼠的脑组织中未检测到伊文思蓝外渗，高接种量组小鼠的嗅球区域显示出明显的蓝色染色，提示BBB破裂。免疫荧光分析显示，感染后第13天，低接种量组小鼠嗅球区域星形胶质细胞和小胶质细胞没有明显增殖，高接种量组小鼠中可观察到明显的胶质细胞活化。结论成功建立VTT感染AG6小鼠的脑病动物模型，其特征是小鼠BBB损伤和特异性定位于嗅球区域的反应性胶质增生（表现为星形胶质细胞和小胶质细胞增生）。

关键词: 痘苗病毒天坛株, 脑病动物模型, 血脑屏障, 胶质细胞, AG6小鼠

Abstract:

Objective A mouse model of vaccinia virus Tiantan strain (VTT)-induced encephalopathy was developed using AG6 mice. Methods VTT was amplified by infecting Vero cells at a multiplicity of infection (MOI) of 0.01, followed by concentration and titration. After 72 h of incubation, virus-containing cells were collected and subjected to concentration. The concentrated viral suspension was serially diluted (10-fold dilutions) and added to 6-well plates containing confluent Vero cell monolayers for plaque assay. The number of plaques formed in each well was counted, and the virus titer was calculated based on the dilution factor. Fourteen 5-6-week-old AG6 mice (half male and half female, housed separately by sex) were randomly divided into a control group (n=3, PBS), a low-dose group (n=6, 1×10? PFU), and a high-dose group (n=5, 5×10? PFU). The mice were anesthetized by isoflurane inhalation and then infected via intranasal instillation. The mental state of the mice in each group was observed daily, and the body weight and mortality were recorded. On day 13 post-infection, 2% Evans Blue (4 mL/kg body weight) was administered via tail vein injection to assess blood-brain barrier (BBB) disruption. Subsequently, brain tissue samples were collected for immunofluorescence analysis to evaluate the activation of astrocytes and microglia. Results The titer of purified VTT was 1×10? PFU/mL. Compared with the control group, mice in the low-dose group showed no significant change in body weight, and no lethality was observed. In contrast, mice in the high-dose group exhibited significant weight loss starting on day 5 post-infection (P<0.05), accompanied by lethality. On day 13 post-infection, no Evans Blue extravasation was detected in the brain tissues of the low-dose group, while the olfactory bulb region of the high-dose group displayed distinct blue staining, indicating disruption of the BBB. Immunofluorescence analysis revealed no significant proliferation of astrocytes and microglia in the olfactory bulb region of the low-dose group on day 13 post-infection. In contrast, marked activation of glial cells was observable in the high-dose group. Conclusion An animal model of VTT-induced encephalopathy in AG6 mice is successfully established, characterized by BBB disruption and reactive gliosis specifically localized to the olfactory bulb region, manifested as astrocytic and microglial proliferation.

Key words: Vaccinia virus Tiantan strain, Animal model of encephalopathy, Blood-brain barrier, Glial cells, AG6 mice

中图分类号:

Q95-33

杨琳,金梦,吴汗青,等. 痘苗病毒天坛株感染AG6小鼠脑病模型的建立与初步分析[J]. 实验动物与比较医学, 2026, 46(1): 3-10. DOI: 10.12300/j.issn.1674-5817.2025.070.

YANG Lin,JIN Meng,WU Hanqing,et al. Establishment and Preliminary Analysis of an AG6 Mouse Encephalopathy Model Induced by Vaccinia Virus Tiantan Strain Infection[J]. Laboratory Animal and Comparative Medicine, 2026, 46(1): 3-10. DOI: 10.12300/j.issn.1674-5817.2025.070.

图/表 3

注：A，痘苗病毒天坛株（VTT）的噬斑实验结果。从左至右，依次为PBS组、稀释10⁵倍组、稀释10⁶倍组、稀释10⁷倍组、稀释10⁸倍组和稀释10⁹倍组。不同滴度VTT感染后AG6小鼠的体重变化曲线（B）和生存曲线（C）。^??P＜0.01，^???P＜0.001，^????P＜0.000 1。
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图1 VTT的噬斑实验结果以及感染后小鼠的体重曲线和生存曲线
注：A，痘苗病毒天坛株（VTT）的噬斑实验结果。从左至右，依次为PBS组、稀释10⁵倍组、稀释10⁶倍组、稀释10⁷倍组、稀释10⁸倍组和稀释10⁹倍组。不同滴度VTT感染后AG6小鼠的体重变化曲线（B）和生存曲线（C）。^??P＜0.01，^???P＜0.001，^????P＜0.000 1。

Figure 1 Plaque assay results， mouse weight change curves， and survival curves following VTT infection
Note： A， The plaque assay results of vaccinia virus Tiantan strain （VTT）. From left to right， the groups are PBS， 10?-fold dilution group， 10?-fold dilution group， 10?-fold dilution group， 10?-fold dilution group， and 10?-fold dilution group. Weight change curves （B） and survival curves （C） of AG6 mice following infection with VTT at different titers. ^??P＜0.01， ^???P＜0.001， ^????P＜0.000 1.

注：A，小鼠实验流程图，从左至右，依次为滴鼻感染，感染后第13天胶质纤维酸性蛋白（GFAP）、离子钙接头蛋白分子-1（IBA-1）的免疫荧光染色和伊文思蓝染色。B，从左至右，依次为滴鼻PBS组、1×10⁵PFU和5×10⁵PFU痘苗病毒天坛株（VTT）感染组的伊文思蓝染色。箭头指示为阳性的嗅球部位。
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图2 伊文思蓝法评价不同滴度VTT感染后AG6小鼠的血脑屏障通透性
注：A，小鼠实验流程图，从左至右，依次为滴鼻感染，感染后第13天胶质纤维酸性蛋白（GFAP）、离子钙接头蛋白分子-1（IBA-1）的免疫荧光染色和伊文思蓝染色。B，从左至右，依次为滴鼻PBS组、1×10⁵PFU和5×10⁵PFU痘苗病毒天坛株（VTT）感染组的伊文思蓝染色。箭头指示为阳性的嗅球部位。

Figure 2 Evaluation of blood-brain barrier permeability in AG6 mice infected with different titers of VTT using the Evans Blue assay
Note： A， Mouse experiment flowchart， from left to right， intranasal infection； glial fibrillary acidic protein （GFAP） and ionized calcium-binding adapter molecule 1（IBA-1） immunofluorescence staining and Evans Blue staining on day 13 post-infection. B， From left to right， intranasal administration of PBS group， 1×10⁵ PFU and 5×10⁵ PFU of vaccinia virus Tiantan strain （VTT） infected groups after injection of Evans Blue. An arrow points to the positive staining in the olfactory bulb.

注：A，小鼠在第13天时嗅球组织的胶质纤维酸性蛋白（GFAP）免疫荧光染色。从左至右，依次为标记细胞核、标记星形胶质细胞、双通道叠加图；从上至下，依次为滴鼻PBS组、1×10⁵PFU和5×10⁵PFU痘苗病毒天坛株（VTT）感染组。B，小鼠在第13天时嗅球组织的离子钙接头蛋白分子1（IBA-1）免疫荧光染色。从左至右，依次为标记细胞核、标记小胶质细胞、双通道叠加图；从上至下，依次为滴鼻PBS组、1×10⁵PFU和5×10⁵PFU VTT感染组。
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图3 GFAP和IBA-1免疫荧光标记显示小鼠嗅球星形胶质细胞及小胶质细胞分布（×20）
注：A，小鼠在第13天时嗅球组织的胶质纤维酸性蛋白（GFAP）免疫荧光染色。从左至右，依次为标记细胞核、标记星形胶质细胞、双通道叠加图；从上至下，依次为滴鼻PBS组、1×10⁵PFU和5×10⁵PFU痘苗病毒天坛株（VTT）感染组。B，小鼠在第13天时嗅球组织的离子钙接头蛋白分子1（IBA-1）免疫荧光染色。从左至右，依次为标记细胞核、标记小胶质细胞、双通道叠加图；从上至下，依次为滴鼻PBS组、1×10⁵PFU和5×10⁵PFU VTT感染组。

Figure 3 Immunofluorescence labeling of GFAP and IBA-1 showing the distribution of astrocytes and microglia in the olfactory bulb of mice（×20）
Note： A， Immunofluorescence staining of glial fibrillary acidic protein （GFAP） in the olfactory bulb tissue of mice on day 13. From left to right， images of nuclei staining， glial fibrillary acidic protein （GFAP）， and the dual-channel merged image； From top to bottom， intranasal administration of PBS group， 1×10⁵ PFU and 5×10⁵ PFU of vaccinia virus Tiantan strain （VTT） infected groups. B， Immunofluorescence staining of ionized calcium-binding adapter molecule 1（IBA-1） in the olfactory bulb tissue of mice on day 13. From left to right， images of nuclei staining， ionized calcium-binding adapter molecule 1（IBA-1）， and the dual-channel merged image； From top to bottom， intranasal administration of PBS group， 1×10⁵ PFU and 5×10⁵ PFU of VTT infected groups.

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痘苗病毒天坛株感染AG6小鼠脑病模型的建立与初步分析

Establishment and Preliminary Analysis of an AG6 Mouse Encephalopathy Model Induced by Vaccinia Virus Tiantan Strain Infection

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