不同品系小鼠侵袭性真菌感染动物模型的构建  

Construction of animal models of invasive fungal infections in different strains of mice

作　　者：王迪 黄劲[3,4] 姚昌昊 王梅竹 程敏 郭鑫瑶 廖万清 康颖倩 WANG Di;HUANG Jing;YAO Changhao;WANG Meizhu;CHENG Min;GUO Xinyao;LIAO Wanqing;KANG Yingqian(Department of Microbiology,School of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,Guizhou,China;Guizhou Talent Base for Research on the Relationship between Microbiology and Human Health&Key Laboratory of Pathogen Biology of Guizhou Higher Education Department,Guiyang 550025,Guizhou,China;Key Laboratory of Environmental Pollution Monitoring and Disease Control of Guizhou Medical University of Ministry of Education,Guiyang 550025,Guizhou,China;Department of Biochemistry and Molecular Biology,Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,Guizhou,China;Department of Dermatology,Changzheng Hospital Affiliated to Navy Medical University,Shanghai 200003,China;Shanghai Key Laboratory of Molecular Medical Mycology,Shanghai 200003,China;Guizhou Academy of Tobacco Science,Guiyang 550009,Guizhou,China)

机构地区：[1]贵州医科大学基础医学院微生物学教研室,贵州贵阳550025 [2]贵州省微生物与人类健康关系研究人才基地&贵州省普通高校病原生物学特色重点实验室,贵州贵阳550025 [3]贵州医科大学环境污染与疾病监控教育部重点实验室,贵州贵阳550025 [4]贵州医科大学基础医学院生物化学与分子生物学教研室,贵州贵阳550025 [5]海军军医大学长征医院皮肤科,上海200003 [6]上海市医学真菌分子生物学重点实验室,上海200003 [7]贵州省烟草科学研究院,贵州贵阳550009 [8]贵州医科大学

出　　处：《贵州医科大学学报》2021年第10期1117-1125,共9页Journal of Guizhou Medical University

基　　金：国家自然科学基金(32060034);国家科技部及教育部“111计划”(D20009);贵州省第六批人才基地项目(RCJD2018-22);贵州省科技厅社会发展攻关支撑项目[黔科合支撑(2019)2873];贵州省科学技术厅国际科技合作基地[黔科合平台人才(2020)4101];贵州省科技计划项目[黔科合支撑(2020)4Y220]。

摘　　要：目的探讨不同品系小鼠侵袭性真菌感染动物模型的构建方法。方法取白念珠菌ATCC10231 T菌株(标准菌株)、GYWu27菌株[临床艾滋病(AIDS)病人来源]、GYF76菌株[临床外阴阴道念珠菌病(VVC)病人来源]、N110菌株(健康人来源)及新生隐球菌H99^(T)菌株(标准菌株)培养至对数生长期,离心重悬于无菌盐水中,采用分光光度法及细胞计数板调整菌液浓度至所需浓度;4~6周龄SPF级KM小鼠和Balb/c小鼠各30只,随机均分为5组,分别注射浓度1×10^(6)、1×10^(7)、1×10^(8)、1×10^(9)及1×10^(10) CFU/L白念珠菌ATCC10231 T菌株,观察14 d内小鼠的死亡速率筛选侵袭性真菌感染的最适浓度;取4~6周龄SPF级KM小鼠和Balb/c小鼠各50只,随机均分为GYWu27组、GYF76组、N110组、H99^(T)组及生理盐水(Saline)组,各菌株感染组小鼠经侧尾静脉注射最适浓度菌液感染、Saline组小鼠注射Saline,记录各组小鼠30 d内的一般状态、体质量及90 d内的生存情况;取GYWu27组、GYF76组及N110组死亡小鼠的肾脏、H99^(T)组小鼠脑组织及定期分批处死的Saline组小鼠肾脏和脑组织分别制作组织匀浆、稀释后接种于含0.05%氯霉素葡萄糖琼脂培养基(SDA)、检测肾脏和脑组织的真菌负荷量,接种于SDA、酵母浸出粉胨葡萄糖培养基(YPD)鉴定分离后菌株;取GYWu27组、GYF76组、N110组两种系小鼠肾脏、肝脏及H99^(T)组两种系小鼠的肾脏、肝脏及脑组织,制作组织切片,观察组织学特征。结果侵袭性真菌感染动物模型Balb/c小鼠和KM小鼠的最适感染浓度分别为1×10^(8) CFU/L和1×10^(9) CFU/L;相同实验条件下,感染后KM小鼠一般状态较Balb/c小鼠好,白念珠菌及新生隐球菌感染的KM小鼠30 d内的体质量变化明显;白念珠菌和新生隐球菌体外培养菌株及感染小鼠体内分离菌株的培养特性无差异;白念珠菌感染的Balb/c小鼠靶器官真菌负荷量明显高于KM小鼠;新生隐球菌感染小鼠病变集中于脑组织Objective To explore the methods of constructing animal models of invasive fungal infections in different strains of mice.Methods Candida albicans strains ATCC10231 T(standard strain),GYWu27(clinical AIDS patient source),GYF76(clinical VVC patient source),N110(healthy human source)and Cryptococcus neoformans strain H99^(T)(standard strain)were cultured to logarithmic growth phase,resuspended by centrifugation in sterile saline,and the concentration of the bacterial solution was adjusted to the desired concentration by spectrophotometric methods and cell counting plates.Thirty 4-6 week-old KM mice and Balb/c mice were randomly divided into five groups and injected with 1×10^(6),1×10^(7),1×10^(8),1×10^(9),and 1×10^(10) CFU/L of C.albicans ATCC10231 T strain,respectively,to observe the death rate of mice within 14 d to screen the optimal concentration for invasive fungal infection.Each of 50 KM mice and Balb/c mice aged 4-6 weeks were randomly divided into GYWu27 group,GYF76 group,N110 group,H99^(T) group,and saline group.The mice in each strain infection group were injected with the aforementioned lateral tail vein.The optimal concentration of bacterial infection obtained in the experiment(determining the inoculation dose of bacterial solution according to body weight),saline group mice were injected with normal saline according to their body weight.The general status,body mass for 30 d and 90 d survival of each group of mice were recorded regularly.The kidneys of the mice in the GYWu27,GYF76,and N110 groups,the brain tissues of the mice in the H99^(T) group,and the kidneys and brain tissues of the mice in saline group that were killed in batches were taken to make tissue homogenates.After dilution,it was inoculated on a SDA plate containing 0.05%chloramphenicol to detect the fungal load of kidney and brain tissue,and then inoculated on SDA and YPD to identify the isolated strains.Take the kidney,liver and brain tissues of KM mice and Balb/c mice in the GYWu27,GYF76,and N110 group that died onset and death,and

关 键 词：念珠菌 白色 隐球菌 新型 动物模型 真菌感染 KM小鼠 BALB/C小鼠 

分 类 号：R379.9[医药卫生—病原生物学]