糖尿病合并新型冠状病毒S蛋白感染小鼠病理生理特征  

Pathophysiological characteristics of mice with diabetes combined with SARS-CoV-2 spike protein infection

作　　者：苏小月 李静璇 林颖 张永祥 肖智勇 周文霞 SU Xiaoyue;LI Jingxuan;LIN Ying;ZHANG Yongxiang;XIAO Zhiyong;ZHOU Wenxia(State Key Laboratory of National Security Specially Needed Medicines,Academy of Military Medical Sciences,Beijing 100850,China)

机构地区：[1]军事医学研究院国家安全特需药品全国重点实验室,北京100850

出　　处：《中国药理学与毒理学杂志》2024年第6期410-419,共10页Chinese Journal of Pharmacology and Toxicology

基　　金：天津市科技发展计划项目(22ZYJDSS00080)。

摘　　要：目的建立糖尿病(DM)合并新型冠状病毒(SARS-CoV-2)感染小鼠模型,研究DM合并SARS-CoV-2感染病程发展中的重要病理生理变化。方法将野生型(WT)小鼠和由细胞角蛋白18基因启动子驱动的人血管紧张素转化酶2受体(K18-hACE2,简称hACE2)的转基因小鼠分别随机分为溶剂对照组、DM组、SARS-CoV-2病毒刺突蛋白感染(S)组以及DM合并S蛋白感染(DM+S)组,每组10~12只。除溶剂对照组及S组外,其余组通过10周高脂饮食后连续3 d ip给予链脲佐菌素(STZ)40 mg·kg^(-1)诱导DM症状,溶剂对照组及S组给予等体积0.1 mol·L^(-1)柠檬酸钠缓冲液。在此基础上,S组及DM+S组小鼠经鼻腔滴入15μg SARS-CoV-2 S蛋白与1 g·L^(-1)聚肌胞苷酸(Poly[I:C])的混合溶液50μL,溶剂对照组滴鼻给予等体积无菌水。在高脂喂养第6周和ip给予STZ 1周后,以口服葡萄糖耐量实验评价小鼠糖耐量水平及胰岛β细胞功能。高脂喂养第6周至ip给予STZ 2周后,每周用血糖仪检测小鼠随机血糖及空腹血糖。DM小鼠S蛋白感染前及感染24,48和120 h后,每组取3只小鼠颌下静脉取血后处死并取肺组织,采用苏木精-伊红染色法观察合并S蛋白感染前后的肺组织病理改变。S组小鼠在感染S蛋白前及感染6,24,48,72和120 h后采血,Luminex液相芯片技术检测小鼠血浆细胞因子白细胞介素1β(IL-1β)、IL-2、IL-6、IL-10、IL-17、干扰素诱导蛋白10(IP-10)、干扰素γ(IFN-γ)、肿瘤坏死因子α(TNF-α)、单核细胞趋化蛋白1(MCP-1)和粒细胞集落刺激因子(G-CSF)水平,ELISA检测血浆硫酸乙酰肝素(HS)水平;将细胞因子水平、HS水平与小鼠感染S蛋白后的肺部病理损伤程度进行Spearman相关性分析。结果STZ合并高脂饮食可诱导小鼠DM样表现,hACE2-DM组随机血糖(P<0.01)和1周后的空腹血糖(P<0.05)均显著高于WT-DM组,且hACE2-DM小鼠胰岛功能损伤程度显著高于WT-DM小鼠(P<0.05)。与DM组相比,DM+S组小鼠均表现出更严重的肺部病�OBJECTIVE To establish a mouse model of diabetes mellitus(DM)combined with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection to investigate the important pathophysiological changes in the development of DM combined with SARS-CoV-2 infection.METHODS Wild-type(WT)mice and transgenic mice expressing the human angiotensin-converting enzyme 2 receptor driven by the cytokeratin-18 gene promoter(K18-hACE2)were randomly divided into the control group,DM group,SARS-CoV-2 spike protein(S)infection group and DM combined with S protein infection group,with 10 to 12 mice in each group.All the mice were induced by 10 weeks of high-fat diet combined with 40 mg·kg^(-1)streptozotocin(STZ)for 3 days by ip,except those in the control group or S protein infection group.The control group was given the same volume of 0.1 mol·L^(-1)sodium citrate buffer.Mice in the S protein infection group and DM+S protein infection group were additionally given 50μL mixture of 15μg SARS-CoV-2 spike protein and 1 g·L^(-1)polyinosinic-polycytidylic acid(poly[I:C])via intranasal drops,while the control group was given an equal volume of sterile water.The glucose tolerance level and pancreatic isletβcell function of mice were evaluated via oral glucose tolerance test at the 6th week of high-fat feeding and 1 week after the administration of STZ by ip.From the 6th week of high-fat feeding to 2 weeks after the administration of STZ,the random blood glucose and fasting blood glucose of mice were measured by a blood glucose meter.Blood samples were taken from subman⁃dibular veins of 3 mice in each group at 24,48 and 120 h after S protein infection,and lung tissues were taken after euthanization.The pathological changes of lungs of DM mice before and after S protein infection were observed by HE staining.Except for the DM group,blood samples were collected before S protein infection and at 6,24,48,72 and 120 h after infection.The levels of plasma interleukin 1β(IL-1β),IL-2,IL-6,IL-10,IL-17,interferon gamma-induced protein 10(IP-10

关 键 词：新型冠状病毒感染 SARS-CoV-2 S蛋白 糖尿病 动物模型 细胞因子 硫酸乙酰肝素 

分 类 号：R96[医药卫生—药理学]