新型冠状病毒感染肺炎小鼠模型的建立与康复研究  

Establishment and rehabilitation of a mouse model of novel coronavirus pneumonia

作　　者：喻柯瑶 汤玲玲 王博寒 孙宪泓 张晓娜 张慧 史潇璐[1] YU Keyao;TANG Lingling;WANG Bohan;SUN Xianhong;ZHANG Xiaona;ZHANG Hui;SHI Xiaolu(Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210029,China;Nanjing University of Chinese Medicine,210023;Nanjing Traditional Chinese Medicine Hospital,210022)

机构地区：[1]南京中医药大学附属医院,江苏南京210029 [2]南京中医药大学,江苏南京210023 [3]南京市中医院,江苏南京210022

出　　处：《广东药科大学学报》2025年第1期117-124,共8页Journal of Guangdong Pharmaceutical University

基　　金：江苏省自然科学基金项目(BK20211391);国家中医药管理局科技项目(2021ZYLCYJ05-3);南京市中医院科技项目(XGJB202202)。

摘　　要：目的 建立一种稳定可复制、高度模拟新型冠状病毒感染肺炎(novel coronavirus pneumonia,NCP)恢复期患者的小鼠模型。方法 将30只Balb/c雌性小鼠,随机分为对照组和感染后第1、3、7、10天组,共5组(n=6),感染组小鼠予SARS-CoV-2病毒棘突(Spike,S)蛋白原液气管滴注进行感染,分别于感染后第1、3、7、10天取材,依次标记为D1S组、D3S组、D7S组、D10S组;对照组(N组)进行假手术,第10天取材。采用免疫荧光双染法观察S蛋白与肺组织ACE2受体结合情况;HE染色观察小鼠的肺组织病理,RT-qPCR测定小鼠肺组织中IL-4、IL-6 mRNA表达;流式细胞术检测小鼠脾脏CD4^(+)T淋巴细胞中Th17/Treg、Th1/Th2细胞比值、DC细胞中cDC、pDC、CD80^(+)、CD86^(+)细胞比例。结果 荧光双染色结果提示S蛋白和血管紧张素转化酶2(ACE2)可在气道上皮细胞中位置重合;HE染色结果显示,与N组相比,D1S组小鼠肺部组织结构紊乱,上皮细胞变性、塌陷,肺泡损伤,伴间质水肿和炎性浸润,而D3S、D7S及D10S组小鼠则呈现逐渐恢复的状态,D10S组小鼠肺组织相较于对照组还存在一些轻微的病理损伤;与N组比较,IL-4 mRNA含量在D1S组小鼠无明显变化(P>0.05),但在D10S组小鼠肺组织中明显升高(P<0.01),而IL-6 mRNA含量在D1S组小鼠显著升高(P<0.01),且在D3S组及D10S组有所降低(P<0.01),但D10S组小鼠IL-6 mRNA水平相较于N组仍有升高(P <0.05);流式细胞术结果显示,与N组比较,D10S组小鼠Th17/Treg显著增加(P<0.01)、Th1/Th2比值显著降低(P<0.05);相较于N组,D10S组小鼠中cDC、CD80^(+)、CD86^(+)细胞比例均有所减少(P<0.05),而D10S组小鼠pDC细胞比例则无明显改变(P>0.05)。结论 D10S组小鼠肺部病理、炎症因子、免疫细胞相关指标变化符合NCP恢复期的临床特征,S蛋白滴鼻可模拟NCP小鼠模型,为新型冠状病毒感染恢复期的研究奠定了基础;NCP恢复期患者仍存在机体损伤及免疫功能紊乱等问题,提示新型冠状病�Objective To establish a stable,reproducible mouse model that highly simulates the recovery period of NCP patients for the research on their rehabilitation.Methods Thirty Balb/c female mice were randomly divided into 5 groups(n=6)on the 1 st,3rd,7th,and 10th days after infection and the control group,and the infected mice were infected by tracheal instillation of SARS-CoV-2 virus spike(S)protein stock solution,and the samples were collected on the 1 st,3rd,7th,and 10th days after infection,and labeled as D1S group,D3S group,D7S group,and D10S group,and the control group(N group)underwent sham surgery,and the samples were collected on the 10th day. The binding of S protein to ACE2 receptor in lung tissue was observed by immunofluorescence doublestaining. The lung histopathology of mice was observed by HE staining. The mRNA expression of IL-4 and IL-6in lung tissue was determined by RT-qPCR. The ratio of Th17/Treg and Th1/Th2 cells in CD4^(+) T lymphocytes andthe proportion of cDC, pDC, CD80^(+) and CD86^(+) cells in DC cells were detected by flow cytometry. Results Thefluorescence double staining results indicated that S protein and ACE2 coincided in position in airway epithelialcells. HE staining showed that compared with the N group, the lung tissue structure of the D1S group wasdisordered, the epithelial cells were degenerated, collapsed, and the alveolar was damaged, accompanied byinterstitial edema and inflammatory infiltration, while the D3S, D7S and D10S groups showed a gradual recovery,and the lung tissue of the D10S group still had some slight pathological damage compared with the control group.Compared with the N group, the content of IL-4 mRNA did not change significantly in the D1S group, but it wassignificantly increased in the lung tissue of the D10S group (P<0.01). The content of IL-6 mRNA wassignificantly increased in the D1S group (P<0.01), decreased in the D3S group and D10S group (P<0.01), butincreased in the D10S group (P<0.05). Flow cytometry results showed that compared with the N group, the

关 键 词：新型冠状病毒感染肺炎 恢复期 免疫紊乱 动物模型 

分 类 号：R-332[医药卫生]