重组鼠IFN-γ腺病毒诱发细胞因子释放综合征小鼠模型的建立和观察  

Establishment and observation of a mouse model of cytokine release syndrome induced by recombinant mouse IFN-γadenovirus

作　　者：杨箐 张玮光 黎陈铖 刘细细 胡中晓 王风楠 陈碧清 田芳 张晓丽 热爱拉·加那提 朱学军 YANG Jing;ZHANG Weiguang;LI Chencheng;LIU Xixi;HU Zhongxiao;WANG Fengnan;Chen Biqing;TIAN Fang;ZHANG Xiaoli;JIANATI Reaila;ZHU Xuejun(The Affiliated Hospital of Nanjing University of Chinese Medicine,the First Clinical Medical College of Nanjing University of Chinese Medicine,Nanjing 210029,Jiangsu,China;Central Laboratory,the Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210029,Jiangsu,China;Department of Hematology,the Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210029,Jiangsu,China)

机构地区：[1]南京中医药大学附属医院、南京中医药大学第一临床医学院,江苏南京210029 [2]南京中医药大学附属医院中心实验室,江苏南京210029 [3]南京中医药大学附属医院血液科,江苏南京210029

出　　处：《中国肿瘤生物治疗杂志》2024年第2期128-134,共7页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金(No.82001206);江苏省中医药科技发展计划(No.ZT202106);江苏省中医院高峰学术人才培养工程(No.y2021rc42);江苏省研究生科研与实践创新计划(No.KYCX23_2122);医学免疫学国家重点实验室开放课题(No.NKMI2021K18)。

摘　　要：目的:通过向C57Bl/6J小鼠腹腔注射IFN-γ腺病毒(Ad-mIFN-γ)建立细胞因子释放综合征(CRS)的动物模型。方法:构建Ad-mIFN-γ及对照Ad-lacZ腺病毒载体,分别以MOI=100体外转染小鼠腹腔巨噬细胞,流式细胞术检测其对细胞mIFN-γ分泌的影响。将40只雌性C57Bl/6J小鼠按随机数字表法分为对照组、载体对照组、病毒低、中、高剂量组(每组8只),分别向各组小鼠腹腔注射PBS(200μL)、Ad-lacZ(2×10^(7)PFU/只)、Ad-mIFN-γ(5×10^(6)PFU/只)、Ad-mIFN-γ(1.5×10^(7)PFU/只)和Ad-mIFN-γ(2×10^(7)PFU/只)。每日观测小鼠的体质量及生存情况;第3天时采用流式细胞术检测小鼠外周血和脾内单核细胞(CD11b^(+))、巨噬细胞(CD11b^(+)/CD86^(+))比例,免疫荧光染色法检测脾内CD11b^(+)的单核细胞比例;第9天时采用流式细胞术检测小鼠血清中细胞因子的分泌水平;第14天,采用颈椎脱臼法处死小鼠,H-E染色法观察小鼠肝、脾、肺和肾的病理和组织学变化。结果:Ad-mIFN-γ体外感染小鼠腹腔巨噬细胞,在第3天检测到巨噬细胞分泌mIFN-γ达到峰值(118.34±2.90)pg/mL,并在一周内持续高分泌mIFN-γ,Ad-lacZ对照组IFN-γ分泌水平较低后,第3天时为(0.17±0.08)pg/mL。小鼠腹腔注射Ad-mIFN-γ后,在14 d内病毒低、中剂量组无小鼠死亡,病毒高剂量组小鼠体质量持续减轻(P<0.001);第3天,病毒高剂量组小鼠外周血和脾组织内单核细胞、巨噬细胞比例较对照组和中剂量组均显著增加(P<0.05或P<0.01);第9天,病毒低、中、高剂量组小鼠血清中mIFN-γ、IL-6、单核细胞趋化蛋白-1(MCP-1)、IL-1、TNF-α等细胞因子的水平均显著升高(P<0.001);10 d内病毒高剂量组小鼠死亡率达100%。组织病理检测可见病毒高剂量组小鼠的肝、脾、肺、肾组织有明显损伤。结论:Ad-mIFN-γ体外感染小鼠原代腹腔巨噬细胞后,可以快速分泌mIFN-γ;腹腔注射高剂量(2×10^(7)PFU/只)Ad-mIFN-γ导致小鼠出现CRS典型表现,可作�Objective:To establish an animal model of cytokine release syndrome(CRS)by intraperitoneal injection of mouse IFN-γadenovirus(Ad-mIFN-γ)into C57Bl/6J mice.Methods:Ad-mIFN-γand Ad-LacZ control adenoviral vectors were constructed,and mouse peritoneal macrophages were transfected with MOI=100 in vitro.The effect on mIFN-γsecretion levels of cells were detected by flow cytometry.Forty female C57Bl/6J mice were divided into the control group,the vector control group,and the low-,medium-,and high-dose virus groups(8 mice in each group)according to the random number table method.The mice were intraperitoneally injected with PBS(200μL/piece),Ad-lacZ(2×10^(7)PFU/piece),Ad-mIFN-γ(5×10^(6)PFU/piece),Ad-mIFN-γ(1.5×10^(7)PFU/piece)and Ad-mIFN-γ(2×10^(7)PFU/piece),respectively.The body weight and survival of the mice were observed daily.On the third day,flow cytometry was used to detect the proportion of monocytes(CD11b^(+))and macrophages(CD11b^(+)/CD86^(+))in the peripheral blood and the spleen,and the proportion of CD11b^(+)monocytes in the spleen was detected by immunofluorescence staining.On the 9th day,flow cytometry was used to detect the secretion of cytokines in the serum of the mice.On the 14th day,the mice were sacrificed by cervical dislocation,and H-E staining was used to observe the pathological and histological changes of the liver,spleen,lungs and kidneys of the mice.Results:Ad-mIFN-γinfected mouse peritoneal macrophages in vitro,and the level of mIFN-γsecreted by macrophages was detected to reach a peak of(118.34±2.90)pg/mL on the third day,and the secretion level of mIFN-γcontinued to be high for one week;while the Ad-lacZ control group secreted a lower level of IFN-γ,with a value of(0.17±0.08)pg/mL on the third day.After intraperitoneal injection of Ad-mIFN-γ,no mice died in the low-and medium-dose virus groups within 14 days,and the body weight of the mice in the high-dose virus group continued to decrease(P<0.001).On the third day,the proportions of monocytes and macrophages in the perip

关 键 词：Γ-干扰素 腺病毒 巨噬细胞 细胞因子释放综合征 动物模型 C57BL/6J小鼠 

分 类 号：R730.51[医药卫生—肿瘤] R73-33[医药卫生—临床医学]