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

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(2×107 PFU/只)、Ad-mIFN-γ(5×106PFU/只)、Ad-mIFN-γ(1.5×107 PFU/只)和Ad-mIFN-γ(2×107 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...

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

Objective: To establish an animal model of cytokine release syndrome (CRS) by intraperitoneal injection of mouse IFN-γ adenovirus (Ad-mIFN- γ) into C57Bl/6J mice. Methods: Ad-mIFN- γ and Ad-LacZ control adenoviral vectors were constructed, and mouse peritoneal macrophages were transfected with MOI=100 in vitro. The effect on mIFN-γ secretion 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 (200 μL/piece), Ad-lacZ (2×107 PFU/piece), Ad-mIFN-γ (5×106 PFU/piece), Ad-mIFN-γ (1.5×107 PFU/piece) and Ad-mIFN-γ (2×107 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-γ infected mouse peritoneal macrophages in vitro, and the level of mIFN-γ secreted 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-γ continued 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 peripheral blood and spleen tissues of the mice in the high-dose virus group were significantly higher than those in the control group and the medium- dose group (P<0.05 or P<0.01). On the 9th day, the serum levels of mIFN-γ, IL-6, monocyte chemotactic protein-1 (MCP-1), IL-1 and TNF-α in the low-, medium- and high-dose groups increased significantly (P<0.001). Within 10 days, the mortality rate of the mice in the high-dose virus group reached 100%. Histopathological examination showed significant damage in the liver, spleen, lung and kidney tissues of the mice in the high-dose virus group. Conclusion: Mouse primary peritoneal macrophages can rapidly secrete mIFN-γ after infection with Ad-mIFN-γ in vitro. Intraperitoneal injection of high-dose Ad-mIFN-γ (2×107 PFU/piece) resulted in typical CRS manifestations in the mice, which can be used as an animal model for CAR-T cell therapy-induced CRS.