目的：通过构建表达IL-12的小鼠CAR-T细胞，探讨经尾静脉将其输注于小鼠体内建立细胞因子释放综合征（CRS）模型的方法。方法：构建基于靶向鼠源CD19的CAR分子，包装逆转录病毒载体并感染小鼠T细胞构建mCD19-CAR-T、mCD19/IL-12-CAR-T 细胞。通过构建小鼠体内胰腺癌 Panc02-CD19 细胞移植瘤模型，检测 mCD19/IL-12-CAR-T 细胞的抗肿瘤活性，ELISA法检测两种CAR-T细胞IL-12和IFN-γ分泌水平；经小鼠尾静脉输注mCD19/IL-12-CAR-T 细胞构建CAR-T细胞CRS小鼠模型，流式细胞术检测小鼠血清中IL-6、MCP-1、IL-1、IL-10、TNF-α、IFN-γ等细胞因子的含量，H-E染色法观察荷瘤小鼠肝、脾、肺和肾的病理组织学变化。结果：经过培养扩增的mCD19/IL-12-CAR-T细胞能有效分泌IL-12，CAR阳性率达（56.9±5.4）%；与非靶细胞Panc02或靶细胞Panc02-CD19共培养时，均能高分泌IFN-γ。成功构建小鼠胰腺癌Panc02-CD19细胞移植瘤模型，经小鼠尾静脉注射1×106个mCD19/IL-12-CAR-T细胞能显著抑制移植瘤的生长，但未能诱发严重CRS；输注2×106个mCD19/IL-12-CAR-T细胞后，小鼠出现体质量减轻、血清炎性因子水平升高、组织损伤，最终导致死亡等一系列典型CRS表现。结论：成功构建IL-12-CAR-T细胞诱发的小鼠CRS模型，其稳定性好、重复性高，具有广泛的应用前景。

Objective：To discuss the method of establishing a cytokine release syndrome (CRS) model by constructing murineoriented chimeric antigen receptor-modified T (CAR-T) cells expressing IL-12 and infusing them back into mice via the tail vein.Methods: CAR molecules targeting murine-derived CD19 were constructed, in which the retroviral vectors were packaged, and then the constructed molecules were used to infect mouse T cells to prepare mCD19-CAR-T and mCD19/IL-12-CAR-T cells. The anti-tumor activity of mCD19/IL-12-CAR-T cells was measured by constructing pancreatic cancer Panc02-CD19 cell transplanted tumor model in mice, and the levels of IL-12 and IFN-γ secreted by two CAR-T cells were detected by ELISA; CRS model was constructed by infusing mCD19/IL-12-CAR-T cells back into the tail vein of the mice. The levels of IL-6, MCP-1, IL-1, IL-10, TNF- α, IFN- γ, and other cytokines in the serum of mice were detected by flow cytometry, and the histopathological changes of liver, spleen, lung, and kidney of the mice were observed by H-E staining. Results: After culture expansion, mCD19/IL-12-CAR-T cells could effectively secrete IL-12,and the CAR positive rate reached (56.9±5.4)%; the modified T cells could efficiently secrete IFN-γ no matter co-cultured with nontargeted Panc02 cells or targeted Panc02-CD19 cells. A mouse pancreatic cancer Panc02-CD19 cell transplanted tumor model was successfully constructed, and the tail vein infusion of 1×106 mCD19/IL-12-CAR-T cells significantly inhibited the growth of the transplanted tumor, but failed to induce severe CRS; after the infusion of 2×106 mCD19/IL-12-CAR-T cells, a series of typical CRS manifestations such as reduced body mass, elevated serum inflammatory factor levels, tissue damage and even death were observed in the mice. Conclusion: The IL-12-CAR-T cell-induced CRS model in mice was successfully constructed, and it is stable and reproducible with wide application prospects.

国家自然科学基金资助项目（No.82001206）；江苏省中医院高峰学术人才培养工程（No.y2021rc42）；医学免疫学国家重点实验室开放课题（No.NKMI2021K18）；江苏省中医药科技发展计划项目（No.ZT202106）；江苏省研究生科研与实践创新计划项目（No. SJCX22_0750,No.KYCX22_1899）