【摘要】 目的:利用C57BL/6 NMU基因敲除小鼠，构建胰腺原位荷瘤模型，为研究胰腺癌发生发展的分子机制并寻找新的免疫治疗策略提供前提基础。方法:根据NMU基因序列结构，在第二个外显子内设计敲除鉴定引物，用PCR以及琼脂糖凝胶电泳手段鉴定基因型。将杂合子小鼠采用雌雄2∶1按比例合笼进行扩繁，以期得到NMU基因敲除纯合子小鼠。将2×10~7的Panc02细胞接种NMU基因敲除纯合子小鼠胰尾，构建原位荷瘤模型。生物信息学分析NMU差异表达、患者生存周期、临床病理分析研究和肿瘤免疫微环境变化。流式细胞术分析NMU敲除后荷瘤小鼠脾脏中CD8~+T细胞浸润情况。结果:C57BL/6 NMU基因敲除杂合子小鼠经过繁育与鉴定得到NMU基因敲除纯合子鼠，其胰尾接种Panc02细胞后成功构建胰腺原位肿瘤模型。生物信息学分析表明NMU作为胰腺癌发展中的早期且持续事件，引起患者预后不良，使肿瘤微环境呈免疫抑制状态。流式细胞术表明NMU敲除后荷瘤小鼠全身免疫景观中CD8~+T细胞表达增多。结论:构建C57BL/6 NMU基因敲除小鼠胰腺原位荷瘤模型，对研究NMU作为胰腺癌发生发展中预后不良事件、胰腺癌免疫抑制机制的研究具有重要意义。更多还原

【Abstract】 Objective:NMU gene knockout C57BL/6 mice were used for establishing orthotopic pancreatic cancer models.It provides a prerequisite foundation for studying the molecular mechanisms of pancreatic cancer development and finding new immunotherapy strategies.Methods:According to the NMU gene sequence structure, knockout identification primers were designed within the second exon, and mouse genotypes were identified using PCR and agarose gel electrophoresis.Heterozygous mice were propagated in a cage with male and female in a ratio of 2∶1,in order to obtain NMU gene knockout homozygous mice.2×10~7 Panc02 cells were inoculated into the pancreatic tail of homozygous NMU gene knockout mice to establish an orthotopic tumor-bearing model.Bioinformatics analysis of the differential expression of NMU,patient survival period, clinical pathological analysis and changes in the tumor immune microenvironment.Flow cytometry analysis of CD8~+T cell infiltration in the spleen of tumor-bearing mice after NMU knockout.Results:After breeding and identification of C57BL/6 NMU gene knockout heterozygous mice, NMU gene knockout homozygous mice were obtained, and the pancreatic tail was inoculated with Panc02 cells to successfully construct a pancreatic orthotopic tumor model.Bioinformatics analysis shows that NMU,as an early and continuous event in the development of pancreatic cancer, causes poor prognosis of patients and makes the tumor microenvironment in an immunosuppressive state.Flow cytometry showed that the expression of CD8~+T cells increased in the systemic immune landscape of tumor-bearing mice after NMU knockout.Conclusion:The C57BL/6 NMU gene knockout mice were established to construct an in situ tumor bearing model of the pancreas, which provides a prerequisite basis for the study of NMU as a prognostic adverse event in the occurrence and development of pancreatic cancer and the study of immunosuppression mechanism of pancreatic cancer.更多还原