目的·通过融合预免疫策略与mVenus-p27K^－细胞G₀期指示系统、DTR-HSV/TK自杀基因系统以及Luc2-tdTomato示踪系统，构建一个无明显转移灶的肿瘤休眠小鼠模型。方法·在KPC1199小鼠胰腺癌细胞系中，依次引入mVenus-p27K^－细胞G₀期指示系统、DTR-HSV/TK自杀基因系统及Luc2-tdTomato示踪系统，构建KPC1199-PDL稳定表达细胞株。KPC1199-PDL细胞在无血清条件下培养后，通过流式细胞术分选为mVenus（+）细胞及mVenus（-）细胞，采用实时荧光定量PCR（qPCR）验证G₀期相关基因的表达。采用CCK-8细胞增殖实验评估KPC1199-PDL细胞对白喉毒素（DTX）和更昔洛韦（GCV）的敏感性。在野生型C57BL/6小鼠中构建经脾-门静脉-肝转移模型，利用免疫荧光技术验证KPC1199-PDL细胞在体内的功能。在C57BL/6小鼠皮下注射KPC1199-PDL细胞，5 d后原位注射DTX和GCV消融皮下瘤，获得预免疫小鼠，并在此基础上构建经脾-门静脉-肝转移模型。采用生物发光成像评估皮下瘤消融和肝转移情况，利用免疫荧光染色检测预免疫小鼠肝脏中肿瘤细胞的分布及休眠状态。结果·KPC1199-PDL细胞稳定表达3种工具系统，且其增殖能力未受影响。在无血清条件培养下，部分KPC1199-PDL细胞表达mVenus蛋白，即进入G₀期；经流式细胞术分选后得到的mVenus（+）细胞G₀期相关基因较mVenus（-）细胞显著高表达（均P<0.05），而增殖相关基因显著低表达（P<0.05）。CCK-8实验显示KPC1199-PDL细胞对DTX和GCV高度敏感。体内实验证实KPC1199-PDL细胞可通过表达tdTomato蛋白有效示踪，以及表达mVenus蛋白提示细胞进入G₀期。经皮下种瘤和药物消融后成功获得预免疫小鼠，在此基础上构建的经脾-门静脉-肝转移模型，生物发光成像未在肝脏观察到转移信号，但肝脏组织切片经免疫荧光检测发现存在单个或小簇状同时表达mVenus和tdTomato，但不表达增殖标志物Ki67的G₀期肿瘤细胞。结论·胰腺癌预免疫小鼠模型结合mVenus-p27 K^－指示系统、DTR-HSV/TK自杀基因系统及Luc2-tdTomato示踪系统，成功得到可识别、可示踪的休眠肿瘤动物模型。

Objective ·To establish a mouse model with dormant cancer and no obvious metastasis by combining the preimmune strategy with the mVenus-p27K^－ cell G₀ phase indicator system, the DTR-HSV/TK suicide gene system, and the Luc2-tdTomato tracer system. Methods ·The KPC1199 mouse pancreatic cancer cell line was transfected with the mVenus-p27K^－ cell G₀ phase indicator system, the DTR-HSV/TK suicide gene system, and the Luc2-tdTomato tracer system to construct a stable expression cell line, KPC1199-PDL. After being cultured in the serum-free condition, KPC1199-PDL cells were sorted into mVenus (+) cells and mVenus (-) cells by flow cytometry, and the expression of G₀ phase-related genes was verified by real-time fluorescence quantitative PCR (qPCR). Sensitivity of KPC1199-PDL cells to diphtheria toxin (DTX) and ganciclovir (GCV) was evaluated by CCK-8 assay. A transsplenic portal vein-hepatic metastasis model was constructed in wild-type C57BL/6 mice to validate the function of KPC1199-PDL cells in vivo by immunofluorescence technology. The KPC1199-PDL cells were injected subcutaneously into C57BL/6 mice, followed by in situ injection of DTX and GCV to ablate subcutaneous tumors 5 d later, to obtain preimmunized mice. The transsplenic portal vein-hepatic metastasis models were constructed in these mice. Bioluminescence imaging was used to evaluate subcutaneous tumor ablation and hepatic metastasis in the mice, and immunofluorescence assay was used to detect the distribution and dormant state of tumor cells in the livers of preimmunize mice. Results ·The three tool systems were stably expressed in KPC1199-PDL cells, and their proliferative ability was not affected. In the serum starving condition, some KPC1199-PDL cells expressed the mVenus protein, indicating entry into the G₀ phase; the mVenus (+) cells sorted out by flow cytometry exhibited significantly higher expression of G₀ phase-related genes (all P<0.05) and significantly lower expression of the proliferation-related gene compared with mVenus (-) cells (P<0.05). The CCK-8 assay demonstrated high sensitivity of KPC1199-PDL cells to DTX and GCV. In vivo experiments confirmed that KPC1199-PDL cells could be effectively traced through tdTomato protein expression, and could indicate entry into the G₀ phase through mVenus protein expression. Following subcutaneous tumor implantation and drug ablation, preimmunized mice were successfully obtained. In the subsequent transsplenic portal vein-hepatic metastasis model, no metastatic signals were observed in the liver by bioluminescence imaging, but single or small clusters of G₀ phase tumor cells expressing both mVenus and tdTomato, not expressing the proliferation marker Ki67, were detected in liver tissue sections by immunofluorescence analysis. Conclusions ·A recognizable and traceable dormant cancer model is constructed with the combination of the preimmune mouse model of pancreatic cancer, the mVeneus-p27K^－ indicator system, the DTR-HSV/TK suicide gene system, and the Luc2-tdTomato tracer system.