【摘要】 目的 探索阿帕替尼联合γ-干扰素对胃癌的治疗作用及其可能机制。方法 以人胃癌细胞系MGC803为研究对象，随机分为对照组[仅给予等量1%二甲基亚砜（dimethyl sulfoxide, DMSO）处理]、阿帕替尼组（给予阿帕替尼8μmol/L）、γ-干扰素组（给予γ-干扰素100 U/ml）、阿帕替尼+γ-干扰素组（γ-干扰素100 U/ml预处理24 h后，加入阿帕替尼8μmol/L）和阿帕替尼+γ-干扰素+铁抑素1（ferrostatin-1,Fer-1）组（γ-干扰素100 U/ml预处理24 h后，加入阿帕替尼8μmol/L和Fer-1 1μmol/L）,72 h后进行后续实验。Transwell实验检测细胞迁移和侵袭；试剂盒检测细胞乳酸脱氢酶（lactatedehydrogenase, LDH）漏出量及丙二醛和还原型谷胱甘肽（glutathione, GSH）含量；Ferro Orange探针检测细胞内Fe²⁺含量；透射电子显微镜检测线粒体结构；JC-1染色检测线粒体膜电位；逆转录聚合酶链反应（reverse transcription polymerase chain reaction, RT-PCR）检测细胞中线粒体DNA（mtDNA）和细胞质dsDNA（cDNA）水平。构建胃癌荷瘤小鼠模型，随机分为对照组、γ-干扰素组[腹腔注射γ-干扰素1.5 U/（kg·d）]、阿帕替尼组[阿帕替尼200 mg/（kg·d）灌胃]和阿帕替尼+γ-干扰素组[同时腹腔注射γ-干扰素1.5 U/（kg·d）和阿帕替尼200 mg/（kg·d）灌胃],均干预20 d,每组5只小鼠。免疫荧光染色检测癌细胞MitoSOX-Red表达及癌组织干扰素基因刺激因子（stimulator of interferon genes, STING）和高尔基体基质蛋白130（golgi matrix protein 130,GM130）共定位；Western blot检测癌细胞人前列腺素内过氧化物合酶2（prostaglandin-endoperoxide synthase 2,PTGS2）、溶质转运蛋白家族7成员11（solute carrier family 7 member 11,SLC7A11）和谷胱甘肽过氧化物酶4（glutathione peroxidase4,GPX4）及癌组织p-STING、STING、磷酸化-TANK结合激酶1（phosphorylated-TANK-binding kinase 1,p-TBK1）和磷酸化-干扰素调节因子3（phosphorylated-interferon regulatory factor 3,p-IRF3）蛋白表达；流式细胞术检测细胞中活性氧和脂质活性氧水平及免疫细胞占比。结果 与阿帕替尼组比较，γ-干扰素+阿帕替尼组细胞存活率降低，在阿帕替尼浓度为8μmol/L时联合γ-干扰素处理达到有效抑制浓度。与对照组MGC803细胞比较，阿帕替尼组LDH漏出量升高，侵袭细胞数和迁移细胞数降低，Fe²⁺、活性氧、脂质活性氧和丙二醛水平及PTGS2表达升高，GSH含量及SLC7A11和GPX4表达降低，线粒体断裂和嵴缺失，J-aggregate/Monomers荧光强度比值和mtDNA水平降低，MitoSOX-Red相对荧光强度和cDNA水平升高，mtDNA水平降低（P均<0.05）。与阿帕替尼组比较，阿帕替尼+γ-干扰素组MGC803细胞LDH漏出量升高，侵袭细胞数和迁移细胞数降低，Fe²⁺、活性氧、脂质活性氧和丙二醛水平及PTGS2表达升高，GSH含量及SLC7A11和GPX4表达降低，线粒体断裂和嵴缺失，J-aggregate/Monomers荧光强度比值和mtDNA水平亦降低，MitoSOX-Red相对荧光强度和cDNA水平亦升高（P均<0.05）。构建胃癌荷瘤小鼠模型，与对照组比较，阿帕替尼组小鼠肿瘤体积和质量均降低，肿瘤组织STING和顺式GM130共定位荧光强度及p-STING、p-TBK1和p-IRF3表达升高，DC、CD8⁺T细胞和中枢记忆T细胞占比增多，耗竭T细胞占比减少（P均<0.05）;与阿帕替尼组比较，阿帕替尼+γ-干扰素组小鼠肿瘤体积和质量均降低，肿瘤组织STING和顺式GM130共定位荧光强度及p-STING、p-TBK1和p-IRF3表达升高，DC、CD8⁺T细胞和中枢记忆T细胞占比增多，耗竭T细胞占比减少（P均<0.05）。结论 γ-干扰素联合阿帕替尼治疗可能促进由铁死亡相关线粒体应激诱导的mtDNA释放，启动环磷酸鸟苷-腺苷酸合成酶（cyclic guanosine monophosphate adenylate synthetase, cGAS）-STING通路，改善肿瘤微环境并促进抗肿瘤免疫反应，增强胃癌细胞对阿帕替尼的敏感性。更多还原

【Abstract】 Objective To explore the therapeutic effect of Apatinib combined with interferon-γ on gastric cancer and its possible mechanism. Methods Taking the human gastric cancer cell line MGC803 as the research objects, the objects were randomly divided into the control group [only given the same amount of 1% dimethyl sulfoxide（DMSO）], Apatinib group（intervened with Apatinib at 8 μmol/L）, interferon-γ group（intervened with interferon-γ at 100 U/ml）, Apatinib + interferon-γ group（after pretreatment with interferon-γ at 100 U/ml for 24 h, 8 μmol/L Apatinib was added） and Apatinib + interferon-γ + ferrostatin-1（Fer-1） group（after pretreatment with interferon-γ 100 U/ml for 24 h, Apatinib 8 μmol/L and Fer-1 1 μmol/L was added）, subsequent experiments were performed 72 h later. Cell migration and invasion were detected by the Transwell assay, the amount of lactatedehydrogenase（LDH） released from cells, malondialdehyde and reduced glutathione（GSH） contents were detected by kits, intracellular Fe²⁺ content was detected by Ferro Orange probe; mitochondrial structure was detected by transmission electron microscopy; mitochondrial membrane potential was detected by JC-1 staining; the levels of mitochondrial DNA（mtDNA） and cytoplasmic dsDNA（cDNA） in cells were detected by reverse transcription polymerase chain reaction（RT-PCR）. A tumor-bearing mouse model of gastric cancer was constructed, and they were randomly divided into control group, interferon-γ group [interferon-γ 1.5 U/（kg·d） intraperitoneally injected], Apatinib group [Apatinib 200 mg/（kg·d） by gavage] and Apatinib + interferon-γ group [interferon-γ 1.5 U/（kg·d） intraperitoneally injected and Apatinib 200 mg/（kg·d） by gavage simultaneously], all groups were treated for 20 d, with 5 mice in each. Immunofluorescence staining was used to detect the expression of MitoSOX-Red in cancer cells and the stimulator of interferon genes（STING） and golgi matrix protein 130（GM130） co-localization in cancer tissues; protein expressions of human prostaglandin-endoperoxide synthase 2（PTGS2）, solute carrier family 7 member 11（SLC7A11） and glutathione peroxidase4（GPX4） and cancer tissues p-STING, STING, phosphorylated-TANK-binding kinase 1（p-TBK1） and phosphorylated-interferon regulatory factor 3（p-IRF3） were detected by Western blot; the levels of reactive oxygen species and lipid reactive oxygen species in cells and the proportion of immune cells were detected by flow cytometry. Results Compared with Apatinib group, the cell survival rate of Apatinib + interferon-γ group reduced, when the concentration of Apatinib was 8 μmol/L, the combined treatment with interferon-γ achieved an effective inhibitory concentration. Compared with the MGC803 cells in the control group, the leakage of LDH in the Apatinib group increased, the number of invasive cells and migratory cells decreased, the levels of Fe²⁺, reactive oxygen species, lipid reactive oxygen species and malondialdehyde and the expression of PTGS2 increased, the content of GSH and the expressions of SLC7A11 and GPX4 decreased, and mitochondrial breaks and crists were absent, the fluorescence intensity ratio of J-aggregate/Monomers and mtDNA level decreased, the relative fluorescence intensity and cDNA level of MitoSOX-Red increased, and the mtDNA level decreased（all P <0.05）. Compared with the Apatinib group, the leakage of LDH in MGC803 cells in the Apatinib +interferon-γ group increased, the number of invasive cells and migratory cells decreased, the levels of Fe²⁺, reactive oxygen species, lipid reactive oxygen species and malondialdehyde and the expression of PTGS2 increased, the content of GSH and the expressions of SLC7A11 and GPX4 decreased, and mitochondrial breaks and crists were absent, the fluorescence intensity ratio of J-aggregate/Monomers and the level of mtDNA also decreased, and the relative fluorescence intensity of MitoSOX-Red and cDNA level also increased（all P <0.05）. A tumor-bearing mouse model of gastric cancer was constructed, compared with the control group, the tumor volume and weight of mice in the Apatinib group decreased, the co-localization fluorescence intensity of STING and cis-GM130 in tumor tissues and the expressions of p-STING, p-TBK1 and p-IRF3 increased, the proportions of DC, CD8⁺T cells and central memory T cells increased, the proportion of exhausted T cells decreased（all P <0.05）; compared with the Apatinib group, the tumor volume and weight of mice in the Apatinib + interferon-γ group both decreased, the co-localization fluorescence intensity of STING and cis-GM130 in tumor tissues and the expressions of p-STING, p-TBK1 and p-IRF3 increased, the proportions of DC, CD8⁺T cells and central memory T cells increased, the proportion of exhausted T cells decreased（all P <0.05）. Conclusion Interferon-γ combined with Apatinib treatment may promote mtDNA release induced by ferroptosis-related mitochondrial stress and initiate the cyclic guanosine monophosphate adenylate synthetase（cGAS）-STING pathway, improve the tumor microenvironment, promote anti-tumor immune responses and enhance the sensitivity of gastric cancer cells to Apatinib.更多还原