抗程序性死亡因子-1与OK432联合治疗在小鼠肝细胞肝癌中的抗肿瘤作用  

Anti-tumor effect of anti-programmed death 1 combined with OK432 in mouse hepatocellular carcinoma model

作　　者：胡丁文 解刚强[1] 袁蒙蒙 胡舜冰 应瑞琼 张永辉 Hu Dingwen;Xie Gangqiang;Yuan Mengmeng;Hu Shunbing;Ying Ruiqiong;Zhang Yonghui(Department of General Surgery,the First Affiliated Hospital of Henan Science and Technology University,Luoyang 471000,China;Department of Gynecology,Henan Provincial People's Hospital,Zhengzhou 450003,China;Henan Science and Technology University,Luoyang 471000,China;Henan Key Laboratory of Stem Cell Differentiation and Modification,Henan University,Zhengzhou 450003,China;Henan International Joint Laboratory of Stem Cell Clinical Translation,Henan Provincial People's Hospital,Zhengzhou 450003,China)

机构地区：[1]河南科技大学第一附属医院肝胆胰外科,洛阳471000 [2]河南省人民医院妇科,郑州450003 [3]河南科技大学医学院,洛阳471000 [4]河南大学河南省干细胞分化与调控重点实验室,郑州450003 [5]河南省人民医院河南省干细胞临床转化国际联合实验室,郑州450003

出　　处：《中华实验外科杂志》2024年第7期1475-1478,共4页Chinese Journal of Experimental Surgery

基　　金：河南省科技攻关(212102310462、222102310119、232102310039);河南省医学科技攻关计划联合共建项目(LHGJ20220046)。

摘　　要：目的观察抗程序性死亡因子-1(PD-1)与OK432联合治疗在小鼠肝细胞肝癌中的抗肿瘤作用, 并探索其抗肿瘤机制。方法 8~10周龄C57BL/6雄性小鼠分别构建H22小鼠肝癌皮下移植瘤模型和腹水瘤模型, 监测小鼠肿瘤大小和生存, 流式细胞术、ELISPOT和酶联免疫吸附试验(ELISA)检测腹水中干扰素γ(IFN-γ)、CD8^(+)T、NK和程序性死亡因子配体1(PD-L1)^(+)细胞, 评价肿瘤微环境中免疫活化与淋巴细胞浸润;使用NK1.1和CD8a抗体清除荷瘤小鼠NK和CD8^(+)T细胞, 监测小鼠生存, 评价CD8^(+)T和NK细胞在治疗中的作用。生存统计使用Log-rank检验, 组间计量数据统计采用One-way ANOVA方法。结果 OK432+抗PD-1组与CON组、OK432组和抗PD-1组比较肿瘤生长显著抑制(F=19.000, P<0.01);OK432+抗PD-1组小鼠中位生存期35 d显著长于CON组的15 d、OK432组的21 d和抗PD-1组的21 d(χ^(2)=21.680, P<0.01)。OK432+抗PD-1组IFN-γ斑点数量[(319.7±48.4)个]远多于CON组[(8.8±8.9)个]、OK432组[(173.2±38.6)个]和抗PD-1组[(172.2±18.9)个, F=82.330, P<0.01];OK432+抗PD-1组腹水中CD8^(+)T和NK细胞比例分别为(14.57±2.52)%和(6.52±1.43)%远高于CON组[(1.35±0.46)%和(0.85±0.54)%]、OK432组[(8.62±2.87)%和(4.04±0.7)%]、抗PD-1组[(5.57±2.23)%和(2.86±1.37)%, CD8^(+)T:F=31.240, P<0.01和NK:F=23.870, P<0.01];OK432+抗PD-1组腹水中IFN-γ浓度为(1 434±407) pg/ml, 显著高于CON组[(306±151) pg/ml]、OK432组[(1 180±565) pg/ml]和抗PD-1组[(649±130) pg/ml, F=9.837, P<0.01];OK432+抗PD-1组腹水中PD-L1^(+)细胞的比例为(22.68±5.43)%, 显著高于CON组[(2.09±0.64)%]、OK432组[(15.16±3.17)%]和抗PD-1组[(9.03±0.97)%, F=37.670, P<0.01]。NK或CD8^(+)T细胞清除后OK432+抗PD-1+抗NK1.1组和OK432+抗PD-1+抗CD8a组小鼠生存期分别为32 d和21 d、CON组18 d、OK432+抗PD-1组34 d, OK432+抗PD-1+抗NK1.1组与OK432+抗PD-1组比较差异无统计学意义(χ^(2)=0.004, P>0.05), OK432+抗PD-1+抗CD8a组与OK432+抗PD-1组比较差异有统�Objective To observe the anti-tumor effect of anti-programmed death 1(PD-1)com-bined with OK432 in mice with hepatocellular carcinoma,and to explore its anti-tumor mechanism.Methods Subcutaneous and ascites tumor models of H22 hepatocellular carcinoma were established in 8-10-week-old C57BL/6 male mice,respectively.Tumor volume and survival were monitored.IFN-,CD8^(+)T,NK,and programmed death ligand 1(PD-LI)+cells in ascites were detected by flow cytometry,ELISPOT,and enzyme linked immunosorbent assay(ELISA)to evaluate immune activation and lymphocyte infiltration in the tumor microenvironment.NK1.1 and CD8a antibodies were used to deplete NK and CD8^(+)T cells from tumor-bearing mice.Mouse survival was monitored to evaluate the role of CD8^(+)T and NK cells in treatment.Survival analysis was performed using the Log-rank test,and measurement data be-tween groups were analyzed using one-way ANOVA.Results The tumor volume of the OK432+anti-PD-1 group was significantly inhibited as compared with the CON group,OK432 group,and anti-PD-1 group(F=19.000,P<0.01).The median survival time of the OK432+anti-PD-1 group(35 days)was signifi-cantly longer than that of the CON group(15 days),OK432 group(21 days),and anti-PD-1 group(21 days)(X2=21.680,P<0.01).The number of IFN-spots in OK432+anti-PD-1 group(319.7±48.4)was much more than that in CON group(8.8±8.9),OK432 group(173.2±38.6)and anti-PD-1 group(172.2±18.9,F=82.330,P<0.01).Proportion of CD8^(+)T and NK cells in ascites of OK432+anti-PD-1 group[(14.57±2.52)%]and[(6.52±1.43)%],respectively was significantly higher than that in CON group[(1.35±0.46)%and(0.85±0.54)%],OK432 group[(8.62±2.87)%and(4.04±0.70)%]and anti-PD-1 group[(5.57±2.23)%and(2.86±1.37)%](CD8^(+)T:F=31.240,P<0.01 and NK:F=23.870,P<0.01).The concentration of IFN-in ascites of OK432+anti-PD-1 group[(1434±407)pg/ml]was significantly higher than that of C0N group[(306±151)pg/ml]and OK432 group[(1180±565)pg/ml]and anti-PD-1 group[(649±130)pg/ml,F=9.837,P<0.01].The proportion of PD-L1^(+)cells in a

关 键 词：肝细胞癌 OK432 抗程序性死亡因子-1 

分 类 号：R735.7[医药卫生—肿瘤]