基于CCL2-CCR2信号轴探索松萝酸对胃癌细胞恶性行为的影响  

Effect of usnic acid on malignant behavior of gastric cancer cells investigated based on CCL2-CCR2 signal axis

作　　者：滕晓丽[1] 时昭红[1] 孟庆彬[2] 周晓黎[1] 廖艳[1] 万莹[1] 杨健[1] TENG Xiaoli;SHI Zhaohong;MENG Qingbin;ZHOU Xiaoli;LIAO Yan;WAN Ying;YANG Jian(Depart-ment of Gastroenterology,the First Hospital of Wuhan,Wuhan 430022,China;Department of Gastrointestinal Surgery,the First Hospital of Wuhan,Wuhan 430022,China)

机构地区：[1]武汉市第一医院消化内科,武汉430022 [2]武汉市第一医院胃肠外科,武汉430022

出　　处：《中国免疫学杂志》2024年第8期1665-1670,共6页Chinese Journal of Immunology

基　　金：湖北省科技计划项目(2016CFB595)。

摘　　要：目的:探讨松萝酸(UA)调节CC趋化因子配体2-CCL2受体(CCR2)信号轴对胃癌细胞恶性行为的影响。方法:以生长良好的人胃癌细胞系SGC-7901细胞为研究对象,通过不同浓度的UA处理,设为UA低浓度(UA-L)组(62.5μmol/L UA)、UA中浓度(UA-M)组(125μmol/L UA)、UA高浓度(UA-H)组(250μmol/L UA);同时对细胞转染CCL2过表达载体(pc DNA3.1 CCL2)、空载体(pc DNA3.1)、沉默CCL2(si CCL2)及阴性对照(si control),并采用250μmol/L UA处理SGC-7901细胞,标记为UA-H+pc DNA3.1 CCL2组、UA-H+pc DNA3.1组、UA-H+si CCL2组、UA-H+si control组,另取未处理的SGC-7901细胞作为对照组。流式细胞术、MTT及qRT-PCR检测细胞凋亡、增殖及CCL2、CCR2 mRNA表达水平;Western blot检测PD-L1、凋亡蛋白(Bax)、增殖蛋白(CyclinD1、CCL2、CCR2)、免疫逃逸相关蛋白(B7H1)表达水平;与体外分离培养的CD8+T细胞共培养后,ELISA检测上清液中IL-4、IFN-γ、IL-10水平。将各组细胞与活化的外周血单个核细胞(PBMC)1∶1共培养72 h,比较各组胃癌细胞对T细胞介导的杀伤敏感性。结果:与对照组相比,UA-L组、UA-M组、UA-H组细胞增殖率、IL-10水平、CyclinD1、PD-L1、CCL2、CCR2、B7H1蛋白及mRNA表达、与活化的PBMC 1∶1共培养72 h后的细胞计数显著降低,细胞凋亡率、IL-4、IFN-γ水平、Bax蛋白表达显著升高(P<0.05);与UA-H+pc DNA3.1组相比,UA-H+pc DNA3.1 CCL2组细胞增殖率、IL-10水平、CyclinD1、PDL1、CCL2、CCR2蛋白及mRNA表达、与活化的PBMC 1∶1共培养72 h后的细胞计数显著增加,细胞凋亡率、IL-4、IFN-γ水平、Bax蛋白表达显著降低(P<0.05);与UA-H+si control组相比,UA-H+si CCL2组细胞增殖率、IL-10水平、CyclinD1、PD-L1、CCL2、CCR2蛋白及mRNA表达、与活化的PBMC 1∶1共培养72 h后的细胞计数显著降低,细胞凋亡率、IL-4、IFN-γ水平、Bax蛋白表达显著升高(P<0.05)。结论:UA可抑制胃癌细胞增殖、免疫逃逸,诱导其凋亡,可能与抑制CCL2-CCR2信号轴有关�Objective:To investigate impacts of usnic acid(UA)on malignant behavior of gastric cancer cells by regulating the chemokine(C-C motif)ligand 2(CCL2)-CCL2 receptor(CCR2)signal axis.Methods:SGC-7901 cells,a well growing human gastric cancer cell line,were treated with different concentrations of UA,which were grouped into low concentration(UA-L)group(62.5μmol/L UA),medium concentration(UA-M)group(125μmol/L UA)and high concentration(UA-H)group(250μmol/L UA);meantime,the cells were transfected with CCL2 overexpression vector(pc DNA3.1 CCL2),empty vector(pc DNA3.1),silenced CCL2(si CCL2)and negative control(si control),and SGC-7901 cells were treated with 250μmol/L UA,labeled as UA-H+pc DNA3.1 CCL2 group,UA-H+pc DNA3.1 group,UA-H+si control group and UA-H+si CCL2 group,another untreated SGC-7901 cells were taken as the control group.Flow cytometry,MTT and qRT-PCR were applied to detect cell apoptosis,proliferation,and expression levels of CCL2 and CCR2 mRNA;Western blot was applied to detect expression levels of PD-L1,apoptotic protein(Bax),proliferative protein(CyclinD1,CCL2,CCR2)and immune escape related protein(B7H1);after co-culturing with CD8+T cells isolated and cultured in vitro,ELISA was applied to detect levels of IL-4,IFN-γand IL-10 in the supernatant.Gastric cancer cells in each group were co-cultured with activated peripheral blood mononuclear cell(PBMC)1∶1 for 72 hours,and the sensitivity of gastric cancer cells in each group to T-cell-mediated killing was compared.Results:Compared with control group,cell proliferation rate,IL-10 level,CyclinD1,PD-L1,CCL2,CCR2 and B7H1 protein and mRNA expressions,cell counts after co-culturing with activated PBMC 1∶1 for 72 hours in UA-L group,UA-M group and UA-H group were obviously reduced,while apoptosis rate,IL-4 and IFN-γlevels,Bax protein expression were obviously increased(P<0.05);compared with UA-H+pc DNA3.1 group,cell proliferation rate,IL-10 level,CyclinD1,PD-L1,CCL2,CCR2 protein and mRNA expressions,cell counts after co-culturing with activated PBMC

关 键 词：松萝酸 CCL2-CCR2信号轴 胃癌细胞 增殖 凋亡 免疫逃逸 

分 类 号：R735.2[医药卫生—肿瘤]