葫芦巴碱调节CD47/SIRPα信号通路对胃癌细胞增殖、凋亡和免疫逃逸的影响  

Impacts of trigonelline on the proliferation,apoptosis and immune escape of gastric cancer cells by regulating CD47/SIRPα signaling pathway

作　　者：冯彩团 陈卿奇 郭殿华 伍远超 FENG Caituan;CHEN Qingqi;GUO Dianhua;WU Yuanchao(Department of Gastroenterology,Hainan Western Central Hospital,Hainan Danzhou 571700,China)

机构地区：[1]海南西部中心医院消化内科,海南儋州571700

出　　处：《现代肿瘤医学》2024年第13期2346-2353,共8页Journal of Modern Oncology

基　　金：海南省卫生健康行业科研项目(编号:21A200294)。

摘　　要：目的:研究葫芦巴碱调节分化簇47(CD47)/信号调节蛋白α(SIRPα)信号通路对胃癌MGC803细胞增殖、凋亡和免疫逃逸的影响。方法:以四甲基偶氮唑盐(MTT)法检测0、20、40、80、160、320μmol/L的葫芦巴碱处理后的人胃癌细胞株MGC803存活率,筛选其最佳作用浓度。将体外培养的MGC803细胞随机分为对照组、葫芦巴碱组(160μmol/L的葫芦巴碱处理)、CD47敲低组[转染CD47小干扰RNA(siRNA)质粒]、空载组(转染CD47空载质粒)、葫芦巴碱+CD47过表达组(160μmol/L的葫芦巴碱干预处理+转染CD47过表达质粒),培养24 h后。采用实时荧光定量PCR和免疫印迹检测各组细胞CD47/SIRPα通路表达;EdU染色和MTT法检测各组细胞增殖;流式细胞术检测各组细胞凋亡;免疫荧光染色检测各组细胞凋亡[B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)]相关蛋白表达;以不同细胞比例共培养人外周血单个核细胞与各组MGC803细胞后以MTT法检测人外周血单个核细胞对各组MGC803细胞的杀伤率;裸鼠体内移植瘤实验验证葫芦巴碱对CD47/SIRPα通路的调控作用。结果:20、40、80、160、320μmol/L的葫芦巴碱均可降低MGC803细胞存活率(P<0.05),且其降低作用随着葫芦巴碱浓度升高而增强,选择接近半抑制浓度值(168.94μmol/L)的160μmol/L葫芦巴碱进行后续实验。与对照组比较,葫芦巴碱组、CD47敲低组细胞和移植瘤组织CD47、SIRPα mRNA及蛋白表达、EdU阳性率、存活率、Bcl-2相对荧光强度、移植瘤重量、移植瘤体积降低,凋亡率、Bax相对荧光强度、人外周血单个核细胞对MGC803细胞杀伤率升高(P<0.05)。与葫芦巴碱组比较,葫芦巴碱+CD47过表达组细胞和移植瘤组织CD47、SIRPα mRNA及蛋白表达、EdU阳性率、存活率、Bcl-2相对荧光强度、移植瘤重量、移植瘤体积升高,凋亡率、Bax相对荧光强度、人外周血单个核细胞对MGC803细胞杀伤率降低(P<0.05)。结论:葫芦巴碱可通过抑制CD4Objective:To investigate the impacts of trigonelline on the proliferation,apoptosis and immune escape of gastric cancer MGC803 cells by regulating the cluster of differentiation 47(CD47)/signal-regulatory protein α(SIRPα) signaling pathway.Methods:Methyl thiazolyl tetrazolium(MTT) assay was applied to detect the survival rate of human gastric cancer cell line MGC803 treated with 0,20,40,80,160,and 320 μmol/L of trigonelline to screen the optimal concentration.MGC803 cells cultured in vitro were randomly grouped into control group,trigonelline group(160 μmol/L trigonelline treatment),CD47 knockdown group [transfected CD47 small interfering RNA(siRNA) plasmid],no-load group(transfected with CD47 no-load plasmid),trigonelline+CD47 overexpression group(160 μmol/L trigonelline treatment+transfection of CD47 overexpression plasmid),and culture for 24 h.Real-time fluorescence quantitative PCR and immunoblotting were applied to detect the expression of CD47/SIRPα pathway in each group of cells.EdU staining and MTT method were applied to detect cell proliferation in each group.Cell apoptosis was detected by flow cytometry in each group.Immunofluorescence staining was applied to detect the expression of apoptosis related proteins [B cell lymphoblastoma-2(Bcl-2),Bcl-2 related X protein(Bax)] in each group.After co culturing human peripheral blood mononuclear cells and MGC803 cells in different cell ratios,the killing rate of human peripheral blood mononuclear cells against MGC803 cells in each group was detected by MTT method.The effect of trigonelline on CD47/SIRRPα pathway was verified by tumor transplantation in nude mice.Results:Trigonelline concentrations of 20,40,80,160 and 320 μmol/L all decreased the survival rate of MGC803 cells(P<0.05),and the decreasing effect was enhanced with the increase of trigonelline concentration,160 μmol/L trigonelline concentration close to the half-suppressed concentration value(168.94 μmol/L) was selected for follow-up experiments.Compared with control group,the expressions o

关 键 词：葫芦巴碱 分化簇47 信号调节蛋白α 胃癌MGC803细胞 增殖 凋亡 免疫逃逸 

分 类 号：R735.2[医药卫生—肿瘤]