多房棘球蚴感染巨噬细胞中CD47和SIRPα表达变化与抑制作用的研究  

Research on the expression changes and inhibitory effects of CD47 and SIRPαin macrophages infected with Echinococcus multilocularis

作　　者：魏盼龙 张耀刚 张涛[3] 杨紫晗 侯静 田美媛 黄登亮 马艳艳[4] WEI Panlong;ZHANG Yaogang;ZHANG Tao;YANG Zihan;HOU Jing;TIAN Meiyuan;HUANG Dengliang;MA Yanyan(Graduate School of Qinghai University,Xining 810000,Qinghai,China;Central Laboratory,Affiliated Hospital of Qinghai University,Xining 810000,Qinghai,China;Affiliated Hospital of Qinghai University,Xining 810000,Qinghai,China;Department of Scientific Research Management,Affiliated Hospital of Qinghai University,Xining 810000,Qinghai,China)

机构地区：[1]青海大学研究生院,青海西宁810000 [2]青海大学附属医院中心实验室,青海西宁810000 [3]青海大学附属医院,青海西宁810000 [4]青海大学附属医院科研管理处,青海西宁810000

出　　处：《中国寄生虫学与寄生虫病杂志》2025年第1期84-90,102,共8页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(81960129,82360128);青海省“昆仑英才·高端创新创业人才”和青海大学附属医院中青年项目(ASRF-2023-ZD-02)。

摘　　要：目的探究多房棘球蚴感染对巨噬细胞CD47与信号调节蛋白α(SIRPα)表达水平及巨噬细胞功能的影响,为探索多房棘球蚴病(AE)发病机制提供依据。方法收集30例AE患者的肝脏病理组织,选取病灶周围0.5 cm处的病灶近旁肝组织(CLT)和超过病灶2 cm以上的病灶远端肝组织(DLT),石蜡切片行免疫组化、冰冻切片行免疫荧光,观察分析CLT和DLT中CD47和SIRPα的表达水平。小鼠单核巨噬细胞白血病细胞(RAW264.7)与多房棘球蚴原头节共培养(细胞数∶原头节数=500∶1),分别于加入原头节前、共培养24和48 h后收集细胞,流式细胞术、细胞免疫荧光和实时荧光定量PCR(qPCR)检测巨噬细胞CD47和SIRPα的表达变化。RAW264.7细胞分为对照组、感染组、抑制剂组和抑制剂感染组(1×10^(5)个/组),感染组和抑制剂感染组加入原头节(200个/组)、抑制剂组和抑制剂感染组加入CD47/SIRPα结合抑制剂(NCGC00138783TFA,10µmol/L),共培养48 h后加入增强型绿色荧光蛋白(EGFP)标记的大肠埃希菌(1×10^(6)个/组),荧光观察后流式细胞术检测巨噬细胞吞噬能力变化,qPCR检测巨噬细胞极化标志物和细胞因子mRNA相对转录水平的变化。两组间比较采用独立样本t检验或配对t检验,多组间比较用单因素方差分析,多重比较使用Tukey’s HSD法。结果免疫组化结果显示,AE患者CLT的CD47、SIRPα阳性细胞分别占(61.99±3.61)%、(54.06±1.85)%,均高于DLT的(57.08±3.38)%、(40.77±1.49)%(t=9.434、58.840,均P<0.01);免疫荧光结果显示,CLT的CD47/SIRPα皮尔逊相关系数为0.66±0.02,高于DLT的0.45±0.01(t=7.624,P<0.01)。流式检测结果显示,共培养24和48 h后的巨噬细胞CD47中位荧光强度分别为8259.00±66.01和9445.00±41.58,均高于加入原头节前的5603.00±193.40(HSD=0.691、0.735,均P<0.01);共培养24和48 h后的巨噬细胞SIRPα中位荧光强度分别为3123.00±184.60和2931.00±54.08,均高于加入原头节前的2508.00±43.15(HSD=0.491�Objective To investigate the effects of Echinococcus multilocularis infection on the expression of CD47 and signal regulatory proteinα(SIRPα)in macrophages and the function of macrophages,and to provide evi-dence for exploring the pathogenesis of alveolar echinococcosis(AE).Methods Liver tissue samples were collected from 30 AE patients.Tissues were categorized into close liver tissue(CLT)located 0.5 cm from the lesion and distal liver tissue(DLT)located more than 2 cm from the lesion.Paraffin sections were used for immunohistochemistry and frozen sections were used for immunofluorescence to analyze the expression levels of CD47 and SIRPαin CLT and DLT.Mouse monocyte-macrophage leukemia cells(RAW264.7)were co-cultured with E.multilocularis protoscoleces at a ratio of 500∶1.Cells were collected before the addition of protoscoleces and after co-culture for 24 and 48 hours,respectively.The expression changes of CD47 and SIRPαin macrophages were detected by flow cytometry,cellular im-munofluorescence and quantitative real-time PCR(qPCR).RAW264.7 cells were divided into control group,infection group,inhibitor group and inhibitor-infection group(1×10^(5) cells per group).The infection group and inhibitor-infection group were supplemented with protoscoleces(200 per group),while the inhibitor group and inhibitor-infection group were treated with a CD47/SIRPαbinding inhibitor(NCGC00138783TFA,10µmol/L).Enhanced green fluorescent pro-tein(EGFP)-labeled Escherichia coli were added to each group(1×10^(6)per group)after co-culture for 48 hourse.Fol-lowing fluorescence observation,the phagocytic capacity of macrophages was assessed by flow cytometry.Additionally,the mRNA relative transcription levels of macrophage polarization markers and cytokines were detected by qPCR.Inde-pendent samples t-test or paired t-test was used for comparisons between two groups,one-way ANOVA was used for multiple groups,and Tukey’s HSD method was used for multiple comparisons.Results Immunohistochemical analy-sis revealed that in AE pat

关 键 词：多房棘球蚴 免疫检查点 吞噬作用 巨噬细胞极化 

分 类 号：R532.32[医药卫生—内科学]