细粒棘球蚴感染诱导巨噬细胞表达CD73和A2AR抑制炎症反应  

Echinococcus granulosus infection induces macrophages to express CD73 and A2AR to suppress inflammatory response

作　　者：逯君霞 许军英 赵彬 王芊文 李文华 耿玉庆 侯隽[1] 吴向未[1,2] 陈雪玲[1] LU Junxia;XU Junying;ZHAO Bin;WANG Qianwen;LI Wenhua;GENG Yuqing;HOU Jun;WU Xiangwei;CHEN Xueling(School of Medicine,Shihezi University,Shihezi 832000,Xinjiang,China;The First Affiliated Hospital,School of Medicine,Shihezi University,Shihezi 832008,Xinjiang,China)

机构地区：[1]石河子大学医学院,新疆石河子832000 [2]石河子大学医学院第一附属医院,新疆石河子832008

出　　处：《中国寄生虫学与寄生虫病杂志》2023年第5期559-566,572,共9页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(82060297);兵团指导性科技计划(2022ZD041);自治区研究生科研创新项目(XJ2022G111);兵团财政科技计划(2021BB006)。

摘　　要：目的探究CD73/腺苷/腺苷A2A受体(A2AR)通路在细粒棘球蚴抑制巨噬细胞炎症反应中的作用及机制。方法取健康C57BL/6小鼠腹腔注射无菌淀粉肉汤(0.5 ml/只),3 d后抽取腹腔液,分离巨噬细胞,以1×10^(6)/ml接种于6孔板中,待细胞贴壁后,加入细粒棘球蚴囊液(终浓度0.8 mg/ml)培养0、6、12、18和24 h后,qRT⁃PCR检测CD73、A2AR、肿瘤坏死因子α(TNF⁃α)和精氨酸酶1(Arg⁃1)的相对转录水平,流式细胞术检测CD73的表达量,蛋白质免疫印迹(Western blotting)检测A2AR、细胞外信号调节激酶1/2(ERK1/2)和磷酸化的ERK1/2(p⁃ERK1/2)的表达量。取分离的巨噬细胞接种于6孔板(1×10^(6)个细胞/孔),囊液组、给药组和对照组(每组3孔)分别加入囊液(终浓度0.8 mg/ml)、囊液(终浓度0.8 mg/ml)和药物(腺苷受体抑制剂SCH58261,终浓度50μmol/L),以及等量培养基,培养24 h后,采用qRT⁃PCR检测TNF⁃α、Arg⁃1的相对转录水平,Western blotting检测ERK1/2、p⁃ERK1/2的表达量。取6~8周龄健康C57BL/6雌鼠,感染组小鼠于肝被膜下注射5000个细粒棘球蚴原头节(20μl/只),健康组小鼠不作处理。1个月后,两组各取6只小鼠,取肝脏,石蜡包埋后制备切片,HE染色观察肝组织病变情况,免疫组织化学染色检测A2AR的表达情况,流式细胞术检测包囊近端和远端肝组织的CD73表达情况。取感染小鼠,给药组(8只)和溶剂组(8只)小鼠分别腹腔注射腺苷受体抑制剂(SCH58261)1 mg/(kg•d)和等量的PBS,健康组小鼠(8只)不作处理,22 d后,称量小鼠的体质量,以及肝脏、脾脏、肾脏和心脏的质量,计算各脏器与体质量的比值;取小鼠肝组织,石蜡包埋后制备切片,HE染色观察包囊周围炎性细胞浸润情况。两组间数据比较采用独立样本t检验,多组间数据比较采用单因素方差分析。结果巨噬细胞经细粒棘球蚴囊液处理后,CD73 mRNA相对转录水平,18 h组(1.66±0.17)和24 h组(2.01±0.15)均高于0 h组(1.00±0.09)(t=3Objective To investigate the role and mechanisms of CD73/adenosine/adenosine A2A receptor(A2AR)pathway in suppression of macrophage inflammatory response of Echinococcus granulosus.Methods Healthy C57BL/6 mice were intraperitoneally injected with aseptic starch broth(0.5 ml per mouse).Ascitic fluid was extracted from the mice on 3 d post⁃injection to separate macrophages,which were inoculated into a 6⁃well plate at a dose of 1×10^(6)/ml.After the cells were attached to the plate wall,E.granulosus cyst fluid was added at a final concentration of 0.8 mg/ml,and then cultured for 0 h,6 h,12 h,18 h and 24 h to examine the relative transcriptional levels of CD73,A2AR,tumor necrosis factor⁃α(TNF⁃α)and arginase 1(Arg⁃1)by qRT⁃PCR.The expression of CD73 was de⁃tected by flow cytometry,and the expression of extracellular signal⁃regulated kinase 1(ERK1/2)and phosphorylated ERK1/2(p⁃ERK1/2)was detected by Western blotting.The separated macrophages were inoculated onto a 6⁃well plate with 1×10^(6) cells per well,and assigned to three groups:cyst fluid group,drug administration group and control group.Each group had 3 wells,to which E.granulosus cyst fluid(final concentration 0.8 mg/ml),E.granulosus cyst fluid(final concentration 0.8 mg/ml)and drugs(adenosine receptor inhibitor SCH58261,final concentration 50μmol/L)and the same amount of medium was added,respectively.After 24 hours of culture,the relative transcription levels of TNF⁃αand Arg⁃1 were detected by qRT⁃PCR.The expression of ERK1/2 and p⁃ERK1/2 was detected by Western blotting.Thirty⁃six healthy C57BL/6 female mice aged 6-8 weeks were selected.5000 protoscolex of E.granulosus(20μl per mouse)were injected under the liver capsule in the infection group,and the healthy group were not treated.One month later,the livers of 6 mice in each group were selected and embedded in paraffin to prepare slices.HE staining was used to observe liver tissue lesions,and the expression of A2AR was detected by immunohis⁃tochemical staining.The expre

关 键 词：细粒棘球蚴 巨噬细胞 CD73 腺苷A2A受体 

分 类 号：R383.3[医药卫生—医学寄生虫学]