体外小鼠巨噬细胞与细粒棘球蚴原头节的相互作用  

Interaction between mouse macrophages and protoscolex of Echinococcus granulosus in vitro

作　　者：黎广 姜慧娇 杜云峰 舒敏 罗雨盟 朱令懿 陈雪玲[3] 吴向未[1,2] LI Guang;JIANG Huijiao;DU Yunfeng;SHU Min;LUO Yumeng;ZHU Lingyi;CHEN Xueling;WU Xiangwei(Department of Hepatobiliary Surgery,The First Affiliated Hospital of Medical College,Shihezi University,Shihezi 832000,Xinjiang,China;School of Medicine,Shihezi University/NHC Key Laboratory of Prevention and Treatment of Central Asia High Incidence Diseases,Shihezi 832000,Xinjiang,China;Department of Immunization,School of Medicine,Shihezi University,Shihezi 832000,Xinjiang,China)

机构地区：[1]石河子大学医学院第一附属医院肝胆外科,新疆石河子832000 [2]石河子大学医学院,国家卫生健康委中亚高发病防治重点实验室,新疆石河子832000 [3]石河子大学医学院免疫教研室,新疆石河子832000

出　　处：《中国寄生虫学与寄生虫病杂志》2025年第1期52-60,共9页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：新疆生产建设兵团促进科技成果转化引导计划(2021BB006);中国医学科学院中央级公益性科研院所基本科研业务费专项资金(2020-PT330-003)。

摘　　要：目的探究小鼠免疫细胞对细粒棘球蚴原头节的杀伤作用,了解发挥功能的免疫细胞类型及其细胞因子的分泌变化。方法分离健康C57BL/6小鼠的腹腔巨噬细胞和脾细胞。收集羊细粒棘球蚴包囊中的原头节并分组(3000个/组)。巨噬细胞共培养组、脾细胞共培养组分别与6×10^(6)个巨噬细胞和脾细胞共培养,选择对原头节具有更强抑制作用的免疫细胞类型;共培养1~5组分别与1.2×10^(6)、2.4×10^(6)、4.8×10^(6)、7.2×10^(6)、9.6×10^(6)个细胞共培养,选择适宜的细胞数量,建立共培养体系。共培养体系中分别加入细粒棘球蚴囊液和肿瘤坏死因子α(TNF-α)抑制剂,观察原头节活性和共培养上清中TNF-α、白细胞介素6(IL-6)、IL-10、转化生长因子β(TGF-β)浓度的变化。伊红染色检测原头节活性,二氯荧光素二乙酸酯(DCFH-DA)法检测活性氧水平,JC-1法检测线粒体膜电位,蛋白质免疫印迹(Western blotting)检测促凋亡蛋白Bcl-2相关X蛋白(Bax)和天冬氨酸蛋白水解酶3(caspase-3)表达水平,ELISA检测培养上清中细胞因子的浓度。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析。结果共培养第6天,巨噬细胞共培养组和脾细胞共培养组的原头节活性分别为(25.07±0.40)%和(76.18±0.31)%,巨噬细胞共培养组各时期的原头节活性低于脾细胞共培养组(F=564.20,P<0.05);共培养第4天,巨噬细胞共培养组的原头节活性氧相对荧光强度为32.20±7.85,高于脾细胞共培养组的12.44±2.93(t=7.07,P<0.05);共培养第6天,巨噬细胞共培养组和脾细胞共培养组的caspase-3蛋白相对表达水平分别为1.28±0.02和1.16±0.02,Bax蛋白相对表达水平分别为1.29±0.01和0.46±0.01,巨噬细胞共培养组各时期的caspase-3、Bax蛋白相对表达水平均高于脾细胞共培养组(F=55.87、167.20,均P<0.05),巨噬细胞对原头节的抑制作用强于脾细胞。共培养第6天,共培养1~5组原头节的线�Objective To explore the cytotoxic effect of mouse immune cells on the Echinococcus granulosus protoscolex and understand the types of immune cells involved and the cytokines secretion changes.Methods Healthy C57BL/6 mice were used to extract splenocytes and peritoneal macrophages.The protoscoleces from sheep E.granulosus cysts were collected and grouped(3000 per group).To select the immune cell type exhibiting a stronger inhibitory effect on the protoscoleces,macrophage co-culture group and splenocyte co-culture group were co-cultured with 6×10^(6) macrophages or splenocytes,respectively.To choose an optimal cell count,co-culture group 1-5 were co-cultured with 1.2×10^(6),2.4×10^(6),4.8×10^(6),7.2×10^(6) and 9.6×10^(6) immune cells,respectively.The co-culture system was established.The E.granulosus cyst fluid and tumor necrosis factor-α(TNF-α)inhibitor were added into the co-culture system respectively,and the activity of protoscoleces and the changes in concentration of TNF-α,interleukin 6(IL-6),IL-10 and transforming growth factor-β(TGF-β)in the co-culture supernatant were observed.Eosin staining was used to detect the activity of protoscoleces,the dichlorodihydrofluorescein diacetate(DCFH-DA)method was used to measure the of reactive oxygen species levels,the JC-1 method was used to assess mitochondrial membrane potential,Western blotting was performed to detect the expression levels of the Bcl-2 associated X protein(Bax)and cysteinyl aspartate specific proteinase 3(caspase-3),and ELISA was used to measure the concentration of cytokines in the cul-ture supernatant.Independent samples t-test was used for comparisons between two groups and one-way ANOVA was used for multiple groups.Results On day 6 of co-culture,the protoscoleces activity in the macrophage co-culture group and the splenocyte co-culture group was(25.07±0.40)%and(76.18±0.31)%,respectively.The protoscoleces ac-tivity in the macrophage co-culture group was lower than that in the splenocyte co-culture group at all time points(F=564.20,P

关 键 词：细粒棘球蚴 巨噬细胞 肿瘤坏死因子Α 天冬氨酸蛋白水解酶3 共培养 

分 类 号：R383.33[医药卫生—医学寄生虫学]