华支睾吸虫感染对小鼠肝纤维化和免疫调节功能的影响  

Effect of Clonorchis sinensis infection on hepatic fibrosis and immune regulation in mice

作　　者：赵磊 李佳 莫刚 李醇 黄国洋 彭小红 ZHAO Lei;LI Jia;MO Gang;LI Chun;HUANG Guoyang;PENG Xiaohong(Guangxi University Key Laboratory of Pathogenic Biology,Guilin 541004,China;Guangxi Key Laboratory of Molecular Medicine in Liver Injury and Repair,the Affiliated Hospital of Guilin Medical University,Guilin 541004,China)

机构地区：[1]广西教育厅病原生物学重点实验室,桂林541004 [2]桂林医学院附属医院广西肝脏损伤与修复分子医学重点实验室,广西桂林541004

出　　处：《中国寄生虫学与寄生虫病杂志》2023年第6期760-765,共6页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(82060376);广西基础医学协同创新研究生联合培养基地建设项目(桂学位[2020]7号)。

摘　　要：为进一步了解小鼠感染华支睾吸虫过程中的肝纤维化情况和免疫调节变化,将BALB/c小鼠随机分成感染组和对照组,每组30只,感染组小鼠胃饲200μl含150个华支睾吸虫囊蚴的生理盐水,对照组小鼠灌胃同体积生理盐水,于感染后第1、2、4、8和16周随机各取6只,称体质量后取肝、脾和血清。取小鼠肝组织,石蜡切片后进行苏木素-伊红(HE)染色和Masson染色,观察胶原纤维沉积情况。小鼠肝组织总RNA逆转录后qRT-PCR检测肌成纤维细胞标志α-肌动蛋白(ACTA2)基因的相对转录水平,蛋白质免疫印迹(Western blotting)检测肝组织ACTA2蛋白的相对表达量。取小鼠脾组织,称重并计算每10 g体质量的脾质量(脾脏指数,mg/10 g)。流式细胞术分析小鼠脾CD4+T细胞、辅助性T1(Th1)细胞、Th2细胞、Th17细胞和调节性T(Treg)细胞的动态变化。取小鼠血清,使用流式细胞术检测肿瘤坏死因子(TNF)、白细胞介素6(IL-6)、γ干扰素(IFN-γ)、IL-2、IL-4、IL-10和IL-17A等细胞因子的分泌水平。使用GraphPad Prism 9.0软件进行数据分析。组间比较采用双向方差分析,两两比较采用t检验。结果显示,小鼠感染华支睾吸虫囊蚴后体质量降低、脾指数升高,感染后各时期肝组织皆可见炎症病灶。HE染色结果显示,小鼠感染华支睾吸虫囊蚴后第1周,肝胆管周围可见华支睾吸虫幼虫,幼虫周围出现炎症细胞浸润。Masson染色结果显示,小鼠感染华支睾吸虫囊蚴后各时期,胶原纤维沉积相对面积均高于对照组(F=20.190,P<0.05)。qRT-PCR结果显示,感染后第2、4和16周,感染组小鼠肝组织ACTA2基因相对转录水平均高于对照组(t=2.042、2.475、1.634,均P<0.01);第8周低于对照组(t=2.758,P<0.05)。Western blotting检测结果显示,感染后各时期,感染组小鼠肝脏ACTA2蛋白相对表达量均高于对照组(F=3.225,P<0.01)。流式细胞术结果显示,感染组小鼠脾CD4+T细胞占比在感染后第2周降低至(13.In order to further understand the liver fibrosis and immune regulatory changes in mice infected with Clonorchis sinensis,BALB/c mice were randomly divided into 2 groups with 30 mice in each group.The infected group were administered 200μl normal saline with 150 C.sinensis metacercariae through oral gavage and the control group received 200μl normal saline.Six mice were randomly selected for dissection at 1,2,4,8 and 16 weeks.The paraffin sections of the liver tissue were stained with hematoxylin-eosin(HE)and Masson to observe the progression of fibrosis.The relative transcription level and actin alpha 2(ACTA2)expression in the liver were detected by qRT-PCR and western blotting separately.The spleen tissue were weighed for calculating the spleen index(spleen weight per 10 g weight,mg/10 g).The flow cytometry was performed to detect the dynamical changes of CD4+T cells,helper T1(Th1)cells,Th2 cells,Th17 cells and regulatory T(Treg)cells in spleen.The secretion levels of tumor necrosis factor(TNF),interleukin-6(IL-6),interferon-γ(IFN-γ),IL-2,IL-4,IL-10,and IL-17A were quantified by cytometric bead array.The data was analyzed by GraphPad Prism 9.0.Two-way ANOVA was used for groups comparison and t test was used for pairwise comparison.The results showed that the mice weight decreased and spleen index increased after infected with C.sinensis and the inflammatory lesions were visible in liver tissue.HE staining showed that C.sinensis larvae and inflammatory cells could be found around the hepatobiliary duct at week 1 after infection.Masson staining revealed that the relative area of collagen fiber deposition was higher than the control group after infection(F=20.190,P<0.05).The qRT-PCR results showed that the hepatic ACTA2 relative transcription level of the infected group were higher than the control group at week 2,4 and 16 after infection(t=2.042,2.475,1.634;all P<0.01)but lower than the control group at week 8(t=2.758,P<0.05).Western blotting results showed that the hepatic ACTA2 protein relative expression l

关 键 词：华支睾吸虫 肝纤维化 脾CD4+ T细胞 动态变化 

分 类 号：R532.23[医药卫生—内科学]