VEGFA/VEGFR2在小鼠肝多房棘球蚴组织血管生成中的表达及作用  

Expression and angiogenic effect of VEGFA/VEGFR2 in mice hepatic metacestode tissue of Echinococcus multilocularis

作　　者：姜慧娇 桂显伟 郭黎姣 杨雄峰 王小义[1] 陈雪玲 吴向未[1] JIANG Hui-jiao;GUI Xian-wei;GUO Li-jiao;YANG Xiong-feng;WANG Xiao-yi;CHEN Xue-ling;WU Xiang-wei(The First Affiliated Hospital,College of Medicine,Shihezi University,Shihezi 832008,China;Department of Immunology,College of Medicine,Shihezi University,Shihezi 832008,China)

机构地区：[1]石河子大学医学院第一附属医院,石河子832000 [2]石河子大学医学院免疫学教研室,石河子832000

出　　处：《中国寄生虫学与寄生虫病杂志》2020年第6期673-681,共9页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(No.81760570.No.81760371);兵团科技发展专项资金(No.2018CB017,No.2019AB031)。

摘　　要：目的检测多房棘球蚴感染后小鼠肝脏和肝细胞血管内皮细胞生长因子A(VEGFA)、血管内皮细胞生长因子受体2(VEGFR2)的mRNA水平及蛋白水平,探讨VEGFA/VEGFR2在多房棘球蚴组织血管生成中的作用。方法将20只雌性C57BL/6小鼠随机分为实验组和对照组(10只/组),实验组小鼠采用肝被膜注射法建立多房棘球蚴感染模型,对照组注射等量生理盐水。于感染后第0、16、37、58、81、105天,收集各组小鼠全血,分离血清。第120天安乐死小鼠后,开腹观察多房棘球蚴生长情况,取小鼠多房棘球蚴组织、多房棘球蚴周围肝组织、对照组小鼠正常肝组织。HE染色观察多房棘球蚴组织血管生成情况,免疫组化法检测各组织中VEGFA、VEGFR2表达及分布。取肝细胞和原头节进行体外培养,36个培养皿均分为共培养组(肝细胞+原头节)、肝细胞组、原头节组,分别于培养后1、2、3 d后取各组上清、肝细胞和原头节。qRT-PCR、蛋白质免疫印迹(Western blotting)检测正常肝组织、多房棘球蚴组织、多房棘球蚴周围4 mm处肝组织、原头节和肝细胞中VEGFA、VEGFR2 mRNA水平和蛋白水平。ELISA检测外周血中VEGFA的含量、正常肝组织、多房棘球蚴组织、多房棘球蚴周围肝组织、共培养组上清、肝细胞组上清和原头节组上清中VEGFA蛋白水平。采用SPSS 20.0软件进行统计学分析。结果qRT-PCR检测结果显示,正常肝组织、多房棘球蚴周围肝组织、多房棘球蚴组织VEGFA mRNA相对转录水平分别为1.033±0.102、1.222±0.501、0.276±0.092,多房棘球蚴组织的转录水平低于正常肝组织与多房棘球蚴周围肝组织(P<0.05);VEGFR2 mRNA相对转录水平分别为1.042±0.071、1.836±0.062、0.226±0.077,多房棘球蚴周围肝组织的转录水平高于正常肝组织(P<0.01),多房棘球蚴组织的转录水平低于正常肝组织与多房棘球蚴周围肝组织(P<0.01)。Western blotting分析结果显示,多房棘球蚴组�Objective To detect the mRNA and protein levels of VEGFA and VEGFR2 in mouse liver tissue and liver cells of mouse infected with Echinococcus multilocularis(Em),in order to explore the roles of vascular endothelial growth factor A(VEGFA)and endothelial growth factor receptor 2(VEGFR2)in tissue angiogenesis.Methods Twenty female C57BL/6 mice were randomly divided into two groups,the experimental group and the control group,10 animals each.Each mouse in the experimental group was directly injected Em metacestodes into the liver,while the control group mice were injected with PBS in the same way.Sera were separated from the whole blood of mice on days 0,16,37,58,81,and 105 after infection.The mice were euthanized on day 120 to observe the growing status of metacestode and collect.Metacestode tissue,liver tissue around metacestode,and normal liver tissue of control group mice for examining the angiogenesis in metacestode tissue by HE staining.The expression and distribution of VEGFA and VEGFR2 in these tissue samples were detected by immunohistochemistry.The liver cells and protoscoleces were sampled for in vitro incubation in three groups in 12 Petri dishes each group,comprising the group of liver cells-protoscoleces co-incubation,liver cells and protoscoleces.The culture supernatant,liver cells and protoscoleces in each group were collected on incubation day 1,2 and 3 to assay the levels of VEGFA,VEGFR2 mRNA and protein in normal liver tissue,metacestode tissue,liver tissues around metacestode within 4 mm,and liver cells after co-incubation with protoscoleces using qRT-PCR and Western blotting.ELISA was performed to detect the content of VEGFA in peripheral blood,as well as the protein levels of VEGFA in normal liver tissue,metacestode tissue,liver tissue around metacestode,as well as in the culture supernatant of the co-incubation group,the liver cell group and the protoscolex group.Results qRT-PCR showed that the relative transcription levels of VEGFA mRNA in healthy liver tissues,liver tissues around metacestode

关 键 词：多房棘球蚴 血管内皮细胞生长因子A 血管内皮细胞生长因子受体2 血管生成 

分 类 号：R532.32[医药卫生—内科学]