基于非依赖性全扫描采集蛋白组学技术初步研究继发性多房棘球蚴病小鼠差异表达蛋白

Preliminary study on differentially expressed proteins in a mouse model of secondary alveolar echinococcosis based on data independent acquisition proteomics

目的通过高分辨质谱数据非依赖性全扫描采集(data independent acquisition,DIA)蛋白组学技术鉴定多房棘球蚴病小鼠不同肝脏组织的差异表达蛋白,寻找其中可能与多房棘球蚴病致病相关的关键蛋白。方法分离多房棘球蚴病青海田鼠体内原头节,用原头节感染雌性昆明小鼠(6~8周龄)进行造模。小鼠分为实验组与对照组,实验组注射约3000个原头节,对照组注射等量生理盐水。两组小鼠培养1年后取实验组和对照组小鼠肝脏组织进行病理学检查,另取实验组小鼠肝脏病灶组织(病灶组)、病旁组织(病旁组)及对照组小鼠正常肝脏组织(正常组)进行蛋白DIA定量分析并筛选差异表达蛋白,对差异蛋白进行生物信息学分析。结果在病灶组与正常组间检出1020种差异表达蛋白,其中671种上调蛋白、349种下调蛋白;在病旁组与正常组间检出495种差异表达蛋白,其中327种上调蛋白、168种下调蛋白。京都基因和基因组百科全书(KEGG通路)富集分析显示,这些差异表达蛋白参与过氧化物酶体、过氧化物酶体增殖激活受体(PPAR)信号通路、脂肪酸降解等重要通路。通过文献检索,发现过氧化物酶体及PPAR信号通路与肝损伤相关,在这两条通路中共筛选出脂肪酸转运蛋白1(Fabp1)、肝衰竭与长链脂酰CoA合成酶(Acsl1)、酰基辅酶A氧化酶1(Acox1)、三羟酰辅酶A脱氢酶(Ehhadh)、乙酰辅酶A酰基转移酶1b(Acaa1b)等几种可能与多房棘球蚴病致病相关的差异表达蛋白,这些差异蛋白在实验组小鼠体内表达量均显著下调。结论多房棘球蚴病小鼠肝脏内有大量蛋白质差异表达,其中Fabp1、Acsl1、Acox1、Ehhadh及Acaa1b等蛋白可能与多房棘球蚴病发病相关。

Objective To identify the differentially expressed proteins in different liver tissues in the mouse model of alveolar echinococcosis using high-resolution mass spectrometry with data independent acquisition(DIA), and to identify the key proteins contributing to the pathogenesis of alveolar echinococcosis. Methods Protoscoleces were isolated from Microtus fuscus with alveolar echinococcosis and the experimental model of alveolar echinococcosis was established in female Kunming mice aged 6 to8 weeks by infection with Echinococcus multilocularis protoscoleces. Mice were divided into the experimental and control groups,and animals in the experimental group was injected with approximately 3 000 protoscoleces, while mice in the control group were injected with the same volume of physiological saline. Mouse liver specimens were sampled from both groups one year post-infection and subjected to pathological examinations. In addition, the lesions(the lesion group) and peri-lesion specimens(the peri-lesion group) were sampled from the liver of mice in the experimental group and the normal liver specimens(the normal group)were sampled from mice in the control group for DIA proteomics analysis, and the differentially expressed proteins were subjected to bioinformatics analysis. Results A total of 1 020 differentially expressed proteins were identified between the lesion group and the normal group, including 671 up-regulated proteins and 349 down-regulated proteins, and 495 differentially expressed proteins were identified between the peri-lesion group and the normal group, including 327 up-regulated proteins and168 down-regulated proteins. The Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis revealed that these differentially expressed proteins were involved in peroxisome, peroxisome proliferator-activated receptor(PPAR) and fatty acid degradation pathways, and the peroxisome and PPAR signaling pathways were found to correlate with liver injury. Several differentially expressed proteins that may contribute to the pathogenesis of alveolar echinococcosis were identified in these two pathways, including fatty acid binding protein 1(Fabp1), Acyl-CoA synthetase long chain family member 1(Acsl1), Acyl-CoA oxidase 1(Acox1), Enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase(Ehhadh) and Acetyl-Coenzyme A acyltransferase1 B(Acaa1 b), which were down-regulated in mice in the experimental group. Conclusion A large number of differentially expressed proteins are identified in the liver of the mouse model of alveolar echinococcosis, and Fabp1, Acsl1, Acox1, Ehhadh and Acaa1 b may contribute to the pathogenesis of alveolar echinococcosis.