论著

人源细粒棘球蚴囊壁与囊液中宿主蛋白的鉴定与分析

  • 李涛 ,
  • 李子华 ,
  • 张翠影 ,
  • 赵巍
展开
  • 1 宁夏医科大学总医院,银川 750004
    2 宁夏医科大学基础医学院,银川 750004
    3 宁夏医科大学科学技术研究中心,银川 750004
    4 宁夏常见传染病重点实验室,银川 750004
李涛(1993-),男,硕士,住院医师,从事外科治疗肝棘球蚴病的研究。E-mail:longtaodaxia123@126.com
* 赵巍(1960-),男,博士,二级教授,从事常见传染病疫苗的基础和转化应用研究、特异性诊断分子的筛选研究及传染病基础免疫分子机制的研究。E-mail:zw-6915@163.com

收稿日期: 2023-06-27

  修回日期: 2023-11-14

  网络出版日期: 2023-12-22

基金资助

银川市科技支撑项目(2023SF10);宁夏医科大学2023年校级科研项目重点项目(XZ2023027)

Identification and analysis of host proteins in the cyst wall and cyst fluid of Echinococcus granulosus in human infection

  • LI Tao ,
  • LI Zihua ,
  • ZHANG Cuiying ,
  • ZHAO Wei
Expand
  • 1 General Hospital of Ningxia Medical University, Yinchuan 750004, China
    2 School of Basic Medicine of Ningxia Medical University, Yinchuan 750004, China
    3 Science and Technology Research Center of Ningxia Medical University, Yinchuan 750004, China
    4 Ningxia Key Laboratory of Common Infectious Diseases, Yinchuan 750004, China

Received date: 2023-06-27

  Revised date: 2023-11-14

  Online published: 2023-12-22

Supported by

Yinchuan Science and Technology Support Project(2023SF10);Ningxia Medical University 2023 University-level Scientific Research Project Key Project(XZ2023027)

摘要

目的 通过对细粒棘球蚴囊壁及囊液中的宿主蛋白成分进行鉴定,进一步阐明细粒棘球绦虫与宿主之间相互作用的现象。方法 收集2022年宁夏医科大学总医院肝胆外科10例细粒棘球蚴病患者手术摘除的具有活性的完整棘球蚴包囊,同时取患者血样。无菌抽取子囊中无色透明的囊液,离心收集上清液;子囊内壁反复冲洗后剪碎,裂解匀浆,收集上清液。用二喹啉甲酸(BCA)蛋白定量试剂盒提取囊液和囊壁全蛋白,经过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)电泳后,切取囊壁全蛋白泳道显著的蛋白条带,进行质谱检测,进行蛋白质免疫印迹(Western blotting)分析。对患者血清和囊液的生化成分浓度进行定量检测。结果 10例细粒棘球蚴病患者的肝脏功能为Child-Pugh A或B级。病灶直径为2.9~16.3 cm,为WHO-IWGE分型的CE2(多子囊型)或CE3(内囊塌陷型)。SDS-PAGE电泳分析结果显示,棘球蚴囊壁蛋白在相对分子质量(Mr)为70 000、50 000、25 000 处显示出清晰的蛋白条带,囊液蛋白在Mr 70 000显示出清晰的蛋白条带。蛋白质谱与Western blotting结果均显示,相对分子质量在约Mr 70 000的蛋白中包含人血白蛋白,约Mr 50 000的目标蛋白为人IgG重链,约Mr 25 000的目标蛋白为人IgG轻链。各生化指标中,囊液和血清中的中K+浓度分别为(5.53 ± 0.86)mmol/L和(3.92 ± 0.33)mmol/L,白蛋白浓度分别为(0.57 ± 0.46)g/L和(39.42 ± 2.77)g/L,胆固醇浓度分别为(0.03 ± 0.02)mmol/L和(3.79 ± 1.53)mmol/L,三者在囊液和血清中的浓度差异有统计学意义(t = 5.56、43.71、7.74,P < 0.05)。结论 宿主来源的血清白蛋白和抗体是细粒棘球蚴囊壁和囊液中含量较高的宿主蛋白。

本文引用格式

李涛 , 李子华 , 张翠影 , 赵巍 . 人源细粒棘球蚴囊壁与囊液中宿主蛋白的鉴定与分析[J]. 中国寄生虫学与寄生虫病杂志, 2023 , 41(6) : 677 -682 . DOI: 10.12140/j.issn.1000-7423.2023.06.003

Abstract

Objective To further elucidate the interaction between Echinococcus granulosus and the host by identifying the host protein components in the cyst wall and cyst fluid of E. granulosus. Methods The active and intact echinococcal cysts removed surgically from 10 patients with E. granulosus were collected from the Department of Hepatobiliary Surgery, Ningxia Medical University General Hospital in 2022. Blood samples were also taken from the same patients. The transparent cyst fluid in the ascus was extracted aseptically, and centrifuged to collect the supernatant; the inner wall of the ascus was repeatedly washed, cut into pieces, lysed and homogenised to collect the supernatant. The bicinchoninic acid (BCA) protein quantification kit was used to extract the cyst fluid and cyst wall total protein. The cyst wall total protein lane was cut out after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis. The significant protein bands were analysed by mass spectrometry and Western blotting analysis. Quantitative analysis was performed to determine the biochemical component concentrations in the patient serum and cyst fluid. Results The liver function of 10 patients with echinococcosis granulosus was Child-Pugh A or B. The diameter of the lesion ranged from 2.9 to 16.3 cm, which were classified as CE2 (multiple subcysts) or CE3 (collapsed internal capsule) according to the WHO-IWGE classification. SDS-PAGE electrophoresis analysis results suggested that the echinococcal cyst wall protein showed clear protein bands at the relative molecular mass (Mr) of 70 000, 50 000, and 25 000, and the cyst fluid protein showed clear bands at Mr 70 000. Protein bands. The protein spectrum and Western blotting results showed that the protein with a relative molecular mass of approximately Mr 70 000 contained human albumin, the target protein with a relative molecular mass of approximately Mr 50 000 was a human IgG heavy chain, and the target protein with a relative molecular mass of approximately Mr 25 000 was a human IgG light chain. Among the various biochemical indicators, the concentrations of K+ in cyst fluid and serum were (5.53 ± 0.86) mmol/L and (3.92 ± 0.33) mmol/L respectively. The albumin concentrations were (0.57 ± 0.46) g/L and (39.42 ± 2.77) g/L. The cholesterol concentrations were (0.03 ± 0.02) mmol/L and (3.79 ± 1.53) mmol/L respectively. The concentration differences between the three in cyst fluid and serum were statistically significant (t = 5.56, 43.71, 7.74; P < 0.05). Conclusion Host-derived serum albumin and antibodies are found of comparatively higher amont in the cyst wall and cyst fluid of E. granulosus.

参考文献

[1] Wu WP, Wang H, Wang Q, et al. A nationwide sampling survey on echinococcosis in China during 2012—2016[J]. Chin J Parasitol Parasit Dis, 2018, 36(1): 1-14. (in Chinese)
  (伍卫平, 王虎, 王谦, 等. 2012—2016年中国棘球蚴病抽样调查分析[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(1): 1-14.)
[2] Wu WP. Prevalence and distribution of two types of echinococcosis in China[J]. China Anim Heath, 2017, 19(7): 7-9. (in Chinese)
  (伍卫平. 我国两型包虫病的流行与分布情况[J]. 中国动物保健, 2017, 19(7): 7-9.)
[3] Kui Y, Xue CZ, Wang X, et al. Progress of echinococcosis control in China, 2021[J]. Chin J Parasitol Parasit Dis, 2023, 41(2): 142-148. (in Chinese)
  (蒉嫣, 薛垂召, 王旭, 等. 2021年全国棘球蚴病防治进展[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(2): 142-148.)
[4] Zhao R, Dong F, Zhao YQ, et al. Analysis of current status and influencing factors of echinococcosis infection among rural residents in three counties of Ningxia in 2018[J]. J Ningxia Med Univ, 2020, 42(8): 795-798. (in Chinese)
  (赵瑞, 董飞, 赵殷奇, 等. 2018年宁夏三县区农村居民包虫病感染现况及影响因素分析[J]. 宁夏医科大学学报, 2020, 42(8): 795-798.)
[5] Tsai IJ, Zarowiecki M, Holroyd N, et al. The genomes of four tapeworm species reveal adaptations to parasitism[J]. Nature, 2013, 496(7443): 57-63.
[6] Pan WQ, Tang LH. Molecular parasitology[M]. Shanghai: Shanghai Scientific & Technical Publishers, 2004. (in Chinese)
  (潘卫庆, 汤林华. 分子寄生虫学[M]. 上海: 上海科学技术出版社, 2004.)
[7] Wang XY, Hu W, Zhang W. Ultrastructural study on the endogenous hair follicle of Echinococcus granulosus[J]. Qinghai Med J, 1995, 25(2): 1-2. (in Chinese)
  (汪晓筠, 王虎, 张伟. 细粒棘球蚴内生发囊的超微结构研究[J]. 青海医药杂志, 1995, 25(2): 1-2.)
[8] Hustead ST, Williams JF. Permeability studies on taenid metacestodes: I. Uptake of proteins by larval stages of Taenia taeniaeformis, T. crassiceps, and Echinococcus granulosus[J]. J Parasitol, 1977, 63(2): 314-321.
[9] Machnicka B, Grzybowski J. Host serum proteins in Taenia saginata metacestode fluid[J]. Vet Parasitol, 1986, 19(1/2): 47-54.
[10] Hayunga EG, Sumner MP, Letonja T. Evidence for selective incorporation of host immunoglobulin by strobilocerci of Taenia taeniaeformis[J]. J Parasitol, 1989, 75(4): 638-642.
[11] Aldridge JR Jr, Jennette MA, Kuhn RE. Uptake and secretion of host proteins by Taenia crassiceps metacestodes[J]. J Parasitol, 2006, 92(5): 1101-1102.
[12] Zheng YD. Proteomic analysis of Taenia hydatigena cyst fluid reveals unique internal microenvironment[J]. Acta Trop, 2017, 176: 224-227.
[13] Liu CS, Cao JP, Zhang HB, et al. Extracellular vesicles secreted by Echinococcus multilocularis: important players in angiogenesis promotion[J]. Microbes Infect, 2023, 25(7): 105147.
[14] Nicolao MC, Rodrigues CR, Coccimiglio MB, et al. Characterization of protein cargo of Echinococcus granulosus extracellular vesicles in drug response and its influence on immune response[J]. Parasit Vectors, 2023, 16(1): 255.
[15] Ahn CS, Kim JG, Han XM, et al. Comparison of Echinococcus multilocularis and Echinococcus granulosus hydatid fluid proteome provides molecular strategies for specialized host-parasite interactions[J]. Oncotarget, 2017, 8(57): 97009-97024.
[16] Monteiro KM, de Carvalho MO, Zaha A, et al. Proteomic analysis of the Echinococcus granulosus metacestode during infection of its intermediate host[J]. Proteomics, 2010, 10(10): 1985-1999.
[17] The Surgical Professional Committee of Hydatid Disease of the Surgical Branch of the Chinese Medical Association. Expert consensus on the diagnosis and treatment of liver two types of echinococcosis(2019)[J]. Chin J Digest Surg, 2019, 18 (8): 711 -721.
  中国医师协会外科医师分会包虫病外科专业委员会. 肝两型包虫病诊断与治疗专家共识(2019版)[J]. 中华消化外科杂志, 2019, 18(8): 711-721.)
[18] Liu F, Hu W, Cui SJ, et al. Insight into the host-parasite interplay by proteomic study of host proteins copurified with the human parasite, Schistosoma japonicum[J]. Proteomics, 2007, 7(3): 450-462.
[19] van Hellemond JJ, Retra K, Brouwers JF, et al. Functions of the tegument of schistosomes: clues from the proteome and lipidome[J]. Int J Parasitol, 2006, 36(6): 691-699.
[20] Tielens AGM, van Hellemond JJ. Unusual aspects of metabolism in flatworm parasites[J]. Parasit Flatworms, 2006: 387-407.
[21] Brehm K, Koziol U. Echinococcus-host interactions at cellular and molecular levels[J]. Adv Parasitol, 2017, 95: 147-212.
[22] Peón AN, Espinoza-Jiménez A, Terrazas LI. Immunoregulation by Taenia crassiceps and its antigens[J]. Biomed Res Int, 2013, 2013: 498583.
[23] McSorley HJ, Hewitson JP, Maizels RM. Immunomodulation by helminth parasites: defining mechanisms and mediators[J]. Int J Parasitol, 2013, 43(3/4): 301-310.
[24] Damian RT. The exploitation of host immune responses by parasites[J]. J Parasitol, 1987, 73(1): 3-13.
[25] Schroeder H, Skelly PJ, Zipfel PF, et al. Subversion of complement by hematophagous parasites[J]. Dev Comp Immunol, 2009, 33(1): 5-13.
[26] Kouoh F, Gressier B, Luyckx M, et al. Antioxidant properties of albumin: effect on oxidative metabolism of human neutrophil granulocytes[J]. Farmaco, 1999, 54(10): 695-699.
[27] Silva-álvarez V, Folle AM, Ramos AL, et al. Echinococcus granulosus antigen B: a hydrophobic ligand binding protein at the host-parasite interface[J]. Prostaglandins Leukot Essent Fatty Acids, 2015, 93: 17-23.
文章导航

/

〈 〉