人源细粒棘球蚴囊壁与囊液中宿主蛋白的鉴定与分析

doi:10.12140/j.issn.1000-7423.2023.06.003

中国寄生虫学与寄生虫病杂志 ›› 2023, Vol. 41 ›› Issue (6): 677-682.doi: 10.12140/j.issn.1000-7423.2023.06.003

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¹(), 李子华²^,³^,⁴, 张翠影²^,³^,⁴, 赵巍²^,³^,⁴^,^*()

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

收稿日期:2023-06-27 修回日期:2023-11-14 出版日期:2023-12-30 发布日期:2023-12-22
通讯作者: * 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
作者简介: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
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 Tao¹(), LI Zihua²^,³^,⁴, ZHANG Cuiying²^,³^,⁴, ZHAO Wei²^,³^,⁴^,^*()

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:2023-06-27 Revised:2023-11-14 Online:2023-12-30 Published:2023-12-22
Contact: * E-mail: zw-6915@163.com
Supported by:
Yinchuan Science and Technology Support Project(2023SF10);Ningxia Medical University 2023 University-level Scientific Research Project Key Project(XZ2023027)

摘要/Abstract

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（M_r）为70 000、50 000、25 000 处显示出清晰的蛋白条带，囊液蛋白在M_r 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M_r 70 000的蛋白中包含人血白蛋白，约M_r 50 000的目标蛋白为人IgG重链，约M_r 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。

关键词: 细粒棘球绦虫, 白蛋白, 抗体, 囊壁, 囊液

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 (M_r) of 70 000, 50 000, and 25 000, and the cyst fluid protein showed clear bands at M_r 70 000. Protein bands. The protein spectrum and Western blotting results showed that the protein with a relative molecular mass of approximately M_r 70 000 contained human albumin, the target protein with a relative molecular mass of approximately M_r 50 000 was a human IgG heavy chain, and the target protein with a relative molecular mass of approximately M_r 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.

Key words: Echinococcus granulosus, Albumin, Antibodies, Cyst wall, Cyst fluid

中图分类号:

R532.32

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.

LI Tao, LI Zihua, ZHANG Cuiying, ZHAO Wei. Identification and analysis of host proteins in the cyst wall and cyst fluid of Echinococcus granulosus in human infection[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2023, 41(6): 677-682.

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参考文献 27

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项目 Project	囊液 Cyst fluid	血清 Serum	t值 t values	P值 P values
渗透压/mOsm Osmotic pressure/mOsm	313.50 ± 73.33	301.59 ± 6.20	0.52	> 0.05
Na⁺浓度/mmol·L^-1Na⁺ concentration/mmol·L^-1	143.40 ± 25.61	141.43 ± 2.42	0.24	> 0.05
K⁺浓度/mmol·L^-1K⁺ concentration/mmol·L^-1	5.53 ± 0.86	3.92 ± 0.33	5.56	< 0.05
Cl^-浓度/mmol·L^-1Cl^- concentration/mmol·L^-1	101.56 ± 12.42	105.35 ± 3.75	0.92	> 0.05
葡萄糖浓度/mmol·L^-1 Glucose concentration/mmol·L^-1	3.07 ± 2.61	4.65 ± 0.26	1.91	> 0.05
白蛋白浓度/g·L^-1 Albumin concentration/g·L^-1	0.57 ± 0.46	39.42 ± 2.77	43.71	< 0.05
胆固醇浓度/mmol·L^-1 Cholesterol concentration/mmol·L^-1	0.03 ± 0.02	3.79 ± 1.53	7.74	< 0.05

人源细粒棘球蚴囊壁与囊液中宿主蛋白的鉴定与分析

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

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