旋毛虫新生幼虫细胞外囊泡的分离鉴定及组学分析

doi:10.12140/j.issn.1000-7423.2024.02.014

中国寄生虫学与寄生虫病杂志 ›› 2024, Vol. 42 ›› Issue (2): 225-233.doi: 10.12140/j.issn.1000-7423.2024.02.014

旋毛虫新生幼虫细胞外囊泡的分离鉴定及组学分析

刘毅¹^,²(), 蔡玉春¹, 陈家旭¹^,^*(), 陈韶红¹, 俞英昉¹

1 中国疾病预防控制中心寄生虫病预防控制所（国家热带病研究中心），传染病溯源预警与智能决策全国重点实验室，国家卫生健康委员会寄生虫病原与媒介生物学重点实验室，世界卫生组织热带病合作中心，科技部国家级热带病国际研究中心，上海 200025
2 上海健康医学院，医学技术学院，上海 201318

收稿日期:2023-11-28 修回日期:2024-03-14 出版日期:2024-04-30 发布日期:2024-04-25
通讯作者: * 陈家旭（1962—），男，博士，研究员，从事新发罕见及血液寄生虫病的免疫病理学、分子生物学、免疫和生物芯片诊断技术研究。E-mail：chenjx@nipd.chinacdc.cn
作者简介:刘毅（1981—），男，博士研究生，从事人体寄生虫学教学与寄生虫感染免疫研究。E-mail：liuy_52@sumhs.edu.cn
基金资助:
国家科技基础条件平台国家寄生虫资源库项目(NPRC-2019-194-30);国家重点研发计划(2021YFC2300800);国家重点研发计划(2021YFC2300801)

Isolation, identification, and omics analysis of extracellular vesicles from Trichinella spiralis newborn larvae

LIU Yi¹^,²(), CAI Yuchun¹, CHEN Jiaxu¹^,^*(), CHEN Shaohong¹, YU Yingfang¹

1 National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention; Chinese Center for Tropical Diseases Research, National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, Key Laboratory on Parasite and Vector Biology, Ministry of Health, WHO Centre for Tropical Diseases, National Center for International Research on Tropical Diseases, Ministry of Science and Technology, Shanghai 200025, China
2 Shanghai University of Medicine & Health Science, The College of Medical Technology, Shanghai 201318, China

Received:2023-11-28 Revised:2024-03-14 Online:2024-04-30 Published:2024-04-25
Contact: * E-mail: chenjx@nipd.chinacdc.cn
Supported by:
National Science and Technology Infrastructure Platform National Parasite Resource Bank Project(NPRC-2019-194-30);National Key R&D Program(2021YFC2300800);National Key R&D Program(2021YFC2300801)

摘要/Abstract

摘要：

目的分离并鉴定旋毛虫新生幼虫分泌的细胞外囊泡（EV）主要成分，了解新生幼虫EV的功能。方法消化旋毛虫保种小鼠肌肉，手机旋毛虫肌幼虫，昆明小鼠灌胃感染肌幼虫（2 000条/鼠）后5 d取小肠，网筛法收集旋毛虫成虫，成虫置于RPMI 1640培养液中培养48 h后收集新生幼虫。采用差速超速离心法提取旋毛虫新生幼虫培养液中的EV，透射电子显微镜观察EV形态，使用纳米颗粒跟踪分析技术检测EV的粒径，蛋白质免疫印迹（Western blotting）验证EV蛋白。对新生幼虫及其EV进行蛋白组质谱分析和miRNA测序分析，采用t检验比较新生幼虫及其EV之间蛋白和miRNA的表达差异。差异表达蛋白和miRNA靶基因进行基因本体论（GO）功能条目富集分析和京都基因与基因组百科全书（KEGG）通路富集分析。使用WoLFPSORT分析蛋白的亚细胞定位。结果透射电子显微镜和纳米颗粒跟踪分析结果显示，旋毛虫新生幼虫分泌的EV为“杯盘”形膜性囊泡，平均粒径为（101.7 ± 1.8） nm。Western blotting结果显示，新生幼虫EV能与旋毛虫感染小鼠血清产生特异性条带，相对分子质量约为65 000。蛋白组质谱分析结果显示，新生幼虫及其EV样品中分别检测到1 424和231种蛋白，有220种蛋白在两组样品中均有表达，其中116种表达有差异（在EV中31种表达上调、85种表达下调）。蛋白亚细胞定位分析结果显示，差异蛋白主要分布于细胞质（30.2%，35/116）、细胞核（19.0%，22/116）和细胞膜（17.2%，20/116）。miRNA测序结果显示，新生幼虫及其EV样品分别检测到183和117种miRNA，有46种miRNA在两组样品中均有表达，其中40种表达有差异（在EV中10种表达上调、30种表达下调）。在EV中表达上调的10种miRNA中，let-7-5p表达最高。GO功能条目富集分析结果显示，差异蛋白有57种属于细胞组成、73种具有分子功能、63种参与生物过程，差异miRNA的靶基因主要与自噬相关12基因转移酶活性、胞质泛素连接酶复合物、细胞对紫外线A的响应和钙离子稳态等条目有关。KEGG通路富集分析结果显示，差异蛋白主要富集在代谢途径、内质网中的蛋白质加工等相关通路，差异miRNA的靶基因仅涉及自噬-其他1条途径。结论本研究分离并鉴定了旋毛虫新生幼虫分泌的EV，检测到新生幼虫及其EV间差异表达的116种蛋白和40种miRNA，主要参与代谢、自噬等途径，推测新生幼虫EV可能在旋毛虫感染早期逃避宿主免疫防御的过程中发挥重要作用。

关键词: 旋毛虫, 新生幼虫, 细胞外囊泡, 蛋白组学, miRNA测序

Abstract:

Objective To isolate and identify the main components of extracellular vesicles (EV) secreted by Trichinella spiralis newborn larvae and elucidate the function of these EVs. Methods T. spiralis muscle larvae were sourced from the muscles of mice, which had been utilized for T. spiralis preservation. The small intestine was excised 5 days after infected with muscle larvae, and T. spiralis adults were obtained through mesh screening. These adults were then cultured in RPMI 1640 medium for 48 hours to yield newborn larvae. EVs were collected by differential ultracentrifugation from newborn larvae culture fluid of T. spiralis, and were observed morphologically under transmission electron microscope, and the diameter of the newborn larvae EVs was measured by nanoparticle tracking analysis. The EV protein components were verified using Western blotting, and analyzed by proteomic mass spectrometry. RNA sequencing technology was applied to analyze the miRNA sequences of the newborn larvae and their secreted EVs. The differences of protein components and differential expression between newborn larvae and their EVs were compared using t-test. The differentially expressed proteins and miRNA target genes were analyzed by gene ontology (GO) functional item enrichment and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment. WoLFPSORT was used to analyze the subcellular localization of proteins. Results Transmission electron microscopy and nanoparticle tracking analysis revealed that T. spiralis newborn larvae produce cup-shaped, membranous vesicles with a diameter of approximately (101.7 ± 1.8) nm. Western blotting demonstrated that the newborn larvae-derived EVs generate specific bands when reacted with sera from mice infected with Trichinella spiralis, with a approximate molecular weight of 65 000. Through proteomics mass spectrometry, 1 424 and 231 proteins were detected in the newborn larvae and EV samples, respectively. Among these, 220 proteins were expressed in both groups, with 116 displaying differential expression patterns (31 upregulated and 85 downregulated in EVs). The subcellular localization analysis revealed that the differentially expressed proteins were predominantly localized in the cytoplasm (30.2%, 35/116), nucleus (19.0%, 22/116), and cell membrane (17.2%, 20/116). miRNA sequencing data showed that 183 and 117 miRNAs were detected in newborn larvae and EV samples, respectively; 46 miRNAs were found in both groups, with 40 showing differential expression patterns (10 upregulated and 30 downregulated in EVs). Notably, let-7-5p, miR-125-5p, miR-10, and miR-92 exhibited higher levels of upregulation in newborn larvae-derived EVs. The GO functional enrichment analysis indicated that the differentially expressed proteins could be categorized into three main groups: cell composition (57), molecular function (73), and biological processes (63). The target genes of differentially expressed miRNA were primarily associated with autophagy-related genes such as transferase activation of autophagy-related 12 and ubiquitin linkage enzyme complex. KEGG pathway enrichment analysis suggested that the differentially expressed proteins were significantly enriched in metabolic pathways and protein processing in endoplasmic reticulum-related pathways, whereas the target genes of differentially expressed miRNA were only involved in the autophagy-other pathway. Conclusion This study successfully isolated and identified the EVs secreted by newborn larvae of T. spiralis. 116 differentially expressed proteins and 40 miRNAs were identified between the newborn larvae and their EVs, predominantly participating in metabolic, autophagic and other pathways, which inferring that newborn larvae EVs may play important roles in evading host immune defence at early Trichinella infection.

Key words: Trichinella spiralis, Newborn larvae, Extracellular vesicles, Proteomics, miRNA sequencing

中图分类号:

R383.15

刘毅, 蔡玉春, 陈家旭, 陈韶红, 俞英昉. 旋毛虫新生幼虫细胞外囊泡的分离鉴定及组学分析[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(2): 225-233.

LIU Yi, CAI Yuchun, CHEN Jiaxu, CHEN Shaohong, YU Yingfang. Isolation, identification, and omics analysis of extracellular vesicles from Trichinella spiralis newborn larvae[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2024, 42(2): 225-233.

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Uniprot 登录号 Uniprot ID	蛋白质名称 Protein name	亚细胞定位 Subcellular location	MISEV 2023分类 MISEV 2023 categories
A0A0V1BH03	镁转运蛋白1 Magnesium transporter protein 1	细胞膜 Plasma membrane	1a
A0A0V1BT16	代谢型谷氨酸受体2 Metabotropic glutamate receptor 2	细胞膜 Plasma membrane	1a
A0A0V1BZ62	P型铜离子转运蛋白 P-type Cu(+) transporter	细胞膜 Plasma membrane	1a
A0A0V1BT87	烯醇化酶 Phosphopyruvate hydratase	线粒体 Mitochondria	2b
A0A0V1AYF7	α-肌动蛋白 Alpha-actinin	细胞质 Cytoplasm	2b
E5SLU1	肌动蛋白5C Actin-5C	细胞骨架 Cytoskeleton	2b
A0A0V1C0Z2	微管蛋白α链 Tubulin alpha chain	线粒体 Mitochondria	2b
A0A0V1BAN8	微管蛋白α-3链 Tubulin alpha-3 chain	线粒体 Mitochondria	2b
E5SWF2	微管β链 Tubulin beta chain	细胞质 Cytoplasm	2b

miRNA名称 miRNA_name	miRNA序列（5'→3'） miRNA sequence (5'→3')	新生幼虫组平均表达量 Average expression level of newborn larvae	EV组平均表达量 Average expression level of EVs	t值 t value	P值 P value
ngi-miR-92a_L + 1_1ss21AG	TATTGCACTTGTCCCGGCCTGT	25	13 582	6.978	< 0.05
mle-miR-92b-3p_1ss11TG	TATTGCACTTGTCCCGGCCTGT	25	13 582	6.978	< 0.05
tca-mir-3885-p3_1ss3AG	GAGGGCGGCGGCGGCGGC	0	623	5.228	< 0.05
tca-miR-10-5p_1ss12TA	TACCCTGTAGAACCGAATTTGT	32	11 644	5.026	< 0.05
lgi-miR-10_1ss12TA	TACCCTGTAGAACCGAATTTGT	32	11 644	5.026	< 0.05
tca-miR-125-5p	TCCCTGAGACCCTAACTTGTGA	208	17 769	4.880	< 0.05
lgi-miR-100_R + 1_1ss16AT	AACCCGTAGATCCGATCTTGTGA	80	4 597	4.625	< 0.05
cel-let-7-5p	TGAGGTAGTAGGTTGTATAGTT	116	85 725	4.501	< 0.05
tca-mir-3885-p5_1ss16CG	GGCGGCGGCGGCGGCGGA	0	31	4.429	< 0.05
tca-mir-3885-p3_1ss16CG	GGCGGCGGCGGCGGCGGA	0	31	4.429	< 0.05

旋毛虫新生幼虫细胞外囊泡的分离鉴定及组学分析

Isolation, identification, and omics analysis of extracellular vesicles from Trichinella spiralis newborn larvae

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