文章摘要
卢荣华,张文雅,张玉茹,杨丽萍,王俊丽,孟晓林,聂国兴.草鱼肝细胞外泌体的分离鉴定及其对肝细胞miR-122/33和免疫相关基因表达的影响[J].水产学报,2020,44(1):1~10
草鱼肝细胞外泌体的分离鉴定及其对肝细胞miR-122/33和免疫相关基因表达的影响
Isolation and identification of hepatocellular exosomes and their effects on the expression of miR-122/33 and immune-related genes in grass carp(Ctenopharyngodon idella)
投稿时间:2018-12-14  修订日期:2019-04-26
DOI:10.11964/jfc.20181211575
中文关键词: 草鱼  肝细胞  外泌体  分离  鉴定  免疫调节
英文关键词: Ctenopharyngodon idella  hepatocytes  exosomes  isolation  identification  immunoregulation
基金项目:国家自然科学基金(31402311,31672671,31872581);NSFC-河南联合基金(U1704109);河南省重点科技攻关项目(国家科技合作领域)(182102410031);河南省自然科学基金(162300410165)
作者单位E-mail
卢荣华 河南师范大学水产学院, 河南 新乡 453007  
张文雅 河南师范大学水产学院, 河南 新乡 453007  
张玉茹 河南师范大学水产学院, 河南 新乡 453007  
杨丽萍 河南师范大学水产学院, 河南 新乡 453007  
王俊丽 河南师范大学生命科学学院, 河南 新乡 453007  
孟晓林 河南师范大学水产学院, 河南 新乡 453007  
聂国兴 河南师范大学水产学院, 河南 新乡 453007 niegx@htu.cn 
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中文摘要:
      外泌体是具有磷脂双分子膜结构的纳米级囊泡,能够参与机体多种生理过程。实验探讨了草鱼肝细胞外泌体的分离鉴定方法,并初步研究外泌体对草鱼肝细胞中miRNAs及免疫相关基因表达的影响。实验以草鱼肝细胞L8824为材料,通过超速离心获得外泌体,利用电子显微镜观察外泌体形态,采用纳米颗粒示踪分析(nanoparticle tracking analysis,NTA)技术检测外泌体粒径和数量,同时利用Western blot分析其标志蛋白CD63的表达,最后用正常肝细胞和油酸诱导的脂肪肝细胞源外泌体孵育草鱼肝细胞,通过Real-time qPCR技术检测两种不同来源的外泌体对草鱼肝细胞中miR-122/33及免疫相关基因(TNF-α,NF-κB,IL-1β,IL-6和IL-10)转录水平的影响。结果显示,草鱼肝细胞外泌体为30~150 nm的不均匀囊泡,呈圆形或椭圆形,有完整的膜结构;外泌体标志蛋白CD63呈阳性表达;NTA技术检测显示外泌体囊泡占所有囊泡的50%以上;脂肪肝细胞源外泌体显著提高了肝细胞中miR-122及炎症因子TNF-α、IL-1β和IL-6的mRNA转录水平。研究表明,通过超速离心法可成功分离草鱼肝细胞外泌体,且脂肪肝细胞源外泌体在草鱼肝细胞免疫调节中可能发挥重要作用。
英文摘要:
      Exosomes are nano-scale vesicles with a phospholipid bimolecular membrane, which can participate in many physiological processes. The aim of this study was to investigate the isolation and identification methods of extracellular exosomes from grass carp hepatocyte and to conduct preliminary study on the effects of exosomes secreted from fatty hepatocyte on the expression of miRNAs and immune-related genes in grass carp hepatocytes. In this study, The exosomes of grass carp hepatocytes L8824 were extracted by ultracentrifugation. The morphology of exosomes was observed by Electron Microscopy, the parcicle size and quantity of exosomes were determined by Nano-particle Tracking Analysis. Meanwhile, we analyzed the expression of the specific protein CD63 using Western blot. Then, we used exosomes,which wwew secreted from normal hepatocytes and fatty hepatocyte induced by oleic acid to incubate grass carp hepatocytes, and the effects of two different exosomes on the transcription levels of miR-122/33 and immune-related genes, which are TNF-α, NF-κB, IL-1β, IL-6 and IL-10, were detected by real-time qPCR. Our results indicated that the exosomes secretion of grass carp hepatocytes culture medium were uneven vesicles of 30-150 nm, round or oval, and had a complete membrane structure. The expression of exosomes marker protein CD63 was positive, and the NTA results showed that the exosome vesicles accounted for more than 50 % of all the vesicles. Additionally, the exosomes of fatty liver cells significantly increased the expression of miR-122 and the immunogenic genes (TNF-α, IL-1β and IL-6) in hepatocytes (P<0.05). These results suggest that the exosomes of grass carp hepatocytes can be isolated successfully by ultracentrifugation. Moreover, exosomes may play a key role in the regulation of immunity of grass carp hepatocytes.
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