文章摘要
草鱼肝细胞外泌体的分离鉴定及对免疫相关基因表达的影响
Isolation and identification of hepatocellular exosomes and its effect on immune-related gene expression in grass carp(Ctenopharyngodon idella)
投稿时间:2018-12-14  修订日期:2019-04-26
DOI:
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: Grass carp hepatocytes  Exosomes  Isolation  Identification  Immunomodulatory
基金项目:国家自然科学基金
作者单位E-mail
卢荣华 河南师范大学水产学院 laoaiyika@hotmail.com 
张文雅 河南师范大学水产学院  
张玉茹 河南师范大学水产学院  
杨丽萍 河南师范大学水产学院  
王俊丽 河南师范大学生命科学学院  
孟晓林 河南师范大学水产学院  
聂国兴 河南师范大学水产学院 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。本文旨在探讨草鱼肝细胞外泌体的分离鉴定方法,并初步研究脂肪肝细胞源外泌体对miRNAs及免疫相关基因表达的影响。本实验以草鱼肝细胞L8824为试验材料,通过超速离心获得外泌体,利用电子显微镜观察外泌体的形态,Western blot分析其标志蛋白CD63的表达,纳米颗粒跟踪分析仪(NTA)检测外泌体粒径和数量,Real-time qPCR技术检测外泌体对miR-122/33及免疫相关基因(TNF-α, NF-κB, IL-1β, IL-6和 IL-10)表达的影响。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结果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(P<0.05)。以上结果表明,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 is a vesicular body formed by the invagination of lysosomal granules in cells, which can participate in many physiological processes. The aim of this study was to investigate the isolation and identification of extracellular exosomes from grass carp liver and the effect of fatty liver cell exosomes on the expression of immune-related genes. In this study, The exosomes of grass carp hepatocytes L8824 were extracted by the method of ultracentrifugation. The morphology of exosomes were observed by Electron Microscopy, the parcicle size were determined by Nano-particle Tracking Analysis. Western blot analyzed the expression of the specific protein CD63, The expression of immune-associated genes (TNF-α, NF- κB, IL-1β, IL-6 and IL-10) was detected by Real-time qPCR. The results indicate that the exosomes secretion of grass carp hepatocyte culture medium was vesicle of 30-150 nm, which was 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 and that exosomes may play an important role in the regulation of immunity of grass carp hepatocytes.
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