摘要
猪流行性腹泻病毒(PEDV)是一种主要感染仔猪小肠的肠道冠状病毒.本研究旨在建立一种用于PEDV体外研究的仔猪小肠类器官模型.首先从PEDV发病猪场的临床仔猪腹泻样品中分离PEDV并进行全基因组测序,结果显示成功分离1株PEDV,命名为PEDV SD株.另外采用肠道类器官的培养技术,从10日龄健康仔猪小肠中分离小肠隐窝组织,经过体外培养7 d左右分化成具有肠道隐窝结构的猪小肠类器官,并能进行连续传代.然后将分离的PEDV SD株感染传代培养的猪小肠道类器官,通过间接免疫荧光试验显示PEDV可以很好地感染猪小肠道类器官,表明成功建立了PEDV感染肠道类器官的模型.猪小肠类器官感染模型可以在体外很好地重现PEDV感染肠道细胞的复杂结构的过程,为深入研究PEDV的致病机制提供一种新的可再生的体外研究模型.
Abstract
Porcine epidemic diarrhea virus(PEDV)is an intestinal coronavirus that infects the small intestines of piglets.The objective of the present study was to develop an in vitro 3D organoid model of porcine small intestines for studying the mechanism of PEDV infection.A PEDV SD strain was isolated from a farm and its whole genome sequence was analyzed in this study.The crypts of small intestines from a 10-day old pig were isolated and embedded in basement membrane extract and covered with the porcine intestinal organoids medium modified from the human system.The organoids with a crypt-villus architecture formed in 7 days.Then the swine organoids were infected with the PEDV SD strain virus.The PEDV infection was determined in indirect immunofluorescence assay.The results showed that the PEDV infection model of swine intestinal organoids were developed in this study.The 3D intestinal organoid model offered a long-term,renewable resource for investigating porcine intestinal infections with PEDV.
基金项目
上海市浦江人才计划(21PJ1416300)
上海市自然科学基金(21ZR1477200)
中央级公益性科研院所基本科研业务费专项(2019JB09)
维普
万方数据