仔猪小肠上皮细胞的体外培养及猪流行性腹泻病毒对其感染性研究

doi:10.16431/j.cnki.1671-7236.2020.12.029

Abstract

Abstract: The study was aimed to establish a simple in vitro culture system for piglet small intestinal epithelial cells,and to provide materials for researches on porcine epidemic diarrhea virus (PEDV).In this study,newborn piglets that did not eat colostrum were used as the initial donors,and the primary cells were separated in vitro by scraping the intestinal mucosa from the intestinal lumen and mechanical separation and dispersion.The piglet intestinal epithelial cells were purified using 0.1% trypsin differential digestion method.Compared the effects of newborn weak piglets and normal piglets as donors on the activity of primary intestinal epithelial cells.MTT method was used to compare the proliferation activity of primary cells of different generations.Immunofluorescence and Real-time RT-PCR were used to detect the infection and proliferation of PEDV strain CV777 in primary intestinal epithelial cells.The results showed that the isolation and culture method in vitro used in this study could obtain primary intestinal epithelial cells with good proliferation activity,with obvious S-type cell proliferation curves.The cells with high purity and single morphology could be obtained by differential digestion,and had good proliferation activity after five consecutive passages.The proliferative activity of intestinal epithelial cells isolated from weak piglets and normal piglets had no obvious difference,and provided new way for reducing the cost of primary cell culture.The results of immunofluorescence and Real-time RT-PCR showed that PEDV could infect the primary intestinal epithelial cells,and replicated and proliferated in them.In this study,we established a simple,practical and low-cost method for in vitro culture of piglet primary small intestinal epithelial cells.The primary cells cultured by this method could be used as basic materials for the isolation and culture of PEDV and related research.

Key words: piglet intestinal epithelial cells; in vitro culture; porcine epidemic diarrhea virus (PEDV); proliferative activity; cell passage

CLC Number:

S852.33

SHEN Xuehuai, ZHAI Yinjian, YIN Lei, PAN Xiaocheng, ZHANG Danjun, ZHAO Ruihong, DAI Yin, HU Xiaomiao, ZHOU Xueli, HOU Hongyan. Culture of Piglet Intestinal Epithelial Cells in vitro and the Infectivity of Porcine Epidemic Diarrhea Virus on the Cells[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(12): 4051-4058.

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Culture of Piglet Intestinal Epithelial Cells in vitro and the Infectivity of Porcine Epidemic Diarrhea Virus on the Cells

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