仔猪肺、肠组织离体感染模型的建立及应用

Establishment and application of piglet lung and intestinal tissue isolated infection model

目前,病毒的研究多局限于细胞水平,精密组织切片(Precision-cut tissue slices,PCTS)与细胞水平相比不仅能够比较完整的保存正常组织结构、细胞间的联系及细胞极性,而且也能保证切片的生存能力,使其能够最大限度地模拟在体状态,为病毒的深入研究带来福音。本研究通过制备仔猪肺、肠的离体感染模型,并对离体模型的可行性进行验证,采用ATP检测试剂盒验证切片活性,并对其感染猪流行腹泻病毒(porcine epidemic diahorrea virus,PEDV) CV777和猪传染性胃肠炎病毒(transmissible gastroenteritis virus,TGEV)GFP,进行RT-PCR检测和免疫荧光分析,结果显示小肠精密切片活性较好,维持在48 h左右,且能成功感染PEDV CV777和TGEV GFP,通过将肺脏气道内填充1.5%的低熔点琼脂糖,然后转移至切片机制作仔猪肺精密切片,观察气道上皮细胞纤毛活力,同时感染猪呼吸道冠状病毒(porcine respiratory coronavirus,PRCoV)后,进行RT-PCR检测和免疫荧光分析,结果显示肺精密切片生存力能够维持5 d左右,PRCoV成功感染肺精密切片且分布于气道上皮细胞周围,且病毒感染组纤毛活力比正常组下降快,结果表明,肺切片纤毛活性可作为病毒感染的重要评价指标。该研究可为模拟体内感染试验的离体模型提供理论依据,也为防制PEDV、TGEV、PRCoV提供新方法。

At present,virus research is mostly confined to cell level.Comparing with the cell level,precision-cut tissue slices can not only preserve the normal tissue structure,cell-cell connection and cell polarity,but also ensure the survival ability of the slice,so that it can simulate the state of the body to the maximum extent,and bring good news for the further study of virus.In this study,in vitro infection models of lung and intestine in piglets were prepared,and the feasibility of in vitro infection models was verified.ATP kit was used to verify the biopsy activity,and RT-PCR and immunofluorescence analysis were performed for PEDV CV777 and TGEV GFP infection.The results showed that the activity of precision-cut intestinal slices was good,which maintained at about 48 hours,and PEDV CV777 and TGEV GFP could be successfully infected.By filling the lung airway with 1.5% low melting point agarose,and then transferring it to the slicing machine,the precision-cut lung slices of piglets were made.The ciliary activity of airway epithelial cells was observed.After PRCoV infection,RT-PCR and immunofluorescence analysis were performed.The results showed that the viability of precision-cut lung slices could be maintained for about five days.PRCoV successfully infected precision-cut lung slices and distributed around airway epithelial cells,and the ciliary activity of virus infected group decreased faster than that of normal group.The results showed that ciliary activity of precision-cut lung slices could be used as an important indicator of viral infection.This study can provide theoretical basis for in vitro model of simulated in vivo infection test,and also provide a new method for the prevention and control of PEDV,TGEV and PRCoV.