猪繁殖与呼吸综合征病毒在猪肺组织精细切片中感染特性的研究

Study on the infection characteristics of porcine reproductive and respiratory syndrome virus in porcine precision-cut lung slices

为研究猪繁殖与呼吸综合征病毒(PRRSV)在猪肺脏组织中的感染特性,本研究建立了高度分化的猪呼吸系统体外培养模型-猪肺组织精细切片(PCLS)。该培养体系包含肺脏组织中多种相关细胞并能够体现出肺脏组织的生理结构和生物学功能。本研究针对制备的猪PCLS进行生物学活性的鉴定,利用评估支气管上皮细胞纤毛摆动百分比的方法检测支气管上皮细胞纤毛活性,结果显示猪PCLS制备良好,在制备10 d以后仍保持95%以上的纤毛活性;利用活/死细胞染色法测定制备的猪PCLS体外培养后活细胞的比例,结果显示制备的猪PCLS在体外培养7 d后支气管上皮细胞和肺泡细胞仍为活细胞;利用间接免疫荧光试验测定猪PCLS中上皮细胞、杯状细胞和肺泡巨噬细胞(PAM)的完整性与分布情况,结果显示制备的猪PCLS上皮细胞和杯状细胞保存完整且猪PCLS中包含丰富的PAM。利用6株不同PRRSV分离株(HuN4、XD-15、WK-34、WK-38、LCL-75和DL-1510)以2.5×105TCID50/片的剂量感染猪PCLS,检测不同PRRSV分离株在猪PCLS中的感染特性,结果表明不同PRRSV分离株在该培养体系中表现出不同的增殖能力。本研究表明猪PCLS可用于PRRSV的体外感染试验。本研究为PRRSV的体外研究提供了新的模型与方法,同时也为其它猪呼吸道病原体提供了新的研究平台。

In order to analyze the infection characteristics of porcine reproductive and respiratory syndrome virus(PRRSV) in porcine lung tissue, a well-differentiated porcine respiratory ex-vivo culture system named as porcine precision-cut lung slices(PCLS) was established in this study. This culture system preserves several related cell types of the lung tissue, which may be able to represent the natural physiological structure and biological functions of lung. Here, the biological activity of porcine PCLS was identified by analyzing the percentage of cilia movement of ciliated epithelial cells to indicate the bronchial ciliary activity. The results showed that the porcine PCLS showed good character and could maintain more than 95% ciliary activity at 10 days postpreparation;The live/dead cell staining method was used to determine the proportion of living cells in porcine PCLS cultured in vitro, and the results showed that the bronchial epithelial cells and alveolar cells were still alive after 7 days of culture;The integrity and distribution of epithelial cells, goblet cells and alveolar macrophages(PAM) in porcine PCLS were determined by indirect immunofluorescence assay, and the results showed that the ciliated epithelial cells and goblet cells remained in a well condition and a great amount of PAM presented in porcine PCLS. Besides, PRRSV infection model was initially established base on this culture system. Porcine PCLS were infected with 6 different PRRSV isolated strains(HuN4, XD-15, WK-34, WK-38, LCL-75, and DL-1510), at 2.5×105 TCID50/slice. The results showed that different PRRSV strains showed distinct replication capacity in this culture system, suggesting that porcine PCLS can be used in analysis of the infection of PRRSV in vitro. This study provides a model and method to investigate PRRSV infection in vitro, as well as a novel research platform for other porcine respiratory pathogens.