ADAM17介导的猪源TNFα抑制PRRSV感染的研究

发布时间：2018-09-08 12:35

【摘要】：2006年,中国爆发了大规模的由高致病性猪繁殖与呼吸综合征病毒(Highly pathogenic porcine reproductive and respiratory syndrome virus,HP-PRRSV)引发的高热病,呈现高热、高发病率和高死亡率特征,严重危害猪只的健康,给养猪业造成了巨大的经济损失。有研究报道,PRRSV感染引发炎症反应时会伴随大量TNFα的分泌产生。然而,迄今为止,有关猪源TNFα分泌机制的研究尚未有报道。此外,随着对TNFα生物学活性的深入研究,TNFα的抗病毒作用也引起人们越来越多的兴趣和关注。为探究TNFα对PRRSV感染的影响,本研究针对HP-PRRSV HuN4毒株,首先采用ELISA方法检测猪原代肺泡巨噬细胞(Porcine alveolar macrophages,PAMs)在HP-PRRSV HuN4毒株感染后不同时间段TNFα的分泌情况,从而直接分析PRRSV感染对PAMs分泌TNFα的影响,结果发现PAMs在PRRSV感染后分泌TNFα的水平显著提高。为进一步从细胞感染角度揭示TNFα在PRRSV感染中的作用,本研究选择Marc-145细胞作为分析TNFα影响PRRSV感染的基础细胞系,然后采用瞬时转染的方法构建能够分泌TNFα的Marc-145细胞模型,接种病毒后,利用荧光定量PCR、TCID50以及间接免疫荧光技术检测病毒感染复制的情况,结果显示TNFα对PRRSV感染具有明显的抑制作用。去整合素-金属蛋白酶17(A disintegrin and metalloprotease 17,ADAM17)是最先发现的剪切酶,可介导多种可溶性蛋白的产生,为进一步探究猪源TNFα的分泌机制,我们展开了对ADAM17介导猪源TNFα分泌的研究。该研究首先在猪原代肺泡巨噬细胞PAMs上采用ADAM17激活剂及抑制剂对其活性进行修饰,结果发现在活化的ADAM17的PAMs细胞上清中TNFα分泌明显上调,而经ADAM17抑制剂预处理的PAMs几乎无TNFα分泌。该结果初步揭示了ADAM17可介导PAMs中TNFα的分泌。为进一步确定ADAM17介导猪源TNFα的分泌,本实验采用瞬转的方法在体外构建了能够表达猪源TNFα的细胞模型(HEK293/TNFα),然后用ADAM17抑制剂及基因过表达技术对ADAM17活性进行修饰。结果显示,在ADAM17活性被抑制的细胞上清中TNFα分泌量显著降低,而在ADAM17活性持续增强的细胞上清中TNFα分泌量明显增加。最后,本研究采用shRNA基因敲除技术构建了将ADAM17基因沉默的HEK293细胞,从而特异性分析ADAM17对猪源TNFα的剪切作用。结果显示在ADAM17基因沉默的HEK293细胞中,几乎无TNFα分泌。综合以上,研究结果证明猪源TNFα的分泌是由ADAM17介导产生的。此外,本研究采用基因定点突变及缺失技术鉴定出78RSS是ADAM17剪切猪源TNFα的关键结构域。本研究从细胞感染的角度揭示了由金属蛋白酶ADAM17介导分泌的TNFα在抗PRRSV感染中的作用,该研究结果为我们后期通过药物调控ADAM17的活性,进而通过控制TNFα的分泌量,从而发挥其抗PRRSV感染的作用,不仅为拮抗病毒感染提供了新思路,而且对开发靶向治疗炎症药物具有指导性作用,对治疗炎症风暴和抗病毒感染具有潜在的应用性价值和良好的应用前景。
[Abstract]:In 2006, a large-scale outbreak of high fever caused by highly pathogenic porcine reproductive and respiratory syndrome virus (Highly pathogenic porcine reproductive and respiratory syndrome virus,HP-PRRSV) in China showed high fever, high morbidity and high mortality, which seriously endangered the health of pigs. Great economic losses have been caused to the pig industry. It has been reported that TNF 伪 secretion is associated with inflammatory response induced by PRRSv infection. So far, however, no studies have been reported on the secretory mechanism of porcine TNF 伪. In addition, with the further study of the biological activity of TNF 伪, the antiviral effect of TNF- 伪 has attracted more and more attention. In order to investigate the effect of TNF 伪 on PRRSV infection, firstly, ELISA method was used to detect the secretion of TNF 伪 in porcine primary alveolar macrophages (Porcine alveolar macrophages,PAMs) at different time points after HP-PRRSV HuN4 infection. The effect of PRRSV infection on the secretion of TNF 伪 by PAMs was analyzed directly. The results showed that the level of TNF 伪 secreted by PAMs increased significantly after PRRSV infection. In order to reveal the role of TNF 伪 in PRRSV infection from the point of view of cell infection, Marc-145 cells were selected as the basic cell lines to analyze the effect of TNF 伪 on PRRSV infection, and then transient transfection was used to construct the Marc-145 cell model which could secrete TNF 伪. After inoculation, the replication of viral infection was detected by fluorescence quantitative PCR,TCID50 and indirect immunofluorescence technique. The results showed that TNF 伪 had obvious inhibitory effect on PRRSV infection. Deintegrin-metalloproteinase-17 (A disintegrin and metalloprotease 17 (ADAM17) is the first shearing enzyme that can mediate the production of many soluble proteins. In order to further explore the secretion mechanism of porcine TNF 伪, we have carried out a study on ADAM17 mediated porcine TNF 伪 secretion. In this study, the activity of PAMs was modified by ADAM17 activator and inhibitor on the primary alveolar macrophage PAMs. The results showed that the secretion of TNF 伪 was up-regulated in the supernatant of activated ADAM17 PAMs cells. However, PAMs pretreated with ADAM17 inhibitor had almost no TNF 伪 secretion. The results showed that ADAM17 could mediate the secretion of TNF 伪 in PAMs. In order to further determine the secretion of porcine TNF 伪 mediated by ADAM17, a cell model (HEK293/TNF 伪) capable of expressing porcine TNF 伪 was constructed by transient method in vitro. Then ADAM17 inhibitor and gene overexpression techniques were used to modify ADAM17 activity. The results showed that the secretion of TNF 伪 in the supernatant of the cells with inhibited ADAM17 activity was significantly decreased, while that in the supernatant of the cell supernatant with the sustained increase of ADAM17 activity was significantly increased. Finally, shRNA gene knockout technique was used to construct HEK293 cells silencing ADAM17 gene, so as to specifically analyze the shear effect of ADAM17 on porcine TNF 伪. The results showed that there was almost no TNF 伪 secretion in HEK293 cells silenced by ADAM17 gene. All above, the results showed that the secretion of TNF 伪 was mediated by ADAM17. In addition, 78RSS was identified as the key domain of ADAM17 shearing porcine TNF 伪 by site-directed mutation and deletion technique. This study revealed the role of TNF 伪 secreted by metalloproteinase-mediated ADAM17 in the treatment of PRRSV infection from the point of view of cell infection. The results showed that the activity of ADAM17 was regulated by drugs, and the secretion of TNF 伪 was controlled by controlling the secretion of TNF 伪. Thus, it not only provides a new way of thinking for antagonizing virus infection, but also has a guiding effect on the development of targeted anti-inflammatory drugs. It has potential application value and good application prospect for the treatment of inflammatory storm and antiviral infection.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.28

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