【摘要】：2006年,中國爆發(fā)了大規(guī)模的由高致病性豬繁殖與呼吸綜合征病毒(Highly pathogenic porcine reproductive and respiratory syndrome virus,HP-PRRSV)引發(fā)的高熱病,呈現(xiàn)高熱、高發(fā)病率和高死亡率特征,嚴重危害豬只的健康,給養(yǎng)豬業(yè)造成了巨大的經(jīng)濟損失。有研究報道,PRRSV感染引發(fā)炎癥反應(yīng)時會伴隨大量TNFα的分泌產(chǎn)生。然而,迄今為止,有關(guān)豬源TNFα分泌機制的研究尚未有報道。此外,隨著對TNFα生物學(xué)活性的深入研究,TNFα的抗病毒作用也引起人們越來越多的興趣和關(guān)注。為探究TNFα對PRRSV感染的影響,本研究針對HP-PRRSV HuN4毒株,首先采用ELISA方法檢測豬原代肺泡巨噬細胞(Porcine alveolar macrophages,PAMs)在HP-PRRSV HuN4毒株感染后不同時間段TNFα的分泌情況,從而直接分析PRRSV感染對PAMs分泌TNFα的影響,結(jié)果發(fā)現(xiàn)PAMs在PRRSV感染后分泌TNFα的水平顯著提高。為進一步從細胞感染角度揭示TNFα在PRRSV感染中的作用,本研究選擇Marc-145細胞作為分析TNFα影響PRRSV感染的基礎(chǔ)細胞系,然后采用瞬時轉(zhuǎn)染的方法構(gòu)建能夠分泌TNFα的Marc-145細胞模型,接種病毒后,利用熒光定量PCR、TCID50以及間接免疫熒光技術(shù)檢測病毒感染復(fù)制的情況,結(jié)果顯示TNFα對PRRSV感染具有明顯的抑制作用。去整合素-金屬蛋白酶17(A disintegrin and metalloprotease 17,ADAM17)是最先發(fā)現(xiàn)的剪切酶,可介導(dǎo)多種可溶性蛋白的產(chǎn)生,為進一步探究豬源TNFα的分泌機制,我們展開了對ADAM17介導(dǎo)豬源TNFα分泌的研究。該研究首先在豬原代肺泡巨噬細胞PAMs上采用ADAM17激活劑及抑制劑對其活性進行修飾,結(jié)果發(fā)現(xiàn)在活化的ADAM17的PAMs細胞上清中TNFα分泌明顯上調(diào),而經(jīng)ADAM17抑制劑預(yù)處理的PAMs幾乎無TNFα分泌。該結(jié)果初步揭示了ADAM17可介導(dǎo)PAMs中TNFα的分泌。為進一步確定ADAM17介導(dǎo)豬源TNFα的分泌,本實驗采用瞬轉(zhuǎn)的方法在體外構(gòu)建了能夠表達豬源TNFα的細胞模型(HEK293/TNFα),然后用ADAM17抑制劑及基因過表達技術(shù)對ADAM17活性進行修飾。結(jié)果顯示,在ADAM17活性被抑制的細胞上清中TNFα分泌量顯著降低,而在ADAM17活性持續(xù)增強的細胞上清中TNFα分泌量明顯增加。最后,本研究采用shRNA基因敲除技術(shù)構(gòu)建了將ADAM17基因沉默的HEK293細胞,從而特異性分析ADAM17對豬源TNFα的剪切作用。結(jié)果顯示在ADAM17基因沉默的HEK293細胞中,幾乎無TNFα分泌。綜合以上,研究結(jié)果證明豬源TNFα的分泌是由ADAM17介導(dǎo)產(chǎn)生的。此外,本研究采用基因定點突變及缺失技術(shù)鑒定出78RSS是ADAM17剪切豬源TNFα的關(guān)鍵結(jié)構(gòu)域。本研究從細胞感染的角度揭示了由金屬蛋白酶ADAM17介導(dǎo)分泌的TNFα在抗PRRSV感染中的作用,該研究結(jié)果為我們后期通過藥物調(diào)控ADAM17的活性,進而通過控制TNFα的分泌量,從而發(fā)揮其抗PRRSV感染的作用,不僅為拮抗病毒感染提供了新思路,而且對開發(fā)靶向治療炎癥藥物具有指導(dǎo)性作用,對治療炎癥風(fēng)暴和抗病毒感染具有潛在的應(yīng)用性價值和良好的應(yīng)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 偽 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 偽. In addition, with the further study of the biological activity of TNF 偽, the antiviral effect of TNF- 偽 has attracted more and more attention. In order to investigate the effect of TNF 偽 on PRRSV infection, firstly, ELISA method was used to detect the secretion of TNF 偽 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 偽 by PAMs was analyzed directly. The results showed that the level of TNF 偽 secreted by PAMs increased significantly after PRRSV infection. In order to reveal the role of TNF 偽 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 偽 on PRRSV infection, and then transient transfection was used to construct the Marc-145 cell model which could secrete TNF 偽. After inoculation, the replication of viral infection was detected by fluorescence quantitative PCR,TCID50 and indirect immunofluorescence technique. The results showed that TNF 偽 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 偽, we have carried out a study on ADAM17 mediated porcine TNF 偽 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 偽 was up-regulated in the supernatant of activated ADAM17 PAMs cells. However, PAMs pretreated with ADAM17 inhibitor had almost no TNF 偽 secretion. The results showed that ADAM17 could mediate the secretion of TNF 偽 in PAMs. In order to further determine the secretion of porcine TNF 偽 mediated by ADAM17, a cell model (HEK293/TNF 偽) capable of expressing porcine TNF 偽 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 偽 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 偽. The results showed that there was almost no TNF 偽 secretion in HEK293 cells silenced by ADAM17 gene. All above, the results showed that the secretion of TNF 偽 was mediated by ADAM17. In addition, 78RSS was identified as the key domain of ADAM17 shearing porcine TNF 偽 by site-directed mutation and deletion technique. This study revealed the role of TNF 偽 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 偽 was controlled by controlling the secretion of TNF 偽. 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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S858.28

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