ADAM17介導(dǎo)的CD163表達調(diào)控對PRRSV感染的影響
發(fā)布時間：2018-12-16 01:07
【摘要】：豬繁殖與呼吸綜合征(Porcine reproductive and respiratory syndrome,PRRS)是由豬繁殖與呼吸綜合征病毒(PRRSV)引起的嚴(yán)重危害養(yǎng)豬業(yè)的傳染病之一。根據(jù)核酸序列的差異,PRRSV被劃分為歐洲型和美洲型毒株。2006年5月以來,我國暴發(fā)的被稱為“高熱綜合癥”的豬病,即為變異的美洲型高致病性豬繁殖與呼吸綜合征病毒(HP-PRRSV)所致。豬肺泡巨噬細(xì)胞(PAM)是PRRSV入侵豬體的靶細(xì)胞,已發(fā)現(xiàn)在PAM上存在三種主要的PRRSV細(xì)胞受體:硫酸乙酰肝素(HS)、唾液酸粘附素(Sn)和清道夫受體CD163。HS的作用是吸附PRRSV到細(xì)胞表面,Sn可以加強吸附作用并介導(dǎo)PRRSV內(nèi)吞進入胞內(nèi),CD163則是隨PRRSV一同進入胞內(nèi)介導(dǎo)病毒的脫衣殼和病毒基因組的釋放。近年研究發(fā)現(xiàn),將豬源CD163分子轉(zhuǎn)染至PRRSV非允許細(xì)胞,使得細(xì)胞穩(wěn)定表達CD163蛋白后就成為PRRSV允許細(xì)胞,能夠成功感染增殖PRRSV,而其他兩種受體則不能,說明CD163在病毒感染細(xì)胞過程中至關(guān)重要。作為清道夫受體超家族的成員,CD163參與機體對異物的免疫應(yīng)答并參與炎癥反應(yīng),其在細(xì)胞表面的表達受多種因素的影響,其中去整合素金屬蛋白酶ADAM17已被證實能夠介導(dǎo)人源CD163的剪切調(diào)控,從而調(diào)節(jié)炎癥反應(yīng)。但是ADAM17能否介導(dǎo)豬源CD163的剪切調(diào)控從而影響病毒的入侵還不得而知,因此本研究闡釋了ADAM17介導(dǎo)CD163的表達調(diào)控機制及其對PRRSV感染的影響。首先,從PAM細(xì)胞中擴增了豬源CD163基因,克隆至真核表達質(zhì)粒pc DNA3.1-CD163,在測序正確后將其轉(zhuǎn)染HEK293細(xì)胞,通過流式細(xì)胞術(shù)和G418加壓篩選,獲得穩(wěn)定表達CD163的HEK293/CD163細(xì)胞系;病毒感染實驗表明PRRSV能夠感染HEK293/CD163細(xì)胞系,為研究病毒的體外感染奠定了基礎(chǔ)。其次,通過抑制劑、激活劑、si RNA基因干擾和基因過表達等方法來抑制或者增強PAM和HEK293/CD163細(xì)胞的ADAM17表達水平或活性,檢測ADAM17對豬源CD163的剪切能力。研究結(jié)果發(fā)現(xiàn),當(dāng)ADAM17表達下調(diào)或者活性受到抑制時,細(xì)胞表面CD163的表達水平升高;當(dāng)ADAM17表達上調(diào)或者被激活時,細(xì)胞表面CD163的表達水平降低,說明ADAM17介導(dǎo)豬源CD163的剪切調(diào)控。最后,在HEK293/CD163細(xì)胞系和PAM進行接毒實驗,測定ADAM17介導(dǎo)的CD163表達對PRRSV感染的影響。實驗結(jié)果發(fā)現(xiàn),當(dāng)用ADAM17抑制劑抑制ADAM17活性或者通過si RNA基因干擾下調(diào)ADAM17表達時,病毒感染量增多;當(dāng)用LPS激活A(yù)DAM17活性或通過基因過表達提高ADAM17表達時,病毒感染量減少。上述研究發(fā)現(xiàn)表明ADAM17通過調(diào)控細(xì)胞表面豬源CD163表達水平從而影響PRRSV入侵細(xì)胞的能力�？傊�,本研究通過流式細(xì)胞術(shù)和G418加壓篩選方法獲得了穩(wěn)定表達CD163的HEK293/CD163細(xì)胞系,并通過間接免疫熒光發(fā)鑒定了PRRSV能夠感染該細(xì)胞系并在HEK293/CD163細(xì)胞中增殖。在HEK293/CD163細(xì)胞系和PAM上證實了ADAM17通過調(diào)控細(xì)胞表面CD163的表達水平,從而影響PRRSV對易感細(xì)胞的感染能力。
[Abstract]:Porcine Reproductive and Respiratory Syndrome (Porcine reproductive and respiratory syndrome,PRRS) is one of the infectious diseases of swine breeding and respiratory syndrome virus (PRRSV). According to nucleic acid sequence differences, PRRSV is divided into European and American strains. Since May 2006, an outbreak of swine disease known as "high fever syndrome" has occurred in China. This is caused by mutant American type highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). Porcine alveolar macrophage (PAM) is the target cell of PRRSV invading porcine body. It has been found that there are three major PRRSV cell receptors on PAM: heparin sulfate (HS), Sialoadin (Sn) and scavenger receptor CD163.HS can adsorb PRRSV to the cell surface. Sn can enhance the adsorption and mediate the endocytosis of PRRSV into the cell. CD163, along with PRRSV, enters the cell to mediate the exfoliation of the virus and the release of the virus genome. In recent years, it has been found that when porcine CD163 molecules are transfected into PRRSV non-permitted cells, cells expressing CD163 protein stably become PRRSV permitted cells, which can successfully infect proliferative PRRSV, and the other two receptors can not. It is suggested that CD163 is very important in the process of virus infection. As a member of the scavenger receptor superfamily, CD163 is involved in the immune response of the body to foreign bodies and in inflammatory response, and its expression on the cell surface is affected by many factors. Among them, desintegrin metalloproteinase (ADAM17) has been proved to be able to mediate the shearing regulation of human CD163 and thus regulate the inflammatory response. However, it is not clear whether ADAM17 can mediate the regulation of porcine CD163 shearing and thus affect virus invasion. Therefore, the mechanism of CD163 expression regulation mediated by ADAM17 and its effect on PRRSV infection have been explained in this study. Firstly, the porcine CD163 gene was amplified from the PAM cells and cloned into the eukaryotic expression plasmid pc DNA3.1-CD163, which was transfected into HEK293 cells after sequencing correctly. The HEK293/CD163 cell line expressing CD163 stably was obtained by flow cytometry and G418 pressurization screening. Virus infection test showed that PRRSV could infect HEK293/CD163 cell line, which laid a foundation for the study of virus infection in vitro. Secondly, inhibitor, activator, si RNA gene interference and gene overexpression were used to inhibit or enhance the ADAM17 expression level or activity of PAM and HEK293/CD163 cells, and to detect the shear ability of ADAM17 to porcine CD163. The results showed that when the expression of ADAM17 was down-regulated or the activity was inhibited, the expression of CD163 on the cell surface increased, and when the expression of ADAM17 was up-regulated or activated, the expression of CD163 on the cell surface decreased, indicating that ADAM17 mediated the shearing regulation of porcine CD163. Finally, the effect of CD163 expression mediated by ADAM17 on PRRSV infection was detected in HEK293/CD163 cell line and PAM. The results showed that when the ADAM17 activity was inhibited by ADAM17 inhibitor or the ADAM17 expression was down-regulated by si RNA gene interference, the viral infection increased, and when the ADAM17 activity was activated by LPS or the ADAM17 expression was increased by gene overexpression, the viral infection decreased. These findings suggest that ADAM17 affects the ability of PRRSV invading cells by regulating the expression of porcine CD163 on cell surface. In conclusion, HEK293/CD163 cell lines expressing CD163 stably were obtained by flow cytometry and G418 pressure screening, and PRRSV could be infected and proliferated in HEK293/CD163 cells by indirect immunofluorescence. It was confirmed in HEK293/CD163 cell lines and PAM that ADAM17 affects the ability of PRRSV to infect susceptible cells by regulating the expression of CD163 on the cell surface.
【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】：2015
【分類號】：S855.3
本文編號：2381597
[Abstract]:Porcine Reproductive and Respiratory Syndrome (Porcine reproductive and respiratory syndrome,PRRS) is one of the infectious diseases of swine breeding and respiratory syndrome virus (PRRSV). According to nucleic acid sequence differences, PRRSV is divided into European and American strains. Since May 2006, an outbreak of swine disease known as "high fever syndrome" has occurred in China. This is caused by mutant American type highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). Porcine alveolar macrophage (PAM) is the target cell of PRRSV invading porcine body. It has been found that there are three major PRRSV cell receptors on PAM: heparin sulfate (HS), Sialoadin (Sn) and scavenger receptor CD163.HS can adsorb PRRSV to the cell surface. Sn can enhance the adsorption and mediate the endocytosis of PRRSV into the cell. CD163, along with PRRSV, enters the cell to mediate the exfoliation of the virus and the release of the virus genome. In recent years, it has been found that when porcine CD163 molecules are transfected into PRRSV non-permitted cells, cells expressing CD163 protein stably become PRRSV permitted cells, which can successfully infect proliferative PRRSV, and the other two receptors can not. It is suggested that CD163 is very important in the process of virus infection. As a member of the scavenger receptor superfamily, CD163 is involved in the immune response of the body to foreign bodies and in inflammatory response, and its expression on the cell surface is affected by many factors. Among them, desintegrin metalloproteinase (ADAM17) has been proved to be able to mediate the shearing regulation of human CD163 and thus regulate the inflammatory response. However, it is not clear whether ADAM17 can mediate the regulation of porcine CD163 shearing and thus affect virus invasion. Therefore, the mechanism of CD163 expression regulation mediated by ADAM17 and its effect on PRRSV infection have been explained in this study. Firstly, the porcine CD163 gene was amplified from the PAM cells and cloned into the eukaryotic expression plasmid pc DNA3.1-CD163, which was transfected into HEK293 cells after sequencing correctly. The HEK293/CD163 cell line expressing CD163 stably was obtained by flow cytometry and G418 pressurization screening. Virus infection test showed that PRRSV could infect HEK293/CD163 cell line, which laid a foundation for the study of virus infection in vitro. Secondly, inhibitor, activator, si RNA gene interference and gene overexpression were used to inhibit or enhance the ADAM17 expression level or activity of PAM and HEK293/CD163 cells, and to detect the shear ability of ADAM17 to porcine CD163. The results showed that when the expression of ADAM17 was down-regulated or the activity was inhibited, the expression of CD163 on the cell surface increased, and when the expression of ADAM17 was up-regulated or activated, the expression of CD163 on the cell surface decreased, indicating that ADAM17 mediated the shearing regulation of porcine CD163. Finally, the effect of CD163 expression mediated by ADAM17 on PRRSV infection was detected in HEK293/CD163 cell line and PAM. The results showed that when the ADAM17 activity was inhibited by ADAM17 inhibitor or the ADAM17 expression was down-regulated by si RNA gene interference, the viral infection increased, and when the ADAM17 activity was activated by LPS or the ADAM17 expression was increased by gene overexpression, the viral infection decreased. These findings suggest that ADAM17 affects the ability of PRRSV invading cells by regulating the expression of porcine CD163 on cell surface. In conclusion, HEK293/CD163 cell lines expressing CD163 stably were obtained by flow cytometry and G418 pressure screening, and PRRSV could be infected and proliferated in HEK293/CD163 cells by indirect immunofluorescence. It was confirmed in HEK293/CD163 cell lines and PAM that ADAM17 affects the ability of PRRSV to infect susceptible cells by regulating the expression of CD163 on the cell surface.
【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】：2015
【分類號】：S855.3
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