ADAM17介导的CD163表达调控对PRRSV感染的影响
发布时间：2018-12-16 01:07
【摘要】：猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)是由猪繁殖与呼吸综合征病毒(PRRSV)引起的严重危害养猪业的传染病之一。根据核酸序列的差异,PRRSV被划分为欧洲型和美洲型毒株。2006年5月以来,我国暴发的被称为“高热综合症”的猪病,即为变异的美洲型高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)所致。猪肺泡巨噬细胞(PAM)是PRRSV入侵猪体的靶细胞,已发现在PAM上存在三种主要的PRRSV细胞受体:硫酸乙酰肝素(HS)、唾液酸粘附素(Sn)和清道夫受体CD163。HS的作用是吸附PRRSV到细胞表面,Sn可以加强吸附作用并介导PRRSV内吞进入胞内,CD163则是随PRRSV一同进入胞内介导病毒的脱衣壳和病毒基因组的释放。近年研究发现,将猪源CD163分子转染至PRRSV非允许细胞,使得细胞稳定表达CD163蛋白后就成为PRRSV允许细胞,能够成功感染增殖PRRSV,而其他两种受体则不能,说明CD163在病毒感染细胞过程中至关重要。作为清道夫受体超家族的成员,CD163参与机体对异物的免疫应答并参与炎症反应,其在细胞表面的表达受多种因素的影响,其中去整合素金属蛋白酶ADAM17已被证实能够介导人源CD163的剪切调控,从而调节炎症反应。但是ADAM17能否介导猪源CD163的剪切调控从而影响病毒的入侵还不得而知,因此本研究阐释了ADAM17介导CD163的表达调控机制及其对PRRSV感染的影响。首先,从PAM细胞中扩增了猪源CD163基因,克隆至真核表达质粒pc DNA3.1-CD163,在测序正确后将其转染HEK293细胞,通过流式细胞术和G418加压筛选,获得稳定表达CD163的HEK293/CD163细胞系;病毒感染实验表明PRRSV能够感染HEK293/CD163细胞系,为研究病毒的体外感染奠定了基础。其次,通过抑制剂、激活剂、si RNA基因干扰和基因过表达等方法来抑制或者增强PAM和HEK293/CD163细胞的ADAM17表达水平或活性,检测ADAM17对猪源CD163的剪切能力。研究结果发现,当ADAM17表达下调或者活性受到抑制时,细胞表面CD163的表达水平升高;当ADAM17表达上调或者被激活时,细胞表面CD163的表达水平降低,说明ADAM17介导猪源CD163的剪切调控。最后,在HEK293/CD163细胞系和PAM进行接毒实验,测定ADAM17介导的CD163表达对PRRSV感染的影响。实验结果发现,当用ADAM17抑制剂抑制ADAM17活性或者通过si RNA基因干扰下调ADAM17表达时,病毒感染量增多;当用LPS激活ADAM17活性或通过基因过表达提高ADAM17表达时,病毒感染量减少。上述研究发现表明ADAM17通过调控细胞表面猪源CD163表达水平从而影响PRRSV入侵细胞的能力。总之,本研究通过流式细胞术和G418加压筛选方法获得了稳定表达CD163的HEK293/CD163细胞系,并通过间接免疫荧光发鉴定了PRRSV能够感染该细胞系并在HEK293/CD163细胞中增殖。在HEK293/CD163细胞系和PAM上证实了ADAM17通过调控细胞表面CD163的表达水平,从而影响PRRSV对易感细胞的感染能力。
[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.
【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】：2015
【分类号】：S855.3
本文编号：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.
【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】：2015
【分类号】：S855.3
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