【作者】 张敏；

【导师】 童光志；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2022， 博士

【摘要】 非洲猪瘟是一种具有严重危害性和高度致死的猪病毒性传染病,死亡率接近100%。2018年非洲猪瘟传入我国,给我国养猪业造成了巨大的经济损失,我国生猪产业遭受了毁灭性的打击。非洲猪瘟病毒是已知最复杂的病毒之一,该病毒的致病机制和免疫逃逸机制尚不清楚,目前尚无研制成功的商品化非洲猪瘟疫苗。非洲猪瘟病毒(ASFV)的包膜蛋白CD2v与宿主的淋巴细胞的CD2蛋白同源,具有免疫调节作用。但是,CD2v参与影响巨噬细胞功能及免疫的分子机制尚不完全清楚。为了研究非洲猪瘟病毒CD2v蛋白对巨噬细胞的影响。本研究首先利用慢病毒过表达系统构建了稳定表达CD2v的传代猪巨噬细胞系(PAM-CD2v),通过IFA和Western Blot鉴定CD2v蛋白表达正常,并具有吸附红细胞形成玫瑰花环的生物学特性。并通过真核转录本m RNA测序(RNA-seq),分析CD2v依赖性转录本的表达谱和基因差异表达,富集分析差异基因的相关信号通路和表型。结果发现,CD2v表达后引起277个基因表达显著差异,其中有64个基因上调和213个基因下调。基因本体(GO)和基因集富集分析表明,CD2v主要参与生长因子结合、细胞内信号转导、细胞外基质组装、细胞外区域和细胞外空间的基因富集;被CD2v负调控的通路的显着富集包括巨噬细胞来源的泡沫细胞分化、细胞迁移、细胞外基质组成和细胞外基质组装。RNA-seq分析发现CD2v蛋白调节猪巨噬细胞的生物过程。之后本研究对CD2v影响PAM细胞迁移进行了验证。Transwell试验和细胞划痕愈合试验的结果表明,CD2v表达后PAM细胞Transwell穿孔细胞数量与GFP对照组相比明显减少,并呈显著性差异(P<0.001);过表达CD2v的PAM细胞的划痕愈合速度明显慢于对照组,在24 h和48 h两者的划痕宽度呈显著性差异(P<0.001),证明CD2v抑制PAM细胞迁移。RNA-seq分析和FPKM统计结果发现CD2v显著抑制迁移相关基因EGR1的表达,荧光定量PCR及Western Blot的结果证实了CD2v抑制EGR1的表达。为了确定CD2v是否通过下调EGR1的表达抑制PAM细胞的迁移。利用CRISPR-Cas9系统构建EGR1基因敲除的PAM细胞系(PAM-EGR1-KO),并通过测序及Western Blot验证。EGR1基因敲除后抑制了PAM细胞的Transwell穿孔和细胞划痕愈合,在24 h和48 h Transwell穿孔细胞数和划痕宽度与野生型PAM细胞相比呈显著性差异(P<0.001)。过表达EGR1后增强了PAM细胞的Transwell穿孔和细胞划痕愈合。同样,在PAM-CD2v细胞中过表达EGR1也增强了细胞的Transwell穿孔和细胞划痕愈合。综上证明,EGR1蛋白参与调控PAM细胞的迁移,CD2v蛋白通过抑制EGR1表达抑制PAM细胞的迁移。通过EGR1蛋白染色质免疫共沉淀及测序(CHIP-seq)分析,结果显示,EGR1与基因组中38.5%基因的结合位点,位于或靠近启动子区域,对EGR1结合峰与最近转录起始位点(TSS)之间距离的进一步分析表明,86%的EGR1结合峰位于最近的TSS上游或下游1 kb内,表明EGR1在猪巨噬细胞中被募集到这些区域直接调节相邻基因的转录活性。进一步IGV软件分析发现EGR1结合在细胞迁移相关基因的启动子区域,通过荧光定量PCR验证了EGR1调控下游迁移相关分子APP、EPHA4和ID2的表达。本研究又通过将PAM-CD2v细胞与淋巴细胞共培养,发现CD2v通过抑制ERK1/2抑制外周淋巴细胞有丝分裂原活化。ERK1/2是EGR1上游,直接调控EGR1的表达,为了研究CD2v蛋白是否通过ERK1/2调控EGR1的表达。进一步通过Western Blot检测显示,CD2v抑制ERK1/2磷酸化,并且ERK1/2磷酸化水平呈剂量依赖性调控EGR1的表达。ERK1/2能够调控PAM细胞的迁移,ERK1/2磷酸化抑制后减少了PAM细胞的Transwell穿孔和细胞划痕愈合,24 h和48 h Transwell穿孔细胞数和划痕宽度与对照细胞相比呈显著性差异(P<0.001)。综上表明,ERK1/2磷酸化水平直接调控PAM细胞EGR1的表达,CD2v蛋白通过抑制ERK1/2磷酸化下调EGR1抑制PAM细胞迁移。接下来本研究对CD2v蛋白影响PAM细胞炎性因子的产生进行了研究。使用LPS分别刺激PAM-CD2v细胞、ERK1/2磷酸化抑制剂处理后的PAM细胞、EGR1基因敲除PAM细胞、EGR1过表达PAM细胞、EGR1过表达PAM-c CD2v细胞后,使用荧光定量PCR检测炎性因子产生的差异。结果表明,CD2v蛋白通过抑制ERK1/2磷酸化下调EGR1表达抑制PAM细胞炎性因子IL1α、IL1β、IL6、IL8、TNFα的表达。吞噬是巨噬细胞的重要功能,为了研究CD2v蛋白表达后PAM细胞吞噬功能的影响。本研究采用荧光微球细胞吞噬试验,通过流式细胞术检测了PAM-CD2v细胞、EGR1缺失PAM细胞、EGR1过表达PAM细胞吞噬微球后荧光细胞比例。结果表明,CD2v表达后促进了PAM细胞的吞噬。同样,EGR1敲除后PAM的吞噬功能增强,而过表达EGR1后PAM和PAMCD2v细胞吞噬减弱。以上的结果证明CD2v通过抑制EGR1表达增强了PAM细胞的吞噬。综上所述,本研究结果阐明了ASFV的CD2v蛋白影响PAM细胞功能的相关机制。CD2v蛋白表达通过抑制ERK1/2的磷酸化,抑制淋巴细胞有丝分裂原的活化;同时CD2v蛋白表达通过抑制ERK1/2磷酸化下调PAM细胞中转录因子EGR1基因的表达,影响EGR1转录结合下游迁移相关基因的转录起始位点,减少迁移相关因子的转录表达,从而影响PAM细胞的迁移。另外,CD2v蛋白抑制ERK1/2磷酸化下调EGR1基因的表达从而抑制PAM细胞炎性因子IL1α、IL1β、IL6、IL8、TNFα的表达。同样的,CD2v蛋白通过下调EGR1基因的表达增强巨噬细胞的吞噬。这些研究揭示了CD2v影响猪巨噬细胞功能的差异表型和机制,为明确CD2v蛋白的功能提供新的线索及数据支持。更多还原

【Abstract】 African swine fever is a globally devastating and highly lethal infectious disease with a mortality rate approaching 100%.The introduction of African swine fever to China in 2018 caused huge economic losses to swine breeding industry suffered a devastating blow.African swine fever viruse is one of the most complex viruses known.There is no commercial vaccine available,and the pathogenic mechanism and immune escape mechanism of the virus are still unclear.The envelope protein CD2 v of African swine fever virus(ASFV)is homologous to the CD2 protein of host lymphocytes and has immunomodulatory effects.However,the molecular mechanism by wherter CD2 v is involved in affecting macrophage function and immunity is not fully understood.To study the effect of African swine fever virus CD2 v protein on macrophages,in this study,a porcine macrophage cell line(PAM-CD2v)stably expressing CD2 v was constructed by using the lentivirus overexpression system.The CD2 v protein expression was normal by IFA and Western Blot,and it had the biological characteristics of adsorbing red blood cells to form rosettes.The expression profiles and gene differential expression of CD2v-dependent transcripts were analyzed by eukaryotic transcript m RNA-sequencing(RNA-seq),and the related signaling pathways and phenotypes of differential genes were enriched and analyzed.It was found that the expression of CD2 v caused a significant difference in the expression of 277 genes,of which 64 genes were up-regulated and 213 genes were down-regulated.Gene ontology(GO)and gene set enrichment analysis indicated that CD2 v was mainly involved in gene enrichment in growth factor binding,intracellular signal transduction,extracellular matrix assembly,extracellular region and extracellular space;pathways negatively regulated by CD2 v were enriched.Significant enrichments included regulation of macrophage-derived foam cell differentiation,cell migration,extracellular matrix organization,and extracellular matrix assembly.RNA-seq analysis found that CD2 v protein regulates biological processes in porcine macrophages.This study then verified the effect of CD2 v on PAM cell migration.The results of Transwell test and cell scratch healing test showed that the number of Transwell perforated cells of PAM cells after CD2 v expression was significantly reduced compared with the GFP control group,and there was a significant difference(P < 0.001);The scratch healing speed of PAM cells overexpressing CD2 v The healing speed was significantly slower than that of the control group,and the scratch width at 24 h and 48 h was significantly different(P < 0.001),which proved that CD2 v inhibited PAM cell migration.RNA-seq analysis and FPKM statistics showed that CD2 v significantly inhibited the expression of migration-related gene EGR1.The results of real-time PCR and Western Blot confirmed that CD2 v inhibited the expression of EGR1.To determine that CD2 v inhibits the migration of PAM cells by downregulating the expression of EGR1.The EGR1 gene-deleted PAM cell line(PAM-EGR1-KO)was constructed using the CRISPR-Cas9 system,and verified by sequencing and Western Blot.EGR1 gene deletion inhibited the transwell perforation and wound healing of PAM cells,and the number of transwell perforated cells and the width of the scratch at 24 h and 48 h were significantly different from those of wildtype PAM cells(P<0.001).Transwell perforation and cell scratch healing of PAM cells were enhanced by overexpression of EGR1.Likewise,overexpression of EGR1 in PAM-CD2 v cells also enhanced cell transwell punching and cell scratch healing.In conclusion,EGR1 protein is involved in regulating the migration of PAM cells,and CD2 v protein inhibits the migration of PAM cells by inhibiting the expression of EGR1.Through the analysis of EGR1 protein chromatin immunoprecipitation and CHIP-seq sequencing,the results showed that the binding sites of EGR1 and 8793 genes out of 30,440 genes in the genome were located in or close to the promoter region,and the EGR1 binding peak was closely related to the nearest transcription start site.Further analysis of the distance between points(TSS)showed that 86% of EGR1-binding peaks were located within1 kb upstream or downstream of the nearest TSS,suggesting that EGR1 is recruited to these regions in porcine macrophages to directly regulate the transcriptional activity of adjacent genes.Further IGV analysis found that EGR1 binds to the promoter region of cell migration-related genes,and it was verified by real-time quantitative PCR that EGR1 regulates the expression of downstream related migration molecules APP,EPHA4 and ID2.In this study,PAM-CD2 v cells were co-cultured with lymphocytes,and it was found that CD2 v inhibited mitogen activation of peripheral lymphocytes by inhibiting ERK1/2.ERK1/2 is upstream of EGR1 and directly regulates the expression of EGR1.We investigated whether CD2 v protein regulates the expression of EGR1 through ERK1/2.Further detection by Western Blot showed that CD2 v inhibited the phosphorylation of ERK1/2 and inhibited the expression of EGR1 in a dosedependent manner.ERK1/2 can regulate the migration of PAM cells.The inhibition of ERK1/2phosphorylation reduces the transwell perforation and cell wound healing of PAM cells.The number of transwell perforated cells and the width of the scratch at 24 h and 48 h were significantly higher than those of the control cells.Sexual differences(P<0.001).In conclusion,the phosphorylation level of ERK1/2 directly regulates the expression of EGR1 in PAM cells,and CD2 v protein inhibits the migration of PAM cells by inhibiting the phosphorylation of ERK1/2 and downregulating EGR1.Next,this study investigated the effect of CD2 v protein on the production of inflammatory factors in PAM cells.Using LPS to stimulate PAM-CD2 v cells,ERK1/2 phosphorylation inhibitortreated PAM cells,EGR1 gene-deficient PAM cells,EGR1-overexpressing PAM cells,and EGR1-overexpressing PAM-c CD2 v cells,fluorescence quantitative PCR was used to detect inflammation.factor differences.The results showed that CD2 v protein inhibited the expression of inflammatory factors IL1α,IL1β,IL6,IL8 and TNFα in PAM cells by inhibiting the phosphorylation of ERK1/2and down-regulating the expression of EGR1.Phagocytosis is an important function of macrophages,in order to study the effect of CD2 v protein expression on the phagocytic function of PAM cells.In this study,the fluorescent microsphere cell phagocytosis assay was used to detect the proportion of fluorescent cells after PAM-CD2 v cells,EGR1-deficient PAM cells,and EGR1-overexpressing PAM cells phagocytosed microspheres by flow cytometry.The results showed that CD2 v expression promoted the phagocytosis of PAM cells.Similarly,the phagocytosis of PAM was enhanced after EGR1 knockdown,whereas the phagocytosis of PAM cells was attenuated after EGR1 overexpression.The above results demonstrated that CD2 v enhanced the phagocytosis of PAM cells by inhibiting EGR1 expression.Taken together,our findings elucidate the relevant mechanisms by which ASFV’s CD2 v protein affects PAM cell function.CD2 v protein expression inhibits the activation of lymphocyte mitogens by inhibiting the phosphorylation of ERK1/2;at the same time,CD2 v protein expression downregulates the expression of the transcription factor EGR1 gene in PAM cells by inhibiting the phosphorylation of ERK1/2,and affects the transcription of EGR1 and downstream genes.Transcriptional expression of migration-related factors at the start site(TSS),thereby affecting the migration of PAM cells.In addition,CD2 v protein inhibits ERK1/2 phosphorylation and downregulates the expression of EGR1 gene,thereby inhibiting the expression of inflammatory factors IL1α,IL1β,IL6,IL8,and TNFα in PAM cells.Similarly,CD2 v protein enhanced macrophage phagocytosis by downregulating EGR1 gene expression.These studies revealed the differential phenotype and mechanism of CD2 v affecting the function of porcine macrophages,and provided new clues and data support for the study of CD2 v protein.更多还原