番鸭呼肠孤病毒感染的分子致病机制的转录组学研究

发布时间：2018-11-18 19:01

【摘要】：番鸭呼肠孤病毒(Muscovy duck reovirus, MDRV)是近10年来发现的引起雏番鸭较高发病率和死亡率的病毒。肝脏和脾脏是MDRV感染的主要靶器官。为探讨MDRV感染的分子致病机制,本研究以肝脏和脾脏为主要研究对象,应用转录组学方法,对10日龄番鸭转录组进行测序、组装和注释；筛选和分析了MDRV感染后肝脏和脾脏的差异表达基因,并对差异表达基因进行了功能分析；最后对MDRV感染激活机体先天性免疫功能,诱导细胞凋亡的分子机制和导致肝脏脂肪变性的分子机制进行了分析和验证。研究结果阐明了MDRV感染的分子致病机理,主要结果如下：1.本研究得到总长度17G的转录组序列,组装得到65,535个Unigenes,平均长度987bp; COG注释共获得41,651个Unigenes的注释,包含25个生物学功能。2. MDRV感染5日龄雏番鸭后第5天,差异表达基因筛选发现,脾脏有1,352个基因表达量上调,4,906个基因表达量下调；GO显著性富集分析表明,这些基因共参与了48个生物学功能,KEGG显著性富集229个Pathway。3.差异表达基因筛选发现肝脏有4,190个基因表达量上调,1,113个基因下调。GO显著性富集分析表明,这些基因共参与了48个生物学功能,KEGG显著性富集237个Pathway。4. MDRV感染肝脏和脾脏差异基因的比较分析发现,脾脏的差异表达基因以下调为主,肝脏以上调为主,两个器官的共有差异表达基因1,350个,GO功能分析表明,这些共有差异表达基因参与了42个生物学功能,KEGG功能分析表明,共有差异表达基因涉及的免疫功能的有炎症因子,先天性免疫功能,抗原提呈过程等。共有差异表达基因的获得将为进一步阐明MDRV感染的致病机理提供了有力的依据。5. KEGG分析表明,MDRV感染,上调了脾脏中模式识别受体RIG-I, MDA5, TLR-1, TLR-2, TLR-4的表达来识别MDRV,活化IRF7促进Ⅰ型IFN的分泌和调节NF-kB信号通路促进炎症因子IL6的分泌,激活JAK-STAT信号,刺激IFN的分泌,增强机体先天性免疫功能。结果经Q-PCR验证,证实转录组数据准确可靠。6. TUNEL法和流式细胞术证实,MDRV感染诱导了肝脏细胞凋亡。KEGG分析表明,MDRV感染通过激活了Fas信号通路,IL-1R信号通路,抑制了PI3K/AKT信号通路诱导细胞凋亡。Western blot和Q-PCR验证表明,转录组数据准确可靠。7. MDRV感染肝脏中脂肪酸含量极显著的增加(P0.01)，，从而诱导肝脏脂肪变性。KEGG分析表明,MDRV感染抑制了肝细胞中胆固醇外排和脂肪酸分解代谢关键酶蛋白基因的表达,从而导致脂肪酸和胆固醇在肝细胞中的蓄积,结果阐明了MDRV感染肝脏发生脂肪变性的分子机制。综上,本研究运用转录组学方法,阐明了MDRV感染肝脏和脾脏的差异表达基因和肝脏、脾脏的共有差异表达基因,分析了这些差异表达基因的生物学功能,同时分析和验证了MDRV激活宿主先天性免疫功能,诱导细胞凋亡和肝脏脂肪变性的分子机制。可见,宿主调动了多个基因和多条通路共同参与调控MDRV感染过程。
[Abstract]:Muscovy duck reovirus (Muscovy duck reovirus, MDRV) is a virus found in recent 10 years to cause higher morbidity and mortality in young muscovy ducks. Liver and spleen are the main target organs of MDRV infection. In order to study the molecular pathogenesis of MDRV infection, the transcriptome of 10-day-old muscovy duck was sequenced, assembled and annotated by transcriptome method. The differentially expressed genes in liver and spleen after MDRV infection were screened and analyzed, and the function of differentially expressed genes was analyzed. Finally, the molecular mechanism of MDRV infection which activates innate immune function, induces apoptosis and leads to hepatic steatosis is analyzed and verified. The main results are as follows: 1. In this study, a total length of 17G transcriptome sequence was obtained, and 65535 Unigenes, average lengths were 987 BP, while 41651 Unigenes annotations were obtained, including 25 biological functions. 2. On the 5th day after MDRV infection, 1352 genes were up-regulated and 4906 genes were down-regulated in spleen. GO significant enrichment analysis showed that these genes were involved in 48 biological functions, and KEGG significantly enriched 229 Pathway.3.. Screening of differentially expressed genes revealed that 4190 genes were up-regulated and 113 genes down-regulated in liver. GO significant enrichment analysis showed that these genes participated in 48 biological functions, and KEGG significantly enriched 237 Pathway.4.. The comparative analysis of differentially expressed genes in liver and spleen of MDRV infection showed that the differential expression genes of spleen were mainly down-regulated and that of liver were up-regulated. There were 1350 differentially expressed genes in the two organs. GO functional analysis showed that there were 1350 differentially expressed genes in the two organs. These co-differentially expressed genes were involved in 42 biological functions. KEGG functional analysis showed that the common differentially expressed genes involved inflammatory factors, congenital immune function, antigen presentation process and so on. The acquisition of common differentially expressed genes will provide a powerful basis for further elucidating the pathogenic mechanism of MDRV infection. KEGG analysis showed that MDRV infection upregulated the expression of pattern recognition receptor (RIG-I, MDA5, TLR-1, TLR-2, TLR-4) in spleen to recognize MDRV,. Activation of IRF7 promoted the secretion of type I IFN and regulated the NF-kB signaling pathway, promoted the secretion of inflammatory factor IL6, activated JAK-STAT signal, stimulated the secretion of IFN, and enhanced the innate immune function of the body. Results the transcriptional data were confirmed to be accurate and reliable by Q-PCR. 6. 6%. TUNEL assay and flow cytometry confirmed that MDRV infection induced apoptosis of liver cells. KEGG analysis showed that MDRV infection activates Fas signaling pathway, IL-1R signal pathway. The inhibition of PI3K/AKT signaling pathway induced apoptosis by. Western blot and Q-PCR showed that the transcriptional data were accurate and reliable. The fatty acid content in the liver of MDRV infection was significantly increased (P0.01), which induced hepatic steatosis. KEGG analysis showed that MDRV infection inhibited the expression of key enzymes in cholesterol excretion and fatty acid catabolism in hepatocytes. The accumulation of fatty acids and cholesterol in liver cells may lead to the molecular mechanism of fatty degeneration in MDRV infected liver. In summary, we used transcriptome method to elucidate the differentially expressed genes in the liver and spleen of MDRV infection, and analyze the biological functions of these differentially expressed genes in the liver and spleen. At the same time, the molecular mechanism that MDRV activates innate immune function, induces apoptosis and steatosis of liver is analyzed and verified. It can be seen that the host involved in the regulation of MDRV infection by multiple genes and multiple pathways.
【学位授予单位】：福建师范大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S858.32

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