PHEV感染对小胶质细胞的激活及其分泌炎症因子的影响研究
本文选题:猪血凝性脑脊髓炎病毒 切入点:小胶质细胞 出处:《吉林大学》2015年硕士论文 论文类型:学位论文
【摘要】:为了探究嗜神经性冠状病毒-猪血凝性脑脊髓炎病毒(PHEV)感染过程中小胶质细胞的应答反应,本研究分别将PHEV接种小鼠和小胶质细胞(BV2细胞)进行了体内和体外实验,以验证PHEV感染对小胶质细胞的激活及其分泌炎症因子的影响,为更全面地揭示PHEV感染过程中小胶质细胞发挥的免疫功能及其内在机制奠定基础。 体内实验以PHEV易感的BALB/c小鼠为实验动物模型,以小胶质细胞表面特异性标记物Iba1蛋白为检测对象,利用荧光定量PCR、Western Blot、免疫组化和间接免疫荧光等方法,分别从基因转录水平、蛋白表达水平和组织学水平对PHEV感染后不同时间点小鼠脑内小胶质细胞的增殖、活化情况进行了检测分析,同时还利用基因芯片技术对PHEV感染后小鼠大脑皮质的多种炎症因子表达情况进行了检测。结果表明,滴鼻途径接种PHEV成功建立PHEV感染小鼠脑组织模型,为探究PHEV感染后脑内小胶质细胞是否发生激活提供坚实基础。与空白对照组相比,接毒组小鼠脑组织内的Iba1基因、蛋白表达量均有升高,且均随着感染时间延长而增大。将采集的小鼠脑组织制作石蜡切片进行免疫组化和间接免疫荧光检测,结果证实PHEV的感染激活了小胶质细胞,表现为细胞数量增多,呈聚集样分布;同时细胞形态变化也很明显,胞体变大并呈阿米巴状。上述实验证实PHEV感染后小鼠脑内小胶质细胞发生了明显增殖、活化现象,且小胶质细胞的激活可能是持续性进行的,伴随PHEV感染的整个发病过程。基因芯片结果证实PHEV感染后小鼠大脑皮质内IL-1β、IL-6、TNF-α、IFN-α、IFN-β、CXCL10、CCL2等炎症因子表达出现不同程度的上调,特别是CXCL10、CCL2表达量在PHEV感染后变化极为显著。 体外实验选择与原代小鼠小胶质细胞特征相似的BV2细胞为研究对象,对PHEV是否感染BV2细胞和PHEV刺激后BV2细胞Iba1、多种炎症因子及多种细胞信号通路相关蛋白表达情况进行了检测,并分析了它们之间的内在联系。间接免疫荧光结果表明接种PHEV后BV2细胞中未检测到明显的PHEV病毒粒子,但标记Iba1的红色荧光强度出现一定程度的增强;荧光定量PCR结果表明,PHEV活毒株、灭活毒株接种BV2细胞后,病毒基因表达均随接种时间延长而逐渐降低,这可能和病毒粒子发生降解和病毒未感染BV2细胞而不能进行复制均有关系;因此,我们认为PHEV不能感染BV2细胞。荧光定量PCR证实PHEV活毒株、灭活毒株均可刺激BV2细胞Iba1基因表达上调;Western Blot结果证实PHEV刺激后BV2细胞Iba1蛋白表达显著性上调;上述实验证实PHEV虽不能感染BV2细胞,但能有效刺激BV2细胞发生活化。荧光定量RCR结果表明,PHEV刺激BV2细胞活化后,IL-1β、IL-6、TNF-α、IFN-α和IFN-β等炎症因子表达出现不同程度的上调,而CXCL10、CCL2表达变化不显著。ELISA结果表明PHEV刺激BV2细胞分泌IL-1β、IL-6、TNF-α、IFN-α增加。此外,对某些细胞信号通路相关蛋白进行了荧光定量PCR检测,,结果显示,虽然PHEV刺激BV2细胞表达TLR4、TLR9和RIG-I变化不显著,但TLR7、MyD88、MDA5、NF-κB、ASC、Caspase-1等表达显著性上调,为揭示其介导BV2细胞表达上述炎症因子的内在机制奠定基础。 PHEV刺激BV2细胞IL-1β、IL-6、TNF-α、IFN-α、IFN-β等炎症因子的表达与PHEV感染后小鼠大脑皮质结果趋于一致,均出现显著性上调,但BV2细胞表达不明显的CXCL10、CCL2在小鼠大脑皮质中表达尤为明显,说明小胶质细胞作为CNS中炎症因子的重要来源,在PHEV引发的病毒性脑炎进程中发挥重要作用;但毕竟小胶质细胞在CNS中含量较少,其他胶质细胞、神经元细胞、甚至某些透过血脑屏障的外周免疫细胞也参与CNS的炎性进程,故阐明小胶质细胞在PHEV引发的病毒性脑炎中发挥的作用还需更深入的研究。
[Abstract]:In order to explore the neurotropic coronavirus hemagglutinating encephalomyelitis virus (PHEV) response of microglia infection in this study were PHEV inoculated mice and microglial cells (BV2 cells) by in vivo and in vitro experiments, to verify the effect of PHEV infection on microglia activation and secretion of inflammatory factors and lay the foundation for more fully reveal the immune function of PHEV infected microglia and their internal mechanism.
In vivo experiments with PHEV susceptible BALB/c mice as experimental animal model to microglial cell surface specific marker protein Iba1 as detection object, using fluorescence quantitative PCR, Western Blot, immunohistochemistry and indirect immunofluorescence method, respectively, from the level of gene transcription, protein expression and histological level on the proliferation of microglia cells at different time points in mice after infection with PHEV activation were detected and analyzed, at the same time using the gene chip technology to a variety of inflammatory cytokines in mouse cerebral cortex expression was detected after PHEV infection. The results show that intranasal inoculation of PHEV was successfully established in brain tissue of mice model of PHEV infection, in order to investigate the infection of PHEV brain microglia is activated to provide a solid foundation. Compared with the control group, Iba1 poisoning group gene in the brain tissue of mice, the protein expression was increased, and Increased with the prolongation of infection. The collected mouse brain tissue paraffin sections by immunohistochemistry and indirect immunofluorescence. Results confirmed that PHEV infection activates microglial cells showed cell number showed aggregation distribution; at the same time, the morphological changes of cells obviously, larger cell body and showed ameboid the experiment demonstrated that PHEV infected microglia in the brain of mice had obvious proliferation, activation, and activated microglia may be sustained by the following PHEV infection in the pathogenesis of gene chip. The results showed that IL-1 beta, within the cerebral cortex of mice infected with PHEV IL-6, TNF- alpha, alpha IFN- IFN-, CXCL10, CCL2 beta, expression of inflammatory factors such as different degrees of increase, especially in CXCL10, the expression of CCL2 in PHEV after infection vary significantly.
In vitro selection and primary mouse microglia features similar to BV2 cells as the research object, the BV2 cell Iba1 PHEV BV2 infection and PHEV cells after stimulation, the expression of a variety of inflammatory factors and many cellular signaling pathway related proteins were detected, and the analysis of the relationship between them. The indirect immunofluorescence results showed that inoculation after PHEV BV2 was not detected in cells of PHEV virus particle appears obvious, but red fluorescence intensity of labeled Iba1 enhanced; fluorescence quantitative PCR results showed that PHEV strain of inactivated virus after inoculation of BV2 cells, and decreased gradually with the prolongation of inoculation of viral gene expression, which may degrade and virus particles and the virus has not infected BV2 cells but not duplicating relations; therefore, we think that PHEV can infect BV2 cells. Fluorescence quantitative PCR confirmed that PHEV live virus, inactivated virus Strains can stimulate BV2 cell expression of Iba1 gene; Western Blot confirmed that BV2 cells Iba1 protein was up-regulated after PHEV stimulation; the experiment demonstrated that PHEV can infect BV2 cells, but can effectively stimulate BV2 cell life. Fluorescence quantitative RCR results showed that PHEV stimulated the activation of BV2 cells, IL-1 beta, IL-6 the expression of IFN-, TNF- alpha, alpha and beta IFN- and other inflammatory factors appear different degrees of increase, and CXCL10, CCL2 expression did not change significantly.ELISA results showed that PHEV stimulated secretion of IL-1 beta, BV2 cell IL-6, TNF- alpha, IFN- increased. In addition, some of the cell signal pathway related proteins were detected by fluorescence quantitative PCR. The results showed that although the expression of TLR4 PHEV and RIG-I TLR9 stimulated BV2 cells did not change significantly, but TLR7, MyD88, MDA5, ASC, NF- kappa B, Caspase-1 expression was significantly elevated, to reveal the internal mediated expression of BV2 cells on the inflammatory factors The system lays the foundation.
PHEV stimulation of BV2 cells IL-6, IL-1 beta, TNF- alpha, IFN- alpha, IFN- beta expression of inflammatory cytokines and PHEV infection in mice after cerebral cortex is consistent, there was a significant increase, but no obvious CXCL10 expression of BV2 cells, the expression of CCL2 is obvious in the cerebral cortex of mice, indicating microglia as an important the source of inflammatory cytokines in CNS, play an important role in PHEV induced viral encephalitis in the process; but after all, microglial cells in CNS were less and the other glial cells, neurons, and even some through blood brain barrier peripheral immune cells are involved in the inflammatory process of CNS, use of viral encephalitis to clarify microglia in PHEV caused by the role needs further research.
【学位授予单位】:吉林大学
【学位级别】:硕士
【学位授予年份】:2015
【分类号】:S855.3
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