發(fā)布時(shí)間：2018-03-18 12:42

  本文選題：豬血凝性腦脊髓炎病毒　切入點(diǎn)：小膠質(zhì)細(xì)胞　出處：《吉林大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：為了探究嗜神經(jīng)性冠狀病毒-豬血凝性腦脊髓炎病毒（PHEV）感染過(guò)程中小膠質(zhì)細(xì)胞的應(yīng)答反應(yīng)，本研究分別將PHEV接種小鼠和小膠質(zhì)細(xì)胞（BV2細(xì)胞）進(jìn)行了體內(nèi)和體外實(shí)驗(yàn)，以驗(yàn)證PHEV感染對(duì)小膠質(zhì)細(xì)胞的激活及其分泌炎癥因子的影響，為更全面地揭示PHEV感染過(guò)程中小膠質(zhì)細(xì)胞發(fā)揮的免疫功能及其內(nèi)在機(jī)制奠定基礎(chǔ)。 體內(nèi)實(shí)驗(yàn)以PHEV易感的BALB/c小鼠為實(shí)驗(yàn)動(dòng)物模型，以小膠質(zhì)細(xì)胞表面特異性標(biāo)記物Iba1蛋白為檢測(cè)對(duì)象，利用熒光定量PCR、Western Blot、免疫組化和間接免疫熒光等方法，分別從基因轉(zhuǎn)錄水平、蛋白表達(dá)水平和組織學(xué)水平對(duì)PHEV感染后不同時(shí)間點(diǎn)小鼠腦內(nèi)小膠質(zhì)細(xì)胞的增殖、活化情況進(jìn)行了檢測(cè)分析，同時(shí)還利用基因芯片技術(shù)對(duì)PHEV感染后小鼠大腦皮質(zhì)的多種炎癥因子表達(dá)情況進(jìn)行了檢測(cè)。結(jié)果表明，滴鼻途徑接種PHEV成功建立PHEV感染小鼠腦組織模型，為探究PHEV感染后腦內(nèi)小膠質(zhì)細(xì)胞是否發(fā)生激活提供堅(jiān)實(shí)基礎(chǔ)。與空白對(duì)照組相比，接毒組小鼠腦組織內(nèi)的Iba1基因、蛋白表達(dá)量均有升高，且均隨著感染時(shí)間延長(zhǎng)而增大。將采集的小鼠腦組織制作石蠟切片進(jìn)行免疫組化和間接免疫熒光檢測(cè)，結(jié)果證實(shí)PHEV的感染激活了小膠質(zhì)細(xì)胞，表現(xiàn)為細(xì)胞數(shù)量增多，呈聚集樣分布；同時(shí)細(xì)胞形態(tài)變化也很明顯，胞體變大并呈阿米巴狀。上述實(shí)驗(yàn)證實(shí)PHEV感染后小鼠腦內(nèi)小膠質(zhì)細(xì)胞發(fā)生了明顯增殖、活化現(xiàn)象，且小膠質(zhì)細(xì)胞的激活可能是持續(xù)性進(jìn)行的，伴隨PHEV感染的整個(gè)發(fā)病過(guò)程�；蛐酒Y(jié)果證實(shí)PHEV感染后小鼠大腦皮質(zhì)內(nèi)IL-1β、IL-6、TNF-α、IFN-α、IFN-β、CXCL10、CCL2等炎癥因子表達(dá)出現(xiàn)不同程度的上調(diào)，特別是CXCL10、CCL2表達(dá)量在PHEV感染后變化極為顯著。 體外實(shí)驗(yàn)選擇與原代小鼠小膠質(zhì)細(xì)胞特征相似的BV2細(xì)胞為研究對(duì)象，對(duì)PHEV是否感染BV2細(xì)胞和PHEV刺激后BV2細(xì)胞Iba1、多種炎癥因子及多種細(xì)胞信號(hào)通路相關(guān)蛋白表達(dá)情況進(jìn)行了檢測(cè)，并分析了它們之間的內(nèi)在聯(lián)系。間接免疫熒光結(jié)果表明接種PHEV后BV2細(xì)胞中未檢測(cè)到明顯的PHEV病毒粒子，但標(biāo)記Iba1的紅色熒光強(qiáng)度出現(xiàn)一定程度的增強(qiáng)；熒光定量PCR結(jié)果表明，PHEV活毒株、滅活毒株接種BV2細(xì)胞后，病毒基因表達(dá)均隨接種時(shí)間延長(zhǎng)而逐漸降低，這可能和病毒粒子發(fā)生降解和病毒未感染BV2細(xì)胞而不能進(jìn)行復(fù)制均有關(guān)系；因此，我們認(rèn)為PHEV不能感染BV2細(xì)胞。熒光定量PCR證實(shí)PHEV活毒株、滅活毒株均可刺激BV2細(xì)胞Iba1基因表達(dá)上調(diào)；Western Blot結(jié)果證實(shí)PHEV刺激后BV2細(xì)胞Iba1蛋白表達(dá)顯著性上調(diào)；上述實(shí)驗(yàn)證實(shí)PHEV雖不能感染BV2細(xì)胞，但能有效刺激BV2細(xì)胞發(fā)生活化。熒光定量RCR結(jié)果表明，PHEV刺激BV2細(xì)胞活化后，IL-1β、IL-6、TNF-α、IFN-α和IFN-β等炎癥因子表達(dá)出現(xiàn)不同程度的上調(diào)，而CXCL10、CCL2表達(dá)變化不顯著。ELISA結(jié)果表明PHEV刺激BV2細(xì)胞分泌IL-1β、IL-6、TNF-α、IFN-α增加。此外，對(duì)某些細(xì)胞信號(hào)通路相關(guān)蛋白進(jìn)行了熒光定量PCR檢測(cè)，，結(jié)果顯示，雖然PHEV刺激BV2細(xì)胞表達(dá)TLR4、TLR9和RIG-I變化不顯著，但TLR7、MyD88、MDA5、NF-κB、ASC、Caspase-1等表達(dá)顯著性上調(diào)，為揭示其介導(dǎo)BV2細(xì)胞表達(dá)上述炎癥因子的內(nèi)在機(jī)制奠定基礎(chǔ)。 PHEV刺激BV2細(xì)胞IL-1β、IL-6、TNF-α、IFN-α、IFN-β等炎癥因子的表達(dá)與PHEV感染后小鼠大腦皮質(zhì)結(jié)果趨于一致，均出現(xiàn)顯著性上調(diào)，但BV2細(xì)胞表達(dá)不明顯的CXCL10、CCL2在小鼠大腦皮質(zhì)中表達(dá)尤為明顯，說(shuō)明小膠質(zhì)細(xì)胞作為CNS中炎癥因子的重要來(lái)源，在PHEV引發(fā)的病毒性腦炎進(jìn)程中發(fā)揮重要作用；但畢竟小膠質(zhì)細(xì)胞在CNS中含量較少，其他膠質(zhì)細(xì)胞、神經(jīng)元細(xì)胞、甚至某些透過(guò)血腦屏障的外周免疫細(xì)胞也參與CNS的炎性進(jìn)程，故闡明小膠質(zhì)細(xì)胞在PHEV引發(fā)的病毒性腦炎中發(fā)揮的作用還需更深入的研究。
[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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S855.3

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