【作者】 莫红缨；

【导师】 萧正伦；

【作者基本信息】 广州医学院 ， 呼吸内科学， 2008， 博士

【摘要】 目的:本研究构建巨噬细胞移动抑制因子(MIF) siRNA,并探讨其对脂多糖/流感病毒介导的急性肺损伤的炎症反应和免疫反应的影响,及相关信号转导机制的作用,探索急性肺损伤治疗的新模式。研究内容和方法:第一部分:MIF siRNA在人肺泡上皮细胞A549的转染和干扰目的基因MIF表达将MIF siRNA用转染剂INTERFERin介导转染体外培养的A549细胞,通过RT- PCR方法观察不同浓度MIF siRNA转染后,对MIF mRNA表达的影响;采用细胞免疫荧光方法分析MIF蛋白表达的强弱;应用MTT方法测定MIF siRNA转染对细胞的毒性,及对LPS刺激A549细胞生存率的影响。第二部分:MIFsiRNA对脂多糖刺激/流感病毒感染人肺泡上皮细胞Toll样受体及其下游信号转导通路的影响离体研究:运用RT-PCR、Western-Blot、细胞免疫荧光和ELISA方法,分别从mRNA、蛋白、介质水平三个层面检测了LPS刺激/流感病毒感染人肺泡上皮细胞A549后TLR2、TLR3、TLR4的mRNA表达;下游MyD88依赖/非依赖信号转导通路元件MyD88和IRF3蛋白表达;核因子NF-κB的核迁移;细胞培养上清炎症介质TNF-α、IL-1β、IL-6,免疫介质IFN-β分泌水平;以及MIF siRNA对其表达的影响。第三部分:MIF siRNA对脂多糖/流感病毒介导的小鼠急性肺损伤的保护作用及相关的信号转导机制动物研究:建立LPS/流感病毒(H1N1)介导的急性肺损伤小鼠模型,运用EMSA、Western-Blot、免疫组化、ELISA和相关病理技术,观察LPS/H1N1介导的急性肺损伤炎症介质TNF-α、IL-1β、IL-6释放的情况,肺组织炎症病理变化,肺组织水通道蛋白AQP1、AQP4表达与肺部感染、水肿关系,以及NF-κB和丝裂原活化蛋白激酶(MAPK)2条信号转导通路中NF-κB、IκB-α、p-p38/p38、p-ERK/ERK、p-JNK/JNK表达;同时探讨MIF siRNA对脂多糖/流感病毒介导的小鼠急性肺损伤的保护作用及相关的信号转导机制。结果:第一部分:MIF siRNA在人肺泡上皮细胞A549的转染和干扰目的基因MIF表达1. MIF siRNA转染体外培养的A549细胞后,可以明显降低MIF mRNA的表达;且不同浓度(0nM、10nM、50nM、100nM)MIF siRNA对细胞进行转染,只有当MIF siRNA浓度大于50nM时,才能明显降低MIF mRNA的表达。2. MIF siRNA转染后,A549细胞MIF的绿色荧光蛋白表达也明显降低,在此基础上,再加入MIF刺激,MIF的蛋白表达又明显增加。3.转染50nM MIF siRNA后,对A549细胞几乎没有毒性;且对LPS刺激A549细胞导致细胞死亡及细胞的生存率下降有一定的保护作用。第二部分:MIF siRNA对脂多糖刺激/流感病毒感染人肺泡上皮细胞Toll样受体及其下游信号转导通路的影响1.1 LPS刺激A549细胞6h时,细胞因子MIF、TLR2、TLR4 mRNA的表达显著增加,分别加入MIF siRNA+DXM、MIF siRNA和DXM的3组干预组,均可下调MIF、TLR2、TLR4 mRNA的表达;而LPS刺激对TLR3 mRNA表达无影响。1.2 H1N1感染A549细胞16h时,细胞因子MIF、TLR3 mRNA的表达显著增加;分别加入MIF siRNA+RBVR、MIF siRNA、RBVR、MIF siRNA+DXM、DXM的5组干预组,均可下调MIF、TLR3 mRNA的表达;而H1N1感染对TLR2、TLR4 mRNA表达无影响。其中MIF siRNA+DXM干预组有协同下调LPS刺激/H1N1感染引起的MIF mRNA高表达的作用。2.1 LPS刺激A549细胞6h时,MyD88和IRF3蛋白表达显著增加;分别加入MIF siRNA+DXM、MIF siRNA和DXM的3组干预组,对MyD88表达均有下调作用,而仅MIF siRNA+ DXM和DXM干预组对IRF3表达有抑制作用,单纯MIF siRNA对IRF3表达无影响。2.2 H1N1感染A549细胞16h时,MyD88和IRF3蛋白表达显著增加;分别加入MIF siRNA+RBVR、MIF siRNA、RBVR、MIF siRNA+DXM、DXM的5组干预组,对MyD88表达均有下调作用,同样仅MIF siRNA+ DXM和DXM干预组对IRF3表达有抑制作用,其余的3组干预组对IRF3表达无影响。3.LPS刺激8h时/或H1N1感染24h时,MyD88在A549细胞的绿色荧光蛋白的表达增多和增强,加入MIF siRNA干预,可下调MyD88蛋白的表达;LPS刺激45min时/或H1N1感染6h时,NF-κB在A549细胞发生核迁移,加入MIF siRNA干预,可部分阻断NF-κB核迁移。4.1 LPS刺激6h时,细胞培养上清TNF-α、IL-1β、IL-6和IFN-β分泌水平显著增加;分别加入MIF siRNA+DXM、MIF siRNA和DXM的3组干预组,均可以不同程度降低炎症介质TNF-α、IL-1β和IL-6的分泌水平。4.2 H1N1感染24h时,细胞培养上清TNF-α、IL-1β、IL-6和IFN-β分泌水平显著增加;分别加入MIF siRNA+RBVR、MIF siRNA、RBVR、MIF siRNA+DXM、DXM的5组干预组,均可以不同程度降低炎症介质TNF-α、IL-1β和IL-6的分泌水平。其中仅MIF siRNA+DXM和DXM2组干预组对LPS刺激/H1N1感染引起的IFN-β高分泌水平有抑制作用。第三部分:MIF siRNA对脂多糖/流感病毒介导的小鼠急性肺损伤的保护作用及相关的信号转导机制1.LPS刺激2h时,或H1N1感染24h时,均可引起小鼠肺组织NF-κB在细胞核的表达增加,IκB-α在细胞浆的表达下降;LPS刺激时加入MIF siRNA、DXM和MIF siRNA +DXM的3组治疗组, H1N1感染时加入MIF siRNA、RBVR、MIF siRNA+RBVR、DXM和MIF siRNA+DXM的5组治疗组,均可不同程度下调NF-κB表达和上调IκB-α表达,其中MIF siRNA+DXM治疗组对IκB-α的上调作用最大。2.1 LPS刺激2h时,磷酸化的p-p38、p-ERK、p-JNK蛋白表达显著增加,对非磷酸化的p38、ERK、JNK蛋白表达无影响;在LPS刺激下,分别加入MIF siRNA、DXM和MIF siRNA+DXM的3组治疗组,均可以不同程度下调p-p38和p-JNK的表达,但3组治疗组均对p-ERK表达无影响。2.2 H1N1感染24h时,仅磷酸化的p-ERK蛋白表达显著增加;在H1N1感染下,分别加入MIF siRNA、RBVR、MIF siRNA+RBVR、DXM和MIF siRNA+DXM的5组治疗组,也只有RBVR和MIF siRNA+RBVR的2组治疗组,可下调p-ERK的表达。3.LPS刺激,或H1N1感染,小鼠肺脏湿重增加,显微镜下可见肺泡正常结构破坏,间质增厚,炎性细胞浸润,上皮细胞和血管内皮细胞的肿胀脱落;LPS刺激时加入MIF siRNA、DXM和MIF siRNA +DXM的3组治疗组,H1N1感染时加入MIF siRNA、RBVR、MIF siRNA+RBVR、DXM和MIF siRNA+DXM的5组治疗组,均能减轻小鼠肺部病理损伤和降低肺组织湿干重比。4.与正常组比较,LPS刺激组和H1N1感染组肺组织血管微血管内皮、支气管周围血管床、气管粘膜上皮(AQP1)及肺组织大小气管上皮和肺泡(AQP4)可见明显减少的棕黄色阳染;经上述治疗组治疗后,AQP1和AQP4的表达有所增强。5.在LPS刺激,或H1N1感染,小鼠肺匀浆的炎症介质TNF-α、IL-1β、IL-6分泌水平均显著增加;经上述治疗组治疗后,均可以不同程度降低炎症介质TNF-α、IL-1β和IL-6的分泌水平。结论:1.线性阳离子聚合物jetPEI能成功转运MIF siRNA到达目的细胞,MIF siRNA能在A549细胞特异沉默目的基因MIF mRNA和蛋白的表达,且对LPS刺激导致细胞死亡有一定的保护作用。2.首次证实MIF siRNA均能阻断LPS刺激A549细胞诱导的TLR4或H1N1感染A549细胞诱导的TLR3高表达,阻断下游信号转导通路元件MyD88,抑制炎症介质TNF-α、IL-1β、IL-6过度分泌。MIF siRNA和地塞米松(DXM)对下游信号转导通路的影响存在差异,DXM均可阻断MyD88和IRF3的合成,既抑制炎症反应又抑制免疫反应;而MIF siRNA仅阻断MyD88合成,对IRF3表达无影响。3.在LPS/H1N1介导的小鼠急性肺损伤实验中,MIF siRNA对LPS/H1N1诱导激活的NF-κB有明显抑制作用,在MAPK信号转导通路,首次证实MIF siRNA对LPS诱导的p-p38和p-JNK高表达有下调作用,从而抑制炎症介质TNF-α、IL-1β、IL-6释放,减轻小鼠肺组织病理损伤。MIF siRNA能减轻脂多糖或流感病毒介导的急性肺损伤,可能是其特异沉默目的基因MIF,遏制肺损伤的多种信号转导通路的激活,抑制炎症反应。更多还原

【Abstract】 Objective:Macrophage migration inhibitory factor (MIF) plays an important role not only in the immune system but also in inflammation response. In this study, we used siRNA to knockdown MIF mRNA for evaluation of the role of MIF siRNA in acute lung injury (ALI) induced by LPS and influenza A virus (H1N1), and further examined the effect of MIF siRNA on the signal transduction system.Contents and methods:Part 1: Transfection of MIF siRNA in human alveolar epithial cells and its interference impact on targeting geneAfter MIF siRNA was transfected into human alveolar epithial cells (A549 cells) with INTERFERin, RT-PCR was carried out to assess the dose-response of MIF siRNA’s effect on MIF mRNA expression, Cell Immumofluorescence Method was used to analyze the expression of MIF protein, MTT Cell Proliferation Assay was performed to detect the cytotoxicity of MIF siRNA to A549 cells, and the survival rate of A549 cells caused by LPS.Part 2: Effect of MIF siRNA on the expression of Toll-like receptors and down stream signaling components in A549 cellsIn vitro: we use following methods, including RT-PCR, Western-Blot, Cell Immunofluorescence Method and ELISA, and with three level, such as mRNA, protein and medium, in A549 cells stimulated by LPS or infected by influenza A virus, to assess the expression of TLR2, TLR3, TLR4 mRNA, to analyse the expression of MyD88 and IRF3 protein in down stream MyD88 -dependent and independent signaling pathways, to observed NF-κB (p65) nuclear translocation, to detect released level of TNF-α, IL-1β, IL-6 in the supernatant, and further evaluated the effect of MIF siRNA on above biological index.Part 3: Effects of MIF siRNA on acute lung injury induced by LPS or H1N1 and the signal pathways involved in the modulationIn vivo: Mouse models of ALI were made by injection of LPS into abdominal cavity, or inoculation of H1N1 into intranasally. We use following methods, including ELISA, EMSA, Western-Blot, Immunohistochemical staining and HE stained for histopathological examination, to detect the released level of TNF-α, IL-1β, IL-6 in lung homogenates, to observed the histopathological manifestation of the lung, to assess the expression of aquapn (AQP1, AQP4) in lung tissue and to correlate with inflammatory cells infiltration, lung edema, then to analyse the expression of NF-κB, IκB-α, p-p38/p38, p-ERK/ERK, p-JNK/JNK protein in signal transcription pathway of NF-κB and MAPK. In addition, we further examined the effect of MIF siRNA on acute lung injury induced by LPS or H1N1, and on these signal transduction system.Results:Part 1: Transfection of MIF siRNA in human alveolar epithial cells and its interference impact on targeting gene1. Once the MIF siRNA was transfected into the A549 cells, it could obviously reduce the MIF mRNA expression. But only the concentration over 50nM, it did.2. After MIF siRNA was transfected into A549 cells, the expression of MIF’s green fluorescent protein reduced greatly, but could be reversed by MIF.3. Almost no toxicity to A549 cells when the concentration of MIF siRNA was low 50nM, but also it could reduce the cell mortality and survival rate caused by LPS. Part 2: Effect of MIF siRNA on the expression of Toll-like receptors and down stream signaling components in A549 cells1. The expression of MIF, TLR2, TLR4 mRNA increase greatly when the LPS stimulated A549 cells up to 6h, and the adding of MIF siRNA+DXM, MIF siRNA and DXM respectively could downregulate the expression of MIF, TLR2, TLR4 mRNA. But LPS did not work to TLR3 mRNA.2. MIF, TLR3 mRNA level increase greatly when the H1N1 infected A549 cells for 16h, and the adding of MIF siRNA+RBVR, MIF siRNA, RBVR, MIF siRNA+DXM, DXM respectively could also downregulate the expression of MIF, TLR3 mRNA. But H1N1 did not work to TLR2, TLR4 mRNA.It is known that the downregulation of MIF mRNA expression is the lowest in the group with the combination of MIF siRNA and DXM among all groups.3. The expression of MyD88 and IRF3 protein increase greatly when the LPS stimulated A549 cells up to 6h, and the adding of MIF siRNA+DXM, MIF siRNA and DXM respectively could downregulate the expression of MyD88. Only MIF siRNA+ DXM and DXM could downregulate the express of IRF3. But MIF siRNA did not work to express of IRF3.4. The expression of MyD88 and IRF3 protein also increase greatly after infection with H1N1 for 16h, and the adding of MIF siRNA+RBVR, MIF siRNA, RBVR, MIF siRNA+DXM, DXM respectively could downregulate the expression of MyD88. Except MIF siRNA+ DXM and DXM could downregulate the expression of IRF3, the other groups no effect on IRF3.5. When LPS stimulated A549 cells to 8h or H1N1 infected A549 cells for 24h, the expression of MyD88 green fluorescent protein was more and strong, but could be reversed by MIF siRNA. If A549 cells stimulated by LPS for 45min or infected by H1N1 for 6h, translocation of NF-κB (P65) took place from cytosol to nucleus, the adding of MIF siRNA could partially knockdown NF-κB translocation.6. The released levels of TNF-α, IL-1β, IL-6 and IFN-βin supernatant increase greatly when the LPS stimulated up to 6h, and the adding of MIF siRNA+DXM、MIF siRNA and DXM respectively could downregulate the released levels of TNF-α, IL-1β, IL-6.7. TNF-α, IL-1β, IL-6 and IFN-βlevel in supernatant were very similar to stimulation with LPS. All of them were markedly increasing by H1N1 infection. The adding of MIF siRNA+RBVR, MIF siRNA, RBVR, MIF siRNA+DXM, DXM respectively could downregulate the released levels of TNF-α, IL-1β, IL-6.Except the siRNA+DXM and DXM2 could downregulate IFN-βlevel, the other groups no effect on IFN-βrelease. Part 3: Effects of MIF siRNA on acute lung injury induced by LPS or H1N1 and the signal pathways involved in the modulation1. Stimulation with LPS for 2h, or infection with N1H1 for 24h, it could increase greatly the expression of NF-κB in nucleus, and decrease markedly the expression of IκB-αin cytosol. Adding of MIF siRNA, DXM, MIF siRNA +DXM during stimulation with LPS, and adding of MIF siRNA, RBVR, MIF siRNA+RBVR, DXM and MIF siRNA+DXM in H1N1 infection could downregulate the expression of NF-κB and upregulate the expression of IκB-α. These data indicate that the upregulation of IκB-αexpression is the highest in the group with the combination of MIF siRNA and DXM among all groups.2. Phosphorylation of p-p38, p-ERK, p-JNK were rapidly increasing by stimulation with LPS for 2h. But no effect on the expression of p38, ERK, JNK, steady-state expression levels were found in them. Adding of MIF siRNA, DXM and MIF siRNA+DXM could downregulate the expression of p-p38 and p-JNK, but did not work to the expression of p-ERK.3. In contrast, there was no significant activation phosphorylation of p-p38, p-JNK in H1N1 infection throughout the time course. Only phosphorylation of p-ERK was rapidly increasing by infection with H1N1 for 24h. Adding MIF siRNA, RBVR, MIF siRNA+RBVR, DXM and MIF siRNA+DXM, the expression of p-ERK was downregulated only by RBVR, MIF siRNA+RBVR.4. Stimulation with LPS or infection with N1H1, It showed that increased lung wet weight, obvious alveolar destruction, mesenchyma infiltration with inflammation cells, epithelial and vascular endothelial swelling and shedding were demonstrated. Adding of MIF siRNA, DXM, MIF siRNA +DXM during stimulation with LPS, and adding of MIF siRNA, RBVR, MIF siRNA+RBVR, DXM and MIF siRNA+DXM in H1N1 infection could attenuate the severity of lung injury and decrease rate of lung wet/dry weight.5. Compare with control, immunostaining examination showed weaker staining against AQP1 in capillary endothelial cells, and against AQP4 in bronchial sub- epithelial basement membrane in mice’s lung by stimulation with LPS or infection with H1N1. After treatment, AQP1 and AQP4 staining were increased to some extent.6. The released levels of TNF-α, IL-1β, IL-6 were increased greatly by stimulation with LPS or infection with H1N1. After treatment, all of them were downregulated in a certain extent.Clonclusion:1. MIF siRNA could be transfected into the target cell by linear cationic polymers jetPEI, and it was special silence the express of target gene MIF mRNA and protein in the A549 cell. Therefore MIF siRNA might exert its safeguard to the death of cell induced by LPS.2. The study provide the first evidence that in A549 cell, MIF siRNA could block the expression both of TLR4 induced by LPS and of TLR3 infected by H1N1, further it could block the expression of MyD88 protein in down stream signaling pathways, and inhibit the secretion of the inflammation cytokine such as TNF-α, IL-1β,IL-6. There were different effect on the down stream signaling pathways between MIF siRNA and Dexamethasone(DXM), DXM could block the expression both of MyD88 and IRF3, and inhibit the reaction of inflammation and immune, but MIF siRNA only blocked the expression of MyD88, and no effect to the expression of IRF3.3. In mouse of acute lung injure induced by LPS/H1N1, MIF siRNA could inhibit the expression of NF-κB. In the MAPK signaling pathways, it was also the first evidence that MIF siRNA was able to downregulate the expression of p-p38 and p-JNK, and lead to inhibit the releasing of inflammation cytokine of TNF-α,IL-1β,IL-6, attenuate the severity of lung injure.MIF siRNA can ease the symptoms of acute lung injure induced by LPS or influenza A virus, such function maybe ground on their special silence gene MIF to inhibit the triggers of mutiplex signal transcription pathway during acute lung injure.更多还原