miR-124介导ICAM-1对MCP-1的调节作用和对巨噬细胞极化的影响

发布时间：2018-06-01 15:15

本文选题：ICAM-1 + miR-124　；参考：《华中农业大学》2017年博士论文

【摘要】：动物肺组织受到细菌和病毒感染时,机体内的免疫细胞会发生应急反应,以抵抗感染。但该应急反应过多,会引起肺组织损伤,导致肺部炎症和急性肺损伤(ALI),甚至会导致急性呼吸窘迫综合症(ARDS)。免疫细胞在肺部的募集是一个有序的过程,中性粒细胞是第一波反应的免疫细胞,感染后1~2小时在肺部募集。募集后的中性粒细胞,跨内皮细胞层迁移进入肺泡,释放可对抗入侵细菌和病毒的物质。单核细胞则是在感染24~48小时后第二波在肺部募集的免疫细胞,进入肺组织后的单核细胞可分化为巨噬细胞和树突细胞。巨噬细胞可通过极化发挥促炎或抗炎作用,但目前在肺损伤中这种作用还不明确。ICAM-1作为细胞的黏附分子,在中性粒细胞募集时表达迅速增加,介导中性粒细胞黏附在毛细血管壁上,穿越血管内皮层而浸润到肺组织中。浸润的中性粒细胞及受影响的内皮细胞和巨噬细胞,分泌单核细胞趋化因子MCP-1进一步诱导单核细胞在肺组织的募集。mi RNA(micro RNA)是小RNA的一种,通过靶向m RNA降解或抑制蛋白的翻译,在多种生物学过程中发挥关键作用。有研究表明,肺组织中mi RNA可以影响MCP-1的表达,而ICAM-1是否能通过mi RNA影响MCP-1的表达,目前还不是很清楚。本研究选取小鼠单核巨噬细胞作为研究对象,试图探讨ICAM-1在肺组织中是否通过mi RNA调节趋化因子MCP-1的表达,及其对巨噬细胞极化的影响。研究内容如下:1.ICAM-1敲除对MCP-1和mi R-124表达的影响首先我们在ICAM-1敲除小鼠的肺组织中,发现MCP-1的蛋白表达量增加。选择小鼠单核巨噬细胞(RAW264.7)转染ICAM-1的si RNA,细胞中ICAM-1的表达被抑制后,提高了MCP-1的蛋白表达。ICAM-1敲除的小鼠肺组织,经小RNA测序后,发现mi R-124的表达显著降低,RT-PCR进一步验证了测序结果。2.mi RNA-124靶基因的验证经生物信息学软件预测mi R-124在小鼠肺组织的靶点,发现mi R-124能与MCP-1的3’UTR序列互补结合,MCP-1是mi R-124潜在的靶基因。构建包含MCP-13’UTR的野生型、突变型和缺失型双荧光素酶质粒,经荧光素酶活性分析,表明MCP-1是mi R-124的靶基因。mi R-124模拟物或抑制剂转染小鼠单核巨噬细胞,RT-PCR和Western blot检测MCP-1的表达,发现mi R-124能抑制MCP-1的转录与翻译。3.转录因子SP-1对mi R-124的转录调控为了研究mi R-124表达的分子机制,克隆小鼠mi R-124的启动子序列,构建于PGL3-Basic载体中,在小鼠单核巨噬细胞中验证其启动子的活性,通过荧光活性分析证实启动子pre-493区域内含有激活mi R-124转录的转录因子。生物信息学分析结合位点,预测SP-1与mi R-124可能结合的位点。经转录位点突变和凝胶迁移实验,确定了SP-1对mi R-124的关键作用位点和两者的相互结合。4.mi R-124、ICAM-1调控巨噬细胞极化LPS(2μg/m L)和IL-4(20ng/m L)分别处理小鼠单核巨噬细胞,构建极化模型。我们发现LPS诱导细胞分化为M1型,IL-4诱导细胞分化为M2型。将mi R-124的模拟物和ICAM-1 si RNA分别转染单核巨噬细胞,检测M1/M2型巨噬细胞的标志分子。我们发现ICAM-1和mi R-124具有促进M2型极化的作用,促进标志分子ym-1、Mrc-1和IL-10的表达。表明mi R-124和ICAM-1具有抗炎作用,能抑制小鼠单核细胞中的炎性反应。5.mi R-124对动物体内肺损伤的作用本研究通过LPS(10mg/kg)诱导体内肺损伤的炎症模型,确认mi R-124对肺损伤的作用。将mi R-124(2OD)尾静脉注射小鼠,RT-PCR检测mi R-124在肺组织中的表达增加。然后用LPS诱导小鼠肺损伤,检测肺髓过氧化物酶活性(MPO),与单独LPS诱导的小鼠相比,mi R-124抑制了MPO的活性,对肺损伤起到了保护的作用。PRRSV感染的猪肺组织中,我们发现ICAM-1和mi R-124的表达上调,MCP-1的表达下调。表明在PRRSV造成的猪肺损伤组织中,ICAM-1、mi R-124与MCP-1的表达负相关,积极参与猪肺组织中的炎症反应。综上所述,本研究通过对比野生型小鼠和ICAM-1敲除小鼠中MCP-1的表达,经mi RNA测序,差异表达分析发现潜在调控MCP-1的mi RNA,阐明ICAM-1能通过mi R-124调节趋化因子MCP-1的蛋白表达来控制单核细胞在肺部的募集。ICAM-1不仅是炎症反应分子,还能调节MCP-1的蛋白表达,影响肺部组织的免疫反应。转录因子SP-1能激活mi R-124的转录。在巨噬细胞中过表达mi R-124能促进巨噬细胞M2型极化,具有抗炎作用,同时对LPS诱导的肺损伤起保护作用,为深入研究与肺部炎症和损伤相关的病理生理机制提供研究基础。
[Abstract]:When the lung tissue is infected by bacteria and viruses, the immune cells in the body can respond to the infection to resist the infection. But the emergency response is too much, causing lung tissue damage, causing lung inflammation and acute lung injury (ALI), and even causing acute respiratory distress syndrome (ARDS). The recruitment of immune cells in the lungs is an orderly way. In the process, neutrophils are the immune cells of the first wave, and they are collected at 1~2 hours after infection. The neutrophils are raised in the lungs. The neutrophils migrate into the alveoli and release the substances that can antagonize the invading bacteria and the virus. The monocytes are the immune cells raised in the lungs by the second wave after 24~48 hours infection and into the lung tissue. The post monocytes can differentiate into macrophages and dendritic cells. Macrophages can play an inflammatory or anti-inflammatory role through polarization, but the role of.ICAM-1 as a cell adhesion molecule is not yet clear in lung injury, and the expression of neutrophils is rapidly increased in the recruitment of neutrophils, and mediates the adhesion of neutrophils on the capillary wall. The vascular endothelium infiltrates into the lung tissue. Infiltrating neutrophils and affected endothelial cells and macrophages, the secretory monocyte chemoattractant factor MCP-1 further induces the recruitment of mononuclear cells in the lung tissue,.Mi RNA (micro RNA) is a small RNA, by targeting m RNA to reduce or inhibit the translation of protein, in a variety of biological processes. Some studies have shown that MI RNA in lung tissue can affect the expression of MCP-1, and whether ICAM-1 can affect the expression of MCP-1 through mi RNA is not yet clear. This study selected mononuclear macrophages in mice as a study to explore whether ICAM-1 can regulate chemokine MCP-1 by Mi RNA in lung tissue. The effect of 1.ICAM-1 on the expression of MCP-1 and MI R-124 in the lung tissue of ICAM-1 knockout mice, we found that the protein expression of MCP-1 was increased. The Si RNA of ICAM-1 in mice was selected, and the ICAM-1 expression in the cells was inhibited and increased. MCP-1 protein expression.ICAM-1 knockout mice lung tissue, after small RNA sequencing, it was found that the expression of MI R-124 decreased significantly. RT-PCR further verified the sequencing results of the.2.mi RNA-124 target gene to predict the target of MI R-124 in the lung tissue of the MI R-124. P-1 is a potential target gene for MI R-124. We construct a wild, mutant and missing double luciferase plasmid containing MCP-13 'UTR. By luciferase activity analysis, it shows that MCP-1 is a.Mi R-124 analogue or inhibitor of the target gene of MI R-124, which is transfected to mononuclear macrophages in mice. Transcriptional regulation of MCP-1 and translation of.3. transcriptional factor SP-1 to MI R-124 in order to study the molecular mechanism of MI R-124 expression, clone the promoter sequence of MI R-124 in mice, construct in PGL3-Basic vector, verify the activity of its promoter in mouse mononuclear macrophages, and confirm the pre-493 region of promoter by fluorescence activity analysis. There are transcriptional factors that activate mi R-124 transcription. Bioinformatics analysis binding sites predict the possible binding sites of SP-1 and MI R-124. Through transcriptional site mutation and gel migration experiments, the key sites of action of SP-1 to MI R-124 and the combination of both.4.mi R-124, ICAM-1 regulated macrophage polarization LPS (2 mu) The mouse mononuclear macrophages were treated respectively, and the polarization model was constructed. We found that LPS induced cells differentiated into M1 type and IL-4 induced cells differentiated into M2 type. Mi R-124 analogue and ICAM-1 Si RNA were transfected to mononuclear macrophages respectively to detect the marker molecules of M1/M2 macrophages. Effect, promote the expression of marker molecules YM-1, Mrc-1 and IL-10. It shows that MI R-124 and ICAM-1 have anti-inflammatory effects and can inhibit the inflammatory response of.5.mi R-124 in mononuclear cells of mice. The effect of LPS (10mg/kg) on lung injury in vivo induced by LPS (10mg/kg) is used to confirm the effect of Mi R-124 on lung injury. After the tail vein injection mice, RT-PCR detected the expression of MI R-124 in the lung tissue. Then LPS induced lung injury and pulmonary myeloperoxidase activity (MPO) was detected. Compared with the LPS induced mice, MI R-124 inhibited the activity of MPO. In the lung tissue of the lung injury that protects the.PRRSV infected lung tissue, we found ICAM-1 and The expression of MI R-124 is up and the expression of MCP-1 is down. It indicates that the expression of ICAM-1, MI R-124 and MCP-1 is negatively related to the expression of ICAM-1, MI R-124 and MCP-1 in the tissues of swine lung injury caused by PRRSV. To sum up, the expression of MCP-1 in the wild type and ICAM-1 knockout mice is compared by Mi RNA sequencing and differential expression analysis. Mi RNA, which regulates the potential regulation of MCP-1, shows that ICAM-1 can regulate the protein expression of chemokine MCP-1 through mi R-124 to control the recruitment of.ICAM-1 not only to the inflammatory response molecules in the lung, but also to regulate the protein expression of MCP-1 and to affect the immune response of the lung tissue. The transcription factor SP-1 activates the MI R-124 transcription. In macrophages, the transcription factor SP-1 can be activated in macrophages. The expression of MI R-124 can promote the M2 polarization of macrophages, have anti-inflammatory effects and protect the lung injury induced by LPS, and provide a basis for the in-depth study of pathophysiological mechanisms associated with lung inflammation and injury.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.4

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