本文選題：ICAM-1 + miR-124��； 參考：《華中農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：動(dòng)物肺組織受到細(xì)菌和病毒感染時(shí),機(jī)體內(nèi)的免疫細(xì)胞會(huì)發(fā)生應(yīng)急反應(yīng),以抵抗感染。但該應(yīng)急反應(yīng)過多,會(huì)引起肺組織損傷,導(dǎo)致肺部炎癥和急性肺損傷(ALI),甚至?xí)䦟?dǎo)致急性呼吸窘迫綜合癥(ARDS)。免疫細(xì)胞在肺部的募集是一個(gè)有序的過程,中性粒細(xì)胞是第一波反應(yīng)的免疫細(xì)胞,感染后1~2小時(shí)在肺部募集。募集后的中性粒細(xì)胞,跨內(nèi)皮細(xì)胞層遷移進(jìn)入肺泡,釋放可對(duì)抗入侵細(xì)菌和病毒的物質(zhì)。單核細(xì)胞則是在感染24~48小時(shí)后第二波在肺部募集的免疫細(xì)胞,進(jìn)入肺組織后的單核細(xì)胞可分化為巨噬細(xì)胞和樹突細(xì)胞。巨噬細(xì)胞可通過極化發(fā)揮促炎或抗炎作用,但目前在肺損傷中這種作用還不明確。ICAM-1作為細(xì)胞的黏附分子,在中性粒細(xì)胞募集時(shí)表達(dá)迅速增加,介導(dǎo)中性粒細(xì)胞黏附在毛細(xì)血管壁上,穿越血管內(nèi)皮層而浸潤(rùn)到肺組織中。浸潤(rùn)的中性粒細(xì)胞及受影響的內(nèi)皮細(xì)胞和巨噬細(xì)胞,分泌單核細(xì)胞趨化因子MCP-1進(jìn)一步誘導(dǎo)單核細(xì)胞在肺組織的募集。mi RNA(micro RNA)是小RNA的一種,通過靶向m RNA降解或抑制蛋白的翻譯,在多種生物學(xué)過程中發(fā)揮關(guān)鍵作用。有研究表明,肺組織中mi RNA可以影響MCP-1的表達(dá),而ICAM-1是否能通過mi RNA影響MCP-1的表達(dá),目前還不是很清楚。本研究選取小鼠單核巨噬細(xì)胞作為研究對(duì)象,試圖探討ICAM-1在肺組織中是否通過mi RNA調(diào)節(jié)趨化因子MCP-1的表達(dá),及其對(duì)巨噬細(xì)胞極化的影響。研究?jī)?nèi)容如下:1.ICAM-1敲除對(duì)MCP-1和mi R-124表達(dá)的影響首先我們?cè)贗CAM-1敲除小鼠的肺組織中,發(fā)現(xiàn)MCP-1的蛋白表達(dá)量增加。選擇小鼠單核巨噬細(xì)胞(RAW264.7)轉(zhuǎn)染ICAM-1的si RNA,細(xì)胞中ICAM-1的表達(dá)被抑制后,提高了MCP-1的蛋白表達(dá)。ICAM-1敲除的小鼠肺組織,經(jīng)小RNA測(cè)序后,發(fā)現(xiàn)mi R-124的表達(dá)顯著降低,RT-PCR進(jìn)一步驗(yàn)證了測(cè)序結(jié)果。2.mi RNA-124靶基因的驗(yàn)證經(jīng)生物信息學(xué)軟件預(yù)測(cè)mi R-124在小鼠肺組織的靶點(diǎn),發(fā)現(xiàn)mi R-124能與MCP-1的3’UTR序列互補(bǔ)結(jié)合,MCP-1是mi R-124潛在的靶基因。構(gòu)建包含MCP-13’UTR的野生型、突變型和缺失型雙熒光素酶質(zhì)粒,經(jīng)熒光素酶活性分析,表明MCP-1是mi R-124的靶基因。mi R-124模擬物或抑制劑轉(zhuǎn)染小鼠單核巨噬細(xì)胞,RT-PCR和Western blot檢測(cè)MCP-1的表達(dá),發(fā)現(xiàn)mi R-124能抑制MCP-1的轉(zhuǎn)錄與翻譯。3.轉(zhuǎn)錄因子SP-1對(duì)mi R-124的轉(zhuǎn)錄調(diào)控為了研究mi R-124表達(dá)的分子機(jī)制,克隆小鼠mi R-124的啟動(dòng)子序列,構(gòu)建于PGL3-Basic載體中,在小鼠單核巨噬細(xì)胞中驗(yàn)證其啟動(dòng)子的活性,通過熒光活性分析證實(shí)啟動(dòng)子pre-493區(qū)域內(nèi)含有激活mi R-124轉(zhuǎn)錄的轉(zhuǎn)錄因子。生物信息學(xué)分析結(jié)合位點(diǎn),預(yù)測(cè)SP-1與mi R-124可能結(jié)合的位點(diǎn)。經(jīng)轉(zhuǎn)錄位點(diǎn)突變和凝膠遷移實(shí)驗(yàn),確定了SP-1對(duì)mi R-124的關(guān)鍵作用位點(diǎn)和兩者的相互結(jié)合。4.mi R-124、ICAM-1調(diào)控巨噬細(xì)胞極化LPS(2μg/m L)和IL-4(20ng/m L)分別處理小鼠單核巨噬細(xì)胞,構(gòu)建極化模型。我們發(fā)現(xiàn)LPS誘導(dǎo)細(xì)胞分化為M1型,IL-4誘導(dǎo)細(xì)胞分化為M2型。將mi R-124的模擬物和ICAM-1 si RNA分別轉(zhuǎn)染單核巨噬細(xì)胞,檢測(cè)M1/M2型巨噬細(xì)胞的標(biāo)志分子。我們發(fā)現(xiàn)ICAM-1和mi R-124具有促進(jìn)M2型極化的作用,促進(jìn)標(biāo)志分子ym-1、Mrc-1和IL-10的表達(dá)。表明mi R-124和ICAM-1具有抗炎作用,能抑制小鼠單核細(xì)胞中的炎性反應(yīng)。5.mi R-124對(duì)動(dòng)物體內(nèi)肺損傷的作用本研究通過LPS(10mg/kg)誘導(dǎo)體內(nèi)肺損傷的炎癥模型,確認(rèn)mi R-124對(duì)肺損傷的作用。將mi R-124(2OD)尾靜脈注射小鼠,RT-PCR檢測(cè)mi R-124在肺組織中的表達(dá)增加。然后用LPS誘導(dǎo)小鼠肺損傷,檢測(cè)肺髓過氧化物酶活性(MPO),與單獨(dú)LPS誘導(dǎo)的小鼠相比,mi R-124抑制了MPO的活性,對(duì)肺損傷起到了保護(hù)的作用。PRRSV感染的豬肺組織中,我們發(fā)現(xiàn)ICAM-1和mi R-124的表達(dá)上調(diào),MCP-1的表達(dá)下調(diào)。表明在PRRSV造成的豬肺損傷組織中,ICAM-1、mi R-124與MCP-1的表達(dá)負(fù)相關(guān),積極參與豬肺組織中的炎癥反應(yīng)。綜上所述,本研究通過對(duì)比野生型小鼠和ICAM-1敲除小鼠中MCP-1的表達(dá),經(jīng)mi RNA測(cè)序,差異表達(dá)分析發(fā)現(xiàn)潛在調(diào)控MCP-1的mi RNA,闡明ICAM-1能通過mi R-124調(diào)節(jié)趨化因子MCP-1的蛋白表達(dá)來控制單核細(xì)胞在肺部的募集。ICAM-1不僅是炎癥反應(yīng)分子,還能調(diào)節(jié)MCP-1的蛋白表達(dá),影響肺部組織的免疫反應(yīng)。轉(zhuǎn)錄因子SP-1能激活mi R-124的轉(zhuǎn)錄。在巨噬細(xì)胞中過表達(dá)mi R-124能促進(jìn)巨噬細(xì)胞M2型極化,具有抗炎作用,同時(shí)對(duì)LPS誘導(dǎo)的肺損傷起保護(hù)作用,為深入研究與肺部炎癥和損傷相關(guān)的病理生理機(jī)制提供研究基礎(chǔ)。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.4

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本文編號(hào)：1964636