本文選題：重癥急性胰腺炎 + 肺損傷�。� 參考：《江蘇大學(xué)》2017年博士論文

【摘要】：研究目的:(1)建立重癥急性胰腺炎(SAP)肺損傷小鼠模型,確定肺損傷程度最嚴(yán)重的時(shí)間點(diǎn)。(2)闡明肺泡巨噬細(xì)胞極化與SAP肺損傷之間的關(guān)系。(3)研究干擾素調(diào)節(jié)因子5 (IRF5)在肺泡巨噬細(xì)胞極化調(diào)控中的作用。(4)進(jìn)一步探討體內(nèi)實(shí)驗(yàn)中慢病毒介導(dǎo)的IRF5干擾(IRF5shRNA)對(duì)SAP肺損傷的干預(yù)作用。研究方法:(1) SAP肺損傷小鼠模型的建立及肺損傷程度的評(píng)估:雨蛙素聯(lián)合脂多糖腹腔注射制備SAP肺損傷小鼠模型,通過(guò)胰腺、肺組織病理學(xué)評(píng)分,血清淀粉酶、肺組織濕干重比(W/D)、肺組織髓過(guò)氧化物酶(MPO)活性、丙二醛(MDA)含量、TNF-α和IL-1β檢測(cè)等方法證實(shí)建模成功并進(jìn)行肺損傷程度評(píng)估,進(jìn)一步確定小鼠肺損傷最嚴(yán)重的時(shí)間點(diǎn)。(2)肺泡巨噬細(xì)胞極化與SAP肺損傷之間的關(guān)系:通過(guò)流式細(xì)胞技術(shù)檢測(cè)SAP肺損傷小鼠肺泡灌洗液中巨噬細(xì)胞及其它炎癥細(xì)胞的含量。實(shí)時(shí)定量PCR檢測(cè)巨噬細(xì)胞相關(guān)炎癥因子TNF-α、 IL-1β的含量。通過(guò)免疫組織化學(xué)染色法及激光共聚焦免疫熒光染色法觀察肺泡M1、M2型巨噬細(xì)胞浸潤(rùn)與肺損傷之間的關(guān)系。(3)研究IRF5在肺泡巨噬細(xì)胞極化調(diào)控中的作用:通過(guò)流式細(xì)胞儀分選出肺泡M1、M2型巨噬細(xì)胞,實(shí)時(shí)定量PCR檢測(cè)IRF5在肺泡M1、M2型巨噬細(xì)胞中mRNA水平的表達(dá)。IRF5小干擾RNA (IRF5 siRNA)轉(zhuǎn)染肺泡M1型巨噬細(xì)胞,通過(guò)實(shí)時(shí)定量PCR、Western blotting檢測(cè)轉(zhuǎn)染后M1、M2型巨噬細(xì)胞標(biāo)志性分子mRNA水平和蛋白水平的表達(dá),鑒定其極化狀態(tài)的變化。(4) IRF5 shRNA對(duì)SAP肺損傷保護(hù)作用的研究:制備慢病毒介導(dǎo)的IRF5干擾病毒,并確定干擾效果最顯著的序列作為后續(xù)IRF5基因的干擾片段。將IRF5 shRNA通過(guò)尾靜脈注射感染小鼠,24h后建模,通過(guò)肺組織病理學(xué)評(píng)分,血清淀粉酶、肺組織濕干重比、MPO活性、MDA含量測(cè)定、TNF-α、 IL-1β檢測(cè)和免疫組織化學(xué)染色法、激光共聚焦免疫熒光染色法等方法評(píng)價(jià)肺組織損傷程度,檢測(cè)肺泡巨噬細(xì)胞極化狀態(tài)的改變,評(píng)價(jià)IRF5 shRNA對(duì)SAP肺損傷是否有保護(hù)作用。研究結(jié)果:(1)成功建立了 SAP肺損傷小鼠動(dòng)物模型,確定了 SAP建模后48h為小鼠急性肺損傷最嚴(yán)重的時(shí)間點(diǎn)。(2)肺泡巨噬細(xì)胞的極化狀態(tài)同SAP肺損傷具有密切的聯(lián)系。肺損傷早期(SAP后24h),此時(shí)處于炎癥進(jìn)展期,以M1型肺泡巨噬細(xì)胞占據(jù)優(yōu)勢(shì);肺損傷后期(SAP后96h),炎癥逐漸過(guò)渡到修復(fù)期,M2型肺泡巨噬細(xì)胞數(shù)量明顯增多。(3) IRF5在肺泡M1型巨噬細(xì)胞中的表達(dá)量明顯高于肺泡M2型巨噬細(xì)胞,有顯著性差異(P0.01)。IRF5 siRNA轉(zhuǎn)染肺泡M1型巨噬細(xì)胞后,TNF-α、 IL-12、iNOS mRNA相對(duì)表達(dá)量較未處理M1型巨噬細(xì)胞顯著降低,有顯著性差異(P0.01)。IL-10、Arg-1 mRNA相對(duì)表達(dá)量較對(duì)未處理M1型巨噬細(xì)胞明顯增加,有顯著性差異(P0.01),與未處理M2型巨噬細(xì)胞比較,無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。IRF5 siRNA轉(zhuǎn)染肺泡M1型巨噬細(xì)胞后,巨噬細(xì)胞極化狀態(tài)向M2型巨噬細(xì)胞轉(zhuǎn)換。(4) IRF5 shRNA組同生理鹽水組及Scramble shRNA組比較,肺損傷程度明顯減輕。肺泡M1型巨噬細(xì)胞明顯減少,M2型巨噬細(xì)胞顯著增多,肺泡巨噬細(xì)胞極化狀態(tài)由M1向M2轉(zhuǎn)換。結(jié)論:重癥急性胰腺炎建模后48h為小鼠急性肺損傷程度最嚴(yán)重的時(shí)間點(diǎn)。肺損傷早期階段,巨噬細(xì)胞以M1極化狀態(tài)為主,肺損傷修復(fù)期M2型巨噬細(xì)胞數(shù)量明顯增多。IRF5是鑒別肺泡M1、M2型巨噬細(xì)胞極化狀態(tài)的重要分子標(biāo)志之一,是調(diào)控肺泡巨噬細(xì)胞極化的關(guān)鍵分子。體內(nèi)實(shí)驗(yàn)中,IRF5shRNA通過(guò)促進(jìn)肺泡M1型巨噬細(xì)胞向M2型巨噬細(xì)胞轉(zhuǎn)換,對(duì)重癥急性胰腺炎肺損傷具有明顯的保護(hù)作用。
[Abstract]:Objective: (1) to establish a mouse model of severe acute pancreatitis (SAP) lung injury to determine the most serious time point of lung injury. (2) to clarify the relationship between alveolar macrophage polarization and SAP lung injury. (3) to study the role of interferon regulator 5 (IRF5) in the polarization regulation of alveolar macrophage cells. (4) to further explore the slow in vivo experiment. The intervention of virus mediated IRF5 interference (IRF5shRNA) on SAP lung injury. (1) establishment of a mouse model of SAP lung injury and evaluation of the degree of lung injury: intraperitoneal injection of LPS combined with lipopolysaccharide to prepare a mouse model of SAP lung injury, through the pancreas, lung tissue disease score, serum amylase, wet dry weight ratio (W/D) of lung tissue (W/D), Lung Group The myeloperoxidase (MPO) activity, the content of malondialdehyde (MDA), TNF- alpha and IL-1 beta test confirmed the success of the modeling and the assessment of the degree of lung injury. (2) the relationship between alveolar macrophage polarization and SAP lung injury: the detection of alveolar alveoli in mice with SAP lung injury by flow cytometry The content of macrophages and other inflammatory cells in the lavage fluid. Real-time quantitative PCR was used to detect the content of macrophage related inflammatory factors TNF- alpha and IL-1 beta. The relationship between pulmonary alveolar M1, M2 macrophage infiltration and lung injury was observed by immunohistochemical staining and laser confocal immunofluorescence staining. (3) the study of IRF5 in alveolar megagocytosis was conducted. The role of the cell polarization regulation: the alveolar M1, M2 type macrophages were selected through the flow cytometry. The real-time quantitative PCR detection of IRF5 in the alveolar M1, the mRNA level of the M2 type macrophages was expressed as.IRF5 small interference RNA (IRF5 siRNA) transfected to the alveolar M1 type macrophages. The expression of mRNA level and protein level of the chronicles to identify the changes in polarization state. (4) the study of the protective effect of IRF5 shRNA on SAP lung injury: preparing the IRF5 interfering virus mediated by the lentivirus, and determining the most significant sequence of the interference effect as the interference segment of the subsequent IRF5 gene. IRF5 shRNA is injected into the tail vein to infect mice, 24h After modeling, the pulmonary tissue pathological score, serum amylase, wet dry weight ratio of lung tissue, MPO activity, MDA content, TNF- alpha, IL-1 beta detection and immunohistochemistry, laser confocal immunofluorescence staining, and laser confocal immunofluorescence staining were used to evaluate the damage of lung tissue, to detect the change of polarization state of alveolar macrophages, and to evaluate the IRF5 shRNA to SAP Whether the lung injury has protective effect. (1) the mice model of SAP lung injury was successfully established, and the most serious time point of acute lung injury after SAP modeling was determined. (2) the polarization state of alveolar macrophages is closely related to the lung injury. The early lung injury (SAP after 24h), at this time in the progression of inflammation, M1 Type of alveolar macrophage occupies the dominant position in the late stage of lung injury (SAP after 96h), the inflammation gradually transition to the period of repair, the number of M2 type alveolar macrophages increased significantly. (3) the expression of IRF5 in the alveolar M1 macrophages is significantly higher than that of the alveolar M2 macrophages, and there is a significant difference (P0.01).IRF5 siRNA transfection to the alveolar M1 macrophages, TNF- alpha, IL- 12, the relative expression of iNOS mRNA was significantly lower than that of the untreated M1 macrophages. There was a significant difference (P0.01).IL-10, and the relative expression of Arg-1 mRNA was significantly higher than that of the untreated M1 type macrophages. There was a significant difference (P0.01). There was no statistical significance compared with the non treated M2 macrophages (P0.05). After that, the polarization of macrophages was converted to M2 type macrophages. (4) compared with the saline group and the Scramble shRNA group, the degree of lung injury was significantly reduced in the IRF5 shRNA group. The alveolar macrophages were significantly reduced, the M2 type macrophages increased significantly, and the polarization state of alveolar macrophages was converted from M1 to M2. Conclusion: 48h after severe acute pancreatitis (SAP) was modeled as 48h. The most serious time point of acute lung injury in mice. At the early stage of lung injury, macrophages were mainly M1 polarization state, and the number of M2 macrophages increased significantly in the repair period of lung injury..IRF5 was one of the important molecular markers to identify the polarization state of alveolar M1 and M2 macrophages. It was the key molecule to regulate the polarization of alveolar macrophages. In the experiment, IRF5shRNA can protect lung alveolar macrophages from severe acute pancreatitis by promoting the transformation of alveolar type M1 macrophages to M2 macrophages.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R576

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