IRF5调控的肺泡巨噬细胞极化在SAP肺损伤中作用的实验研究

发布时间：2018-07-02 09:53

本文选题：重症急性胰腺炎 + 肺损伤　；参考：《江苏大学》2017年博士论文

【摘要】：研究目的:(1)建立重症急性胰腺炎(SAP)肺损伤小鼠模型,确定肺损伤程度最严重的时间点。(2)阐明肺泡巨噬细胞极化与SAP肺损伤之间的关系。(3)研究干扰素调节因子5 (IRF5)在肺泡巨噬细胞极化调控中的作用。(4)进一步探讨体内实验中慢病毒介导的IRF5干扰(IRF5shRNA)对SAP肺损伤的干预作用。研究方法:(1) SAP肺损伤小鼠模型的建立及肺损伤程度的评估:雨蛙素联合脂多糖腹腔注射制备SAP肺损伤小鼠模型,通过胰腺、肺组织病理学评分,血清淀粉酶、肺组织湿干重比(W/D)、肺组织髓过氧化物酶(MPO)活性、丙二醛(MDA)含量、TNF-α和IL-1β检测等方法证实建模成功并进行肺损伤程度评估,进一步确定小鼠肺损伤最严重的时间点。(2)肺泡巨噬细胞极化与SAP肺损伤之间的关系:通过流式细胞技术检测SAP肺损伤小鼠肺泡灌洗液中巨噬细胞及其它炎症细胞的含量。实时定量PCR检测巨噬细胞相关炎症因子TNF-α、 IL-1β的含量。通过免疫组织化学染色法及激光共聚焦免疫荧光染色法观察肺泡M1、M2型巨噬细胞浸润与肺损伤之间的关系。(3)研究IRF5在肺泡巨噬细胞极化调控中的作用:通过流式细胞仪分选出肺泡M1、M2型巨噬细胞,实时定量PCR检测IRF5在肺泡M1、M2型巨噬细胞中mRNA水平的表达。IRF5小干扰RNA (IRF5 siRNA)转染肺泡M1型巨噬细胞,通过实时定量PCR、Western blotting检测转染后M1、M2型巨噬细胞标志性分子mRNA水平和蛋白水平的表达,鉴定其极化状态的变化。(4) IRF5 shRNA对SAP肺损伤保护作用的研究:制备慢病毒介导的IRF5干扰病毒,并确定干扰效果最显著的序列作为后续IRF5基因的干扰片段。将IRF5 shRNA通过尾静脉注射感染小鼠,24h后建模,通过肺组织病理学评分,血清淀粉酶、肺组织湿干重比、MPO活性、MDA含量测定、TNF-α、 IL-1β检测和免疫组织化学染色法、激光共聚焦免疫荧光染色法等方法评价肺组织损伤程度,检测肺泡巨噬细胞极化状态的改变,评价IRF5 shRNA对SAP肺损伤是否有保护作用。研究结果:(1)成功建立了 SAP肺损伤小鼠动物模型,确定了 SAP建模后48h为小鼠急性肺损伤最严重的时间点。(2)肺泡巨噬细胞的极化状态同SAP肺损伤具有密切的联系。肺损伤早期(SAP后24h),此时处于炎症进展期,以M1型肺泡巨噬细胞占据优势;肺损伤后期(SAP后96h),炎症逐渐过渡到修复期,M2型肺泡巨噬细胞数量明显增多。(3) IRF5在肺泡M1型巨噬细胞中的表达量明显高于肺泡M2型巨噬细胞,有显著性差异(P0.01)。IRF5 siRNA转染肺泡M1型巨噬细胞后,TNF-α、 IL-12、iNOS mRNA相对表达量较未处理M1型巨噬细胞显著降低,有显著性差异(P0.01)。IL-10、Arg-1 mRNA相对表达量较对未处理M1型巨噬细胞明显增加,有显著性差异(P0.01),与未处理M2型巨噬细胞比较,无统计学意义(P0.05)。IRF5 siRNA转染肺泡M1型巨噬细胞后,巨噬细胞极化状态向M2型巨噬细胞转换。(4) IRF5 shRNA组同生理盐水组及Scramble shRNA组比较,肺损伤程度明显减轻。肺泡M1型巨噬细胞明显减少,M2型巨噬细胞显著增多,肺泡巨噬细胞极化状态由M1向M2转换。结论:重症急性胰腺炎建模后48h为小鼠急性肺损伤程度最严重的时间点。肺损伤早期阶段,巨噬细胞以M1极化状态为主,肺损伤修复期M2型巨噬细胞数量明显增多。IRF5是鉴别肺泡M1、M2型巨噬细胞极化状态的重要分子标志之一,是调控肺泡巨噬细胞极化的关键分子。体内实验中,IRF5shRNA通过促进肺泡M1型巨噬细胞向M2型巨噬细胞转换,对重症急性胰腺炎肺损伤具有明显的保护作用。
[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.
【学位授予单位】：江苏大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R576

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