IL-33参与烧冲复合伤人与小鼠肺损伤机制的研究

发布时间：2018-05-23 06:20

  本文选题：烧冲复合伤 + 冲击伤　； 参考：《南开大学》2013年博士论文

【摘要】：目的：烧冲复合伤是军事冲突、恐怖袭击、交通事故等突发事件中最常见的损伤类型之一。烧冲复合伤后肺脏损伤导致即时死亡的发生率高达47%。急性肺损伤(ALI)和急性呼吸窘迫综合症(ARDS)是烧冲复合伤早期死亡的主要原因之一,烧冲复合伤后即刻引起低血容量性休克与炎症级联反应。大量研究结果显示在ALI/ARDS的发病过程中,细胞因子与中性粒细胞发挥了关键性的作用。IL-33是新近发现的一种细胞因子,属于IL-1家族。IL-33mRNA表达于人类与小鼠的多个器官和不同类型的细胞,在蛋白水平,IL-33主要表达在上皮细胞、内皮细胞和纤维细胞。IL-33能够促进中性粒细胞趋化至感染部位减轻脓毒症。然而,在烧冲复合伤后肺组织IL-33与中性粒细胞是否存在这种机制仍不清楚。因此,我们建立重度烧冲复合伤动物模型,探讨在烧冲复合伤动物模型与烧冲复合伤患者中IL-33是否参与肺损伤。 方法：利用野生型C57BL/6小鼠与IL-33转基因小鼠分别建立烧冲复合伤、冲击伤、烧伤动物模型,小鼠随机分为正常对照组,烧伤组,冲击伤组,烧冲复合伤组。通过免疫组织化学法分别检测肺组织髓过氧化物酶(myeloperoxidase,MPO)、白介素-33(IL-33)、G蛋白偶联受体-2(GRK2)、趋化因子CXCR2的表达。透射电镜(transmission electron microscopy)观察肺组织超微结构的变化。real-time RT-PCR检测IL-33mRNA与GRK2mRNA的表达。酶联免疫吸附实验(ELISA)检测血浆IL-6与TNF-a浓度。Micro-CT观察肺组织的变化。同时,通过免疫组织化学法检测烧冲复合伤患者伤后24小时肺组织MPO、IL-33的表达。酶联免疫吸附实验检测烧冲复合伤患者血浆IL-6与TNF-a浓度。胸部X-ray观察肺组织的变化。 结果： 1.我们以5g8701炸药为爆炸源,距离爆炸源43cm,53cm,63cm分别建立冲击伤动物模型,压力值分别为200Kpa,94Kpa,88.3Kpa,根据冲击伤诊断标准确定中度冲击伤距离为53cm。 2.我们的动物模型表明烧冲复合伤后肺组织严重损伤。与正常对照组小鼠肺组织肉眼观相比,烧伤组小鼠肺组织轻度充血；冲击伤组小鼠肺组织中度充血,可见约3处点灶状出血；复合伤组小鼠肺组织明显充血,可见约5处斑点状出血；烧冲复合伤组小鼠伤后24小时与正常对照组、烧伤组、冲击伤组相比,肺组织湿干比重、出血面积明显增大。 3.与各实验组相比,在致伤后24小时烧冲复合伤小鼠的呼吸频率(Rr)、潮气量(Tv)、每分通气量(Mv)、最大呼气流量(PEF)明显降低降低,然而呼气时间(Te)与吸气时间(Ti)显著延长。 4.伤后24小时micro-CT检查显示：正常对照组与烧伤组小鼠肺组织未见病变；冲击伤组小鼠左侧肺组织可见轻度的毛玻璃样变区域；烧冲复合伤组小鼠左侧肺组织可见中度毛玻璃样变区域。 5.烧冲复合伤野生型C57BL/6小鼠伤后24小时肺组织髓过氧化物酶(MPO)、白介素一33(IL-33)、G-蛋白偶联受体-2(GRK2)表达明显增强。与烧冲复合伤后IL-33转基因小鼠伤后24小时相比,烧冲复合伤野生型C57BL/6小鼠趋化因子(CXCR2)明显减少；与各实验组相比,烧冲复合伤组小鼠血浆IL-6与TNF-α浓度均明显升高。 6.在烧伤、冲击伤、烧冲复合伤小鼠肺组织中IL-33的阳性细胞数与GRK2阳性细胞数、MPO阳性细胞数均有显著的正相关(r=0.65,p=0.042；r=0.638, p=0.0047；r=0.764,p=0.001)(r=0.716,p=0.02；r=0.661,p=0.0037；r=0.755, p=0.012),与Mv呈负相关(r=-0.677,p=0.031;r=-0.68,p=0.03;r=-0.816,p=0.004). 7.烧冲复合伤尸检见伤侧肺组织表面可见散在的斑片状出血,HE染色观察见大面积肺泡内出血。肺组织标本免疫组化染色见IL-33阳性细胞数高表达区域MPO阳性细胞数也高表达。 8.烧冲复合伤患者伤侧肺组织呈中度毛玻璃样变。 结论： 1.成功的建立了一种烧冲复合伤小鼠动物模型,具体参数为：野生型C57BL/6小鼠距离爆炸源53cm,压力峰值94Kpa致中度冲击伤,即刻将中度冲击伤小鼠背部脱毛区置入90℃沸水中持续9s,造成小鼠背部25%TBSAⅢ度烫伤。 2. IL-33通过激活GRK2通路阻断趋化因子CXCR2下调促进中性粒细胞迁移参与烧冲复合伤小鼠肺损伤。 3.高表达的IL-33参与烧冲复合伤患者肺损伤。
[Abstract]:Objective: burn and impulse compound injury is one of the most common types of injuries in military, terrorist, and traffic accidents. The incidence of immediate death caused by lung injury after burn and impulse compound injury is as high as 47%. acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), one of the main causes of early death of burning and punching complex injuries. A number of results show that cytokine and neutrophils play a key role in the pathogenesis of ALI/ARDS, and.IL-33 is a newly discovered cytokine in the pathogenesis of ALI/ARDS, which belongs to the multiple organs and different types of human and mice in the family.IL-33mRNA. Type of cells, at protein level, IL-33 is mainly expressed in epithelial cells, endothelial cells and fibrous cell.IL-33 to promote neutrophil chemotaxis to sepsis. However, it is not clear whether the mechanism of IL-33 and neutrophils exists in the lung tissue after burn and blast injury. Objective to investigate whether IL-33 is involved in lung injury in patients with combined burn blast combined injury and combined burn blast injury.
Methods: the wild type C57BL/6 mice and IL-33 transgenic mice were used to establish the burning and punching complex injury, the impact injury and the burn animal model. The mice were randomly divided into the normal control group, the burn group, the impact injury group and the burn and flush compound injury group. The pulmonary myeloperoxidase (myeloperoxidase, MPO) and interleukin -33 (IL-33) were detected by immunohistochemistry. G protein coupling receptor -2 (GRK2), the expression of chemokine CXCR2. Transmission electron microscopy (transmission electron microscopy) observed changes in the ultrastructure of the lung tissue and the expression of.Real-time RT-PCR detection IL-33mRNA and GRK2mRNA. The expression of MPO and IL-33 in the lung tissue was detected by the method of immunohistochemistry for 24 hours after injury. The plasma concentration of IL-6 and TNF-a was detected by enzyme linked immunosorbent assay (ELISA). The changes of lung tissue were observed by chest X-ray.
Result锛，

本文编号：1923605