SecinH3对小鼠脓毒症导致的肺损伤的治疗作用以及机制的研究

发布时间：2018-04-24 12:48

本文选题：SecinH3 + 小鼠　；参考：《第二军医大学》2013年博士论文

【摘要】：研究背景急性肺损伤是常见于ICU的临床综合征,并导致较高的死亡率。它常常发生在sepsis患者中。在脓毒症导致的急性肺损伤的发病过程中,微血管渗漏,以及肺水肿,能够加重疾病的严重程度。在这个过程中,一个显著的特征是血管内皮细胞屏障的破坏,通透性增高。内皮细胞之间的紧密连接和细胞粘附在内皮屏障中起着重要的作用,很多研究者针对细胞连接,发现了很多重要的信号通路,以及改善通透性的药物,但是在严重的肺损伤中,不仅是细胞连接遭到破坏,内皮细胞本身的损伤或者凋亡也会导致通透性增加。因此保护内皮细胞免受损伤,是降低通透性的一项基础的手段。一种cytohesin Arf-GEFs的抑制剂,secinH3,通过阻断Arf6的激活,从而增强了细胞之间的连接。但是SecinH3对内皮细胞是否有保护作用,则没有文献报道,因此,在本文中,我们在小鼠脓毒症导致肺损伤的模型中,应用cytohesion的抑制剂,secinH3,来治疗脓毒症导致的肺损伤,观察他是否对内皮细胞有保护作用,以及它对血管通透性以及肺损伤的影响。研究方法小鼠分成2组,假手术组和CLP组,研究CLP导致的脓毒症对肺的影响,以确定CLP导致肺损伤的模型的效果。观察小鼠的一般情况,血气分析,肺的顺应性,以及肺的病理切片,和死亡率。 2小鼠随机分成4组：假手术组,CLP组,SecinH3低剂量组,SecinH3高剂量组。以确定SecinH3是否能降低通透性,减轻肺水肿。观察制表位：小鼠血气分析,肺的顺应性,以及肺的病理切片,和死亡率。蛋白,细胞,水分的泄露的情况。以及肺内的促炎因子的水平。 3.上述四个组在术后24小时取材后检测肺脏的内皮细胞的状态,透射电镜和血液中vWF的水平。超氧化物歧化酶,丙二醛等氧化应激的指标,同时检测Nrf-2的表达。在体外培养人脐静脉内皮细胞,用LPS作为损伤因素,研究SecinH3对LPS处理的内皮细胞的影响,流式细胞术检测细胞的凋亡。并且在体内和体外研究用SOD的抑制剂DDC研究SecinH3的作用。结果 1.CLP术后,小鼠的呼吸功能,肺的顺应性大幅恶化。死亡率大幅增加,肺的HE染色显示,CLP术后,小鼠的肺内病理损伤严重,肺泡腔内渗出大量的细胞。肺泡间隔增宽。 2.在CLP导致的肺损伤模型中,SecinH3可以提高了术后小鼠的肺呼吸功能,小鼠的血气和顺应性都得到改善,并且肺的病理切片也得到了改善。肺的白细胞,全细胞渗出,以及湿干比。促炎因子的检测表明：肺内的TNF和IL-6没有下降。 3.在术后肺的电镜显示：小鼠肺脏的内皮细胞的内皮细胞在CLP术后,肿胀严重。但是SecinH3可以改善内皮细胞的肿,保护内皮屏障。在SecinH3使用后,血液中vWF的水平也出现大幅下降。进一步的研究表明,SecinH3可以增加SOD的活性,减轻MDA的量。减少了内皮细胞的凋亡。免疫印迹实验表明：SecinH3是通过增强Nrf-2的表达从起到减轻氧化应激的效果,Nrf-2是抗氧化中的重要的因子。SOD的抑制剂DDC的实验证明：DDC可以阻断SecinH3的抗氧化作用。结论 1.CLP模型可以导致急性肺损伤,该损伤模型对肺的大体功能和微观病理都有较大的影响,是较好的肺损伤的模型。 2.SecinH3可以减轻急性肺损伤后的细胞渗出蛋白渗出以及肺水肿,降低肺的通透性。但是不能减低肺内促炎因子的水平。 3.SecinH3通过调节Nrf-2的表达,增加SOD的活性,减轻了氧化应激,从而保护了内皮细胞,减少了内皮细胞的凋亡。
[Abstract]:Research background
Acute lung injury is a clinical syndrome common to ICU and leads to higher mortality. It often occurs in patients with sepsis. Microvascular leakage and pulmonary edema can aggravate the severity of the disease during the pathogenesis of acute lung injury caused by sepsis. In this process, a significant feature is the vascular endothelial cell barrier. The damage and permeability increase.
The close connections and cell adhesion between endothelial cells play an important role in the endothelial barrier. Many researchers have discovered many important signaling pathways and improved permeable drugs for cell connections, but in severe lung injuries, not only are cell connections damaged, endothelial cells themselves are damaged or apoptotic, too. It can lead to increased permeability. Therefore, protecting endothelial cells from injury is a basic means to reduce permeability.
An inhibitor of cytohesin Arf-GEFs, secinH3, enhances the connection between cells by blocking the activation of Arf6. But there is no literature on whether SecinH3 has protective effects on endothelial cells. Therefore, in this article, we used a cytohesion inhibitor, secinH3, in the model of lung injury caused by sepsis in mice. Sepsis induced lung injury, to observe whether he has a protective effect on endothelial cells, and its effect on vascular permeability and lung injury.
research method
The mice were divided into 2 groups, sham operation group and CLP group, to study the effect of sepsis on lung caused by CLP in order to determine the effect of CLP model on lung injury. Observe the general condition of the mice, blood gas analysis, lung compliance, lung pathological section, and mortality.
2 mice were randomly divided into 4 groups: the sham operation group, the CLP group, the SecinH3 low dose group, the high dose group of SecinH3, to determine whether SecinH3 could reduce the permeability and reduce the pulmonary edema. The observation of the surface position of the mice: the blood gas analysis, lung compliance, and the pathological section of the lung, and the mortality. Protein, cell, water leakage and the proinflammatory factors in the lung The level of the son.
3. the above four groups were selected 24 hours after the operation to detect the state of the endothelial cells in the lungs, transmission electron microscopy and the level of vWF in the blood. The indexes of oxidative stress such as superoxide dismutase, malondialdehyde and other oxidative stress were used to detect the expression of Nrf-2. Human umbilical vein endothelial cells were cultured in vitro, and LPS was used as a damage factor to study the endothelium of LPS treated by SecinH3 Cell apoptosis was detected by flow cytometry. In vivo and in vitro, SOD inhibitor DDC was used to study the role of SecinH3.
Result
After 1.CLP, the respiratory function and lung compliance of the mice were greatly deteriorated. The mortality rate increased significantly. The lung HE staining showed that after CLP, the pathological damage of lung in mice was serious and the alveolar cavity exuded a large number of cells. The alveolar septum was widened.
2. in CLP induced lung injury model, SecinH3 could improve lung respiratory function in mice after operation. The blood gas and compliance of mice were improved, and the lung pathological sections were also improved. Lung white blood cells, whole cell exudation, and wet dry ratio. The detection of pro-inflammatory factors showed that TNF and IL-6 in lung did not decline.
3. the electron microscope after the operation showed that the endothelial cells in the lungs of the mice were swollen seriously after CLP, but SecinH3 could improve the swelling of the endothelial cells and protect the endothelial barrier. After SecinH3, the level of vWF in the blood decreased significantly. Further studies showed that SecinH3 could increase the activity of SOD and reduce the amount of MDA. The apoptosis of endothelial cells was reduced. The immunoblotting experiment showed that SecinH3 was effective by enhancing the expression of Nrf-2 and alleviating oxidative stress. Nrf-2 was an important inhibitor of.SOD, the inhibitor of DDC, the experiment proved that DDC could block the antioxidant effect of SecinH3.
conclusion
The 1.CLP model can lead to acute lung injury. This damage model has a greater impact on the general function and microscopic pathology of the lung, and is a better model for lung injury.
2.SecinH3 can reduce exudation of the exudates and pulmonary edema after acute lung injury, and reduce the permeability of the lung. However, it can not reduce the level of proinflammatory factors in the lung.
3.SecinH3 can reduce the oxidative stress by regulating the expression of Nrf-2 and increasing the activity of SOD, thereby protecting the endothelial cells and reducing the apoptosis of endothelial cells.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R459.7

【共引文献】