HIF-1α在海水淹溺性肺损伤机制中作用的研究

发布时间：2018-06-13 20:02

本文选题：海水淹溺 + 渗透压　；参考：《第四军医大学》2012年硕士论文

【摘要】：研究背景：淹溺是一个重大公共卫生问题，淹溺性肺损伤是淹溺对机体造成的最主要的伤害之一，其中海水淹溺性肺损伤较淡水淹溺肺损伤更为严重，也更容易发展成为急性呼吸窘迫综合症，目前还没有关于海水淹溺肺损伤机制所特有的比较系统的理论。肺组织缺氧环境是海水淹溺性肺损伤与其它急性肺损伤（如缺氧性肺损伤和LPS导致的肺损伤）所共有的特点，而高渗环境是海水淹溺性肺损伤所特有的影响因素，海水淹溺肺损伤中同时存在缺氧和高渗因素，而这两个因素又都可能影响肺组织的HIF-1α表达，同时鉴于HIF-1α在炎症反应和肺血管通透性变化中的重要作用，因此本课题着重研究了HIF-1α在海水淹溺性肺损伤机制中的作用。目的：研究HIF-1α在海水淹溺性肺损伤机制中的作用。方法：SD大鼠随机分成6组，每组8只，分别用海水（1300mOsm/kgH2O）、高渗山梨醇（1300mOsm/kgH2O）、等渗海水（300mOsm/kgH2O）、等渗山梨醇（300mOsm/kgH2O）和淡水（0mOsm/kgH2O）气管内缓慢滴入，并设立空白对照组（只麻醉并暴露气管不滴入液体），各组滴入液体保持相同的温度（25℃）和pH值（8.2）。分别在指定时间点测量每组大鼠PaO2、PaCO2以及肺组织炎症因子(TNF-α、IL-1β和IL-6)含量、支气管肺泡灌洗液(BALF)白细胞计数、肺血管通透性、肺组织湿/干重比值（W/D）、肺组织HIF-1α和VEGF蛋白及mRNA含量。另外，分别用海水（1300mOsm/kgH2O）、高渗山梨醇溶液（1300mOsm/kgH2O）、高渗白蛋白溶液（1300mOsm/kgH2O）和双蒸水（0mOsm/kgH2O）处理NR8383细胞，各组溶液保持相同的温度（25℃）和pH值（8.2），观察不同渗透压梯度和时间点下NR8383细胞HIF-1α蛋白和mRNA含量变化，并观察缺氧和高渗联合作用对上述细胞HIF-1α蛋白和mRNA含量变化的影响。此外，应用ATM、PI3K、p38的抑制剂KU55933、LY294002、SB203580及CHX研究高渗调节HIF-1α表达的可能机制。最后，应用HIF-1α的小干扰RNA技术和测量单层细胞通透性的方法研究高渗环境下HIF-1α在炎症反应和肺血管通透性调节中的作用。结果：用高渗液体（海水和高渗山梨醇）气管内滴入组大鼠肺组织缺氧、炎症因子水平、肺血管通透性、肺水肿都明显高于用等渗和低渗液体（等渗海水、等渗山梨醇和淡水）滴入组，同时伴有HIF-1α和VEGF蛋白及mRNA含量的明显增加。在NR8383细胞上证明了是海水中的高渗因素而不是离子、温度、pH值等其它因素导致的HIF-1α蛋白和mRNA含量的增加；同时，发现高渗和缺氧可以协同作用增加HIF-1α蛋白。此外，实验表明高渗通过增加ATM和PI3K的磷酸化水平上调了HIF-1α mRNA的表达进而使HIF-1α蛋白含量增加，并且通过增加p38的磷酸化水平抑制了HIF-1α蛋白的降解进而使可检测到的HIF-1α蛋白含量增加。最后，我们验证了高渗环境下HIF-1α促进NR8383细胞产生炎症因子，同时HIF-1α通过增加VEGF蛋白表达使RLMVEC单层细胞通透性增加。结论：海水淹溺导致的肺部高渗和缺氧环境协同增加了肺组织HIF-1α表达，，增加了的HIF-1α通过提高肺组织炎症因子水平和肺血管通透性进而加重了肺部炎症反应和肺水肿。
[Abstract]:Background: drowning is a major public health problem. Drowning lung injury is one of the most important injuries caused by drowning, in which seawater drowning lung injury is more serious than that of fresh water drowning, and it is more likely to develop into acute respiratory distress syndrome. The hypoxic environment of lung tissue is the common characteristic of seawater drowning lung injury and other acute lung injury (such as hypoxic lung injury and LPS induced lung injury), while hypertonic environment is a special factor in seawater drowning lung injury, and there are also hypoxia and hypertonic factors in seawater drowning injury. The two factors may also affect the expression of HIF-1 alpha in lung tissue, and in view of the important role of HIF-1 alpha in the changes of inflammatory response and pulmonary vascular permeability, this topic focuses on the role of HIF-1 alpha in the mechanism of seawater drowning induced lung injury.
Objective: To study the role of HIF-1 alpha in the mechanism of lung injury induced by seawater drowning.
Methods: SD rats were randomly divided into 6 groups, with 8 rats in each group, with seawater (1300mOsm/kgH2O), hypertonic sorbitol (1300mOsm/kgH2O), isosotic seawater (300mOsm/kgH2O), isososmotic sorbitol (300mOsm/kgH2O) and fresh water (0mOsm/kgH2O) endotracheal endotracheal, and an empty white control group (only anaesthetized and exposed to the liquid of the trachea). Each group was instilled in liquid. At the same temperature (25 degrees centigrade) and pH (8.2), the content of PaO2, PaCO2, inflammatory factors (TNF-, IL-1, and IL-6) in each group of rats, the white cell count, pulmonary vascular permeability, the ratio of lung tissue wet / dry weight (W/D), the HIF-1 and VEGF protein and mRNA content of lung tissue, and the concentration of HIF-1 and VEGF protein and mRNA in lung tissue were measured at the designated time points. NR8383 cells were treated with seawater (1300mOsm/kgH2O), hypertonic sorbitol solution (1300mOsm/kgH2O), hypertonic albumin solution (1300mOsm/kgH2O) and double steamed water (0mOsm/kgH2O). The solution of each group kept the same temperature (25) and pH value (8.2). The changes of HIF-1 a protein and mRNA content in NR8383 cells were observed at different osmotic pressure and time points, and the deficiency was observed. The effect of the combination of oxygen and hypertonic on the changes of HIF-1 alpha protein and mRNA in the above cells. In addition, the possible mechanism of hypertonic HIF-1 alpha expression was studied by using ATM, PI3K, p38 inhibitor KU55933, LY294002, SB203580 and CHX. Finally, the application of HIF-1 alpha's small interference RNA Technology and measurement of the permeability of monolayer cells was used to study the hypertonic environment. The role of F-1 alpha in inflammatory response and regulation of pulmonary vascular permeability.
Results: hypertonic liquid (sea water and high osmotic sorbitol) was used in the endotracheal drip of rats' lung tissue, the level of inflammatory factors, pulmonary vascular permeability, and pulmonary edema were significantly higher than that of isosmotic and hypotonic fluid (isosotic sorbitol and fresh water), accompanied by a significant increase in the content of HIF-1 alpha and VEGF protein and mRNA. In NR8383 It is shown that the hypertonic factor in the sea is not the ion, the temperature, the pH value and other factors resulting in the increase of the HIF-1 alpha protein and the mRNA content. At the same time, it is found that hypertonic and anoxia can synergistically increase the HIF-1 alpha protein. Furthermore, the experiment shows that hypertonic increases the expression of HIF-1 a mRNA by increasing the phosphorylation level of ATM and PI3K. Increase the content of HIF-1 alpha protein and inhibit the degradation of HIF-1 alpha protein by increasing the phosphorylation level of p38 and then increase the detected HIF-1 alpha protein content. Finally, we verify that HIF-1 alpha promotes the production of inflammatory factors in NR8383 cells under hypertonic environment, and HIF-1 alpha is permeable by increasing the expression of VEGF protein to make RLMVEC monolayer cells permeable. Sex increases.
Conclusion: the pulmonary hyperosmotic and anoxic environment caused by seawater drowning synergistically increased the expression of HIF-1 alpha in lung tissue, and increased HIF-1 alpha by increasing the level of inflammatory factors and pulmonary vascular permeability in lung tissue and aggravating the pulmonary inflammatory response and pulmonary edema.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R649.3

【引证文献】