NFAT5在海水淹溺性肺损伤中作用机制的研究

发布时间：2018-05-16 19:16

本文选题：海水淹溺型肺损伤 + 海水淹溺型肺水肿　；参考：《第四军医大学》2013年硕士论文

【摘要】：研究背景：海水淹溺是航海作业环境常见的意外事故，也是军队海上作战战斗减员的主要原因之一。人体吸入海水后，引起肺顺应性改变、海水淹溺型肺水肿、肺内分流、低氧血症及酸中毒，若不及时处理可导致全身脏器不可逆的损害。其中海水淹溺后造成的肺水肿(pulmonary edema induced by seawater drowning,PE-SWD)和炎症，经过海水淹溺型急性肺损伤(seawater aspiration induced acute lung injury，SWD-ALI)阶段进一步发展为海水淹溺型急性呼吸窘迫综合征(seawater aspiration induced acuterespiratory distress syndrome，SWD-ARDS),救治起来极为困难，病死率极高。有研究报道，NFAT5是哺乳动物体内唯一已知的渗透压转录相关因子，高渗可以导致细胞皱缩，线粒体去极化，DNA损伤，最终造成细胞凋亡，NFAT5则是负责在这种条件下维持细胞内外渗透压的平衡。最近研究表明，NFAT5通过刺激前炎因子的表达，在炎症疾病中扮演着重要的角色。现广泛报道的海水淹溺型肺损伤的发病机制并不完善，治疗也具有一定的限制性，效果并不十分理想。因此，正确认识海水淹溺导致肺损伤的机理是及时有效治疗海水吸入所致急性肺损伤的基础，对海水淹溺型肺损伤发病机制的进一步研究具有十分重要的临床指导意义。本课题结合海水本身的高渗特征和肺损伤发病的普遍机理，致力于找到海水这一特殊液体引起急性肺损伤的具体发病机制。实验目的：本实验研究了NFAT5在海水淹溺型肺损伤中所扮演的重要角色，通过复制SWD-ALI大鼠模型，观察NFAT5及有机渗透物质在海水淹溺发生后表达的变化，以及其对山梨醇转运蛋白，牛磺酸转运蛋白，醛糖还原酶等有机渗透物质转运蛋白的调控，探讨NFAT5在海水淹溺型肺损伤发生后对肺水肿的减轻机制。并探索NFAT5在海水淹溺发生后炎症环节中，所扮演的重要角色。实验方法：体内实验：复制SD大鼠海水淹溺肺损伤模型，将56只180-200gSD大鼠随机分为7组，对照组，海水淹溺1h组，2h组，3h组，4h组，5h组，6h组，每组8只。按1ml/kg给予大鼠3%戊巴比妥钠进行麻醉后，经气管注入新鲜配制海水(4ml/kg)。根据不同时间点的选择，处死动物后，取左下肺组织行HE染色，观察病理变化。右下肺组织称重，烘干，进行湿/干比值的检测。大鼠处死后的肺组织分块并保存于-80°C冰箱，分别用于ELISA检测炎症因子变化，WesternBlot、免疫组化检测NFAT5蛋白含量变化，实时定量PCR在mRNA水平检测NFAT5及渗透压相关转运蛋白的变化，有机渗透物质如山梨醇、牛磺酸、甜菜碱变化，由液相质谱联用法进行检测。体外实验： A549细胞的培养及处理：使用RPMI1640培养液培养A549细胞，在进行海水干预前行MTT试验，向培养液中加入不同比例海水，观察不同浓度海水对细胞的抑制率，选择合适浓度进行实验。（1）观察不同渗透压对A549细胞的影响：选择20%，30%，40%，50%浓度梯度海水干预细胞，培养6h，行WesternBlot及实时定量PCR观察细胞NFAT5在蛋白及mRNA水平的变化。（2）观察不同海水干预时间对A549细胞的影响:选择30%浓度海水，分为4h，6h，8h，10h，12h组，行WesternBlot及实时定量PCR观察细胞NFAT5在蛋白及mRNA水平的变化。（3）ELISA检测不同渗透压及时间对A549细胞进行干预后IL-6,IL-1β,TNF-α在细胞上清液中的变化。实验结果：体内实验：大鼠注入海水后，肺组织W/D,ELI较对照组显著增加（p0.01）,高渗刺激后渗透压保护基因NFAT5蛋白及mRNA表达量增加，同时，，有机渗透物质和渗透压相关转运蛋白含量也随NFAT5的增加而增加。随时间的变化，肺水肿程度减轻。根据ELISA,HE染色，实时定量PCR结果，发现炎症反应在4h时开始加重。体外实验：通过Western blot和qRT-PCR检测，发现随渗透压的升高及培养时间的延长，NFAT5及渗透压相关转运蛋白表达量均增加。ELISA结果显示，随NFAT5的升高，IL-6,IL-1β,TNF-α前炎因子的生成量增多。实验结论： NFAT5在海水淹溺型肺损伤的发生机制中发挥着重要作用，在肺水肿发生过程中，NFAT5通过对SMIT、BGT1、AR等渗透压相关转运蛋白的调控，致使山梨醇，牛磺酸，甜菜碱等有机渗透物质的增加。NFAT5通过在高渗条件下维持细胞内外渗透压的平衡，减轻了肺水肿。但随着时间的变化，在4h时，IL-6,IL-1β,TNF-α等前炎因子的生成量显著增多，这与NFAT5的过度表达有着密切的关系。
[Abstract]:Background of Study :

The study shows that NFAT5 is the only known osmotic pressure transcription related factor in mammals . The mechanism of pulmonary edema induced by seawater drowning is not perfect . It is very important to study the pathogenesis of acute lung injury caused by seawater drowning .

In this paper , the role of NFAT5 in seawater drowning induced lung injury was studied . By means of the model of SWD - ALI rats , the changes of NFAT5 and its expression after seawater drowning were observed , and the mechanism of NFAT5 in the pathogenesis of pulmonary edema after seawater drowning was discussed .

In vivo experiment :

The rats were randomly divided into seven groups : control group , seawater drowning 1h group , 2h group , 3h group , 4h group , 5h group and 6h group .

Culture and treatment of A549 cells : The A549 cells were cultured with RPMI1640 medium and dried in seawater .

The effects of different osmotic pressures on A549 cells were observed . ( 1 ) The effects of different osmotic pressures on A549 cells were observed . ( 1 ) The effects of different osmotic pressures on A549 cells were observed : ( 2 ) The changes of the levels of NFAT5 protein and mRNA were observed at 6h , 6h , 8h , 10h , 12h , Western Blot and real - time quantitative PCR .

Experimental results :

In vivo experiment :

The expression of NFAT5 protein and mRNA in lung tissue were increased significantly after injection of seawater into seawater . The contents of NFAT5 and NFAT5 increased with the increase of NFAT5 . At the same time , the levels of organic and osmotic pressure - related transport proteins increased with the increase of NFAT5 . The results of ELISA , HE staining and real - time quantitative PCR showed that inflammatory reaction began to aggravate at 4h .

In vitro experiments :

By Western blot and qRT - PCR , the expression of NFAT5 and osmotic pressure - related transport proteins increased with the increase of osmotic pressure and the incubation time . The results showed that IL - 6 , IL - 1尾 , TNF - 伪 pro - inflammatory factors increased with the increase of NFAT5 . The results showed that :

NFAT5 plays an important role in the pathogenesis of seawater drowning type lung injury . During the course of pulmonary edema , NFAT5 can regulate osmotic pressure related transport protein such as SMIT , BG T1 , AR and so on . NFAT5 reduces pulmonary edema by maintaining the balance of osmotic pressure inside and outside of cells under hyperosmotic condition . However , the amount of pro - inflammatory factors such as IL - 6 , IL - 1尾 , TNF - 伪 is significantly increased at 4 h , which is closely related to the overexpression of NFAT5 .
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R649.3

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