FXR诱导肺泡细胞死亡在胎粪吸入综合征发病机制中的作用研究

发布时间：2018-06-14 21:28

本文选题：胎粪吸入综合征 + 法尼醇X受体　；参考：《第三军医大学》2017年硕士论文

【摘要】：背景与目的胎粪吸入综合征(Meconium aspiration syndrome,MAS)是指新生儿在子宫内或产时吸入胎粪污染的羊水,导致气道阻塞及全身炎症反应,可累及全身各个脏器及系统,症状因羊水污染程度及吸入的羊水量而表现不同。MAS发病机制复杂,尚未完全明确,重症MAS并发症多,治疗困难,故MAS仍是产科医师及新生儿科医师重点关注疾病。细胞死亡是生命现象不可逆停止以及生命的结束,是细胞代谢的生物学过程,与细胞生长、增殖、分化一样都受到机体调控。前期实验证明:胆酸可诱导A549细胞死亡,低浓度胆酸诱导细胞凋亡,高浓度的胆酸诱导坏死性凋亡。但其具体的作用机制如何?我们将采用人肺癌上皮细胞株A549细胞作为工具细胞,A549细胞与肺泡上皮细胞功能相似,进一步探讨肺损伤因素如胎粪、胆酸等如何诱导细胞死亡。法尼醇X受体(Farnesoid X Receptor,FXR)是几个代谢途径的重要监管者。FXR作为一种胆汁酸受体和胆汁酸生物合成的生物感受器,有助于影响胆汁酸在新陈代谢、炎症及细胞周期控制中的作用。近年来,多项临床研究发现FXR在急性肺损伤(Acute Lung Injury,ALI)/急性呼吸窘迫综合征(Acute Respiratory Distress Syndrome,ARDS)发生中发挥重要作用,但原因不清楚。前期有研究中发现FXR可能作为胆酸的核受体介导了胆酸诱导肺泡上皮细胞(Alveolar Epithelial Cells,AEC)死亡和抑制PS分泌的作用。具有调节炎症及保护内皮细胞功能的FXR为何在新生儿呼吸系统疾病中发挥损伤放大效应,其机制有待深入研究。方法第一部分:通过气管插管,气管内注入胎粪制备新生大鼠胎粪吸入综合征的动物模型;第二部分:采用不同浓度胎粪混悬液刺激A549细胞,检测细胞凋亡及坏死情况,并采用Wester blot免疫印迹法、SDS-PAGE凝胶电泳法测定经胎粪刺激后的肺组织及细胞FXR及RIPK3的表达。探索FXR在新生大鼠胎粪吸入综合征发病机制中的作用;第三部分:临床选择机械通气新生儿,分为肺源性插管患者组、非肺源性插管患者的对照组,收集肺泡灌洗液细胞行流式细胞学检查,分析肺泡细胞死亡比例及类型。结果(一)气管导管内注入胎粪混悬液2ml/kg的MAS组出现了相应的肺损伤。(二)1.A549细胞经过不同浓度的胎粪刺激后,细胞死亡比例明显增加,呈浓度依赖性,胎粪混悬液浓度越高,A549细胞死亡比例越高。2.MAS与C组和NS组比较,MAS组动物肺组织FXR及RIPK3蛋白表达水平有增加趋势。3.不同浓度胎粪刺激A549细胞后,FXR、RIPK3的蛋白表达水平增加。(三)1.肺源性气管插管组细胞死亡平均数为25%(凋亡21%)左右,非肺源性插管患者细胞死亡平均数为5%左右。2.肺源性气管插管组血液中IL-6的平均值为533,明显高于非肺源性插管患者。结论1.通过气管导管内注入胎粪混悬液2ml/kg可成功制作胎粪吸入综合征模型,为研究胎粪吸入综合征及其相关疾病提供更好的动物模型。2.胎粪可诱导A549细胞死亡比例增加,呈浓度依赖性,20mg/ml组刺激24小时为最适浓度组,细胞死亡比例在39%左右。随着胎粪混悬液浓度的增加,FXR、RIPK3的蛋白表达水平明显增加。3.肺源性气管插管组患者中,细胞死亡比例明显大于非肺源性气管插管患者,且死亡方式以凋亡为主。
[Abstract]:Background and objective Meconium aspiration syndrome (MAS) refers to the neonate inhalation of meconium contaminated amniotic fluid in the uterus or during birth, resulting in airway obstruction and systemic inflammatory reaction, which may involve the various organs and systems of the whole body. The symptoms are complex because of the degree of amniotic fluid pollution and the amount of amniotic fluid inhaled. It is not completely clear that the complications of severe MAS are many and the treatment is difficult, so MAS is still an obstetrician and newborn pediatrician. Cell death is an irreversible stop of life phenomenon and the end of life. It is the biological process of cell metabolism, which is controlled by the organism as well as cell growth, proliferation and differentiation. Can induce A549 cell death, low concentration of cholic acid to induce apoptosis and high concentration of cholic acid to induce necrotic apoptosis. But how is its specific mechanism? We will use human lung cancer epithelial cell line A549 cells as tool cells, A549 cells similar to alveolar epithelial cells, and further explore lung injury factors such as meconium, cholic acid and so on. What induces cell death. Farnesoid X Receptor (FXR) is an important regulator of several metabolic pathways,.FXR, as a biosynthesis of bile acid receptor and bile acid biosynthesis, which contributes to the effect of bile acids on metabolism, inflammation and cell cycle control. In recent years, a number of clinical studies have found FXR in a number of clinical studies. The cause of acute lung injury (Acute Lung Injury, ALI) / acute respiratory distress syndrome (Acute Respiratory Distress Syndrome, ARDS) plays an important role, but the reason is unclear. In previous studies, it was found that FXR may be used as a nuclear receptor for cholic acid to induce cholic acid induced alveolar epithelial cells (Alveolar Epithelial) death and inhibition. The role of secretory function. The mechanism of FXR, which regulates inflammation and protecting endothelial cell function, plays a damaging magnification effect in neonatal respiratory diseases. The first part: the first part: the animal model of neonatal meconium inhalation syndrome was prepared by intratratracheal intubation and intratracheal injection of meconium; the second part: different The concentration of meconium suspension was used to stimulate A549 cells to detect cell apoptosis and necrosis. The Wester blot immunoblotting method was used to determine the expression of FXR and RIPK3 in the lung tissues and cells stimulated by meconium by SDS-PAGE gel electrophoresis. The role of FXR in the pathogenesis of neonatal meconium inhalation syndrome was explored. The third part: clinical selection of machinery. The newborns were divided into pulmonary intubation group, non pulmonary intubation group and control group without pulmonary intubation. The cell line flow cytology of alveolar lavage fluid was collected to analyze the proportion and type of alveolar cell death. Results (1) MAS group of 2ml/kg in the endotracheal tube injected with meconium suspension showed corresponding lung injury. (two) 1.A549 cells passed different concentrations. After the meconium stimulation, the proportion of cell death increased significantly, the concentration depended, the higher the concentration of meconium suspension, the higher the proportion of A549 cell death, the higher the ratio of.2.MAS to C and NS, the expression level of FXR and RIPK3 protein in the lung tissue of MAS group was increased, and.3. at different concentration of meconium, the FXR, RIPK3 protein expression level was increased. (three) the average number of cell deaths in the 1. pulmonary endotracheal intubation group was 25% (apoptosis 21%), and the average number of cell deaths in non pulmonary intubation patients was 5%.2. pulmonary endotracheal intubation group, the mean value of IL-6 in the blood of pulmonary endotracheal intubation group was 533, obviously higher than that of non pulmonary intubation patients. Conclusion 1. through intratracheal catheter injection of meconium suspension 2ml/kg can be successfully made. The meconium aspiration syndrome model provides a better animal model for the study of meconium aspiration syndrome and related diseases..2. meconium can induce the increase of A549 cell death ratio, which is concentration dependent. The 20mg/ml group is stimulated for 24 hours as the optimum concentration group, the proportion of cell death is about 39%. With the increase of meconium suspension concentration, FXR, RIPK3 protein In the patients with.3. pulmonary endotracheal intubation, the proportion of cell death was significantly greater than that of non pulmonary endotracheal intubation patients, and the death mode was mainly apoptosis.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R722.1

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