本文選題：胎糞吸入綜合征 + 法尼醇X受體　； 參考：《第三軍醫(yī)大學》2017年碩士論文

【摘要】：背景與目的胎糞吸入綜合征(Meconium aspiration syndrome,MAS)是指新生兒在子宮內或產時吸入胎糞污染的羊水,導致氣道阻塞及全身炎癥反應,可累及全身各個臟器及系統(tǒng),癥狀因羊水污染程度及吸入的羊水量而表現(xiàn)不同。MAS發(fā)病機制復雜,尚未完全明確,重癥MAS并發(fā)癥多,治療困難,故MAS仍是產科醫(yī)師及新生兒科醫(yī)師重點關注疾病。細胞死亡是生命現(xiàn)象不可逆停止以及生命的結束,是細胞代謝的生物學過程,與細胞生長、增殖、分化一樣都受到機體調控。前期實驗證明:膽酸可誘導A549細胞死亡,低濃度膽酸誘導細胞凋亡,高濃度的膽酸誘導壞死性凋亡。但其具體的作用機制如何?我們將采用人肺癌上皮細胞株A549細胞作為工具細胞,A549細胞與肺泡上皮細胞功能相似,進一步探討肺損傷因素如胎糞、膽酸等如何誘導細胞死亡。法尼醇X受體(Farnesoid X Receptor,FXR)是幾個代謝途徑的重要監(jiān)管者。FXR作為一種膽汁酸受體和膽汁酸生物合成的生物感受器,有助于影響膽汁酸在新陳代謝、炎癥及細胞周期控制中的作用。近年來,多項臨床研究發(fā)現(xiàn)FXR在急性肺損傷(Acute Lung Injury,ALI)/急性呼吸窘迫綜合征(Acute Respiratory Distress Syndrome,ARDS)發(fā)生中發(fā)揮重要作用,但原因不清楚。前期有研究中發(fā)現(xiàn)FXR可能作為膽酸的核受體介導了膽酸誘導肺泡上皮細胞(Alveolar Epithelial Cells,AEC)死亡和抑制PS分泌的作用。具有調節(jié)炎癥及保護內皮細胞功能的FXR為何在新生兒呼吸系統(tǒng)疾病中發(fā)揮損傷放大效應,其機制有待深入研究。方法第一部分:通過氣管插管,氣管內注入胎糞制備新生大鼠胎糞吸入綜合征的動物模型;第二部分:采用不同濃度胎糞混懸液刺激A549細胞,檢測細胞凋亡及壞死情況,并采用Wester blot免疫印跡法、SDS-PAGE凝膠電泳法測定經(jīng)胎糞刺激后的肺組織及細胞FXR及RIPK3的表達。探索FXR在新生大鼠胎糞吸入綜合征發(fā)病機制中的作用;第三部分:臨床選擇機械通氣新生兒,分為肺源性插管患者組、非肺源性插管患者的對照組,收集肺泡灌洗液細胞行流式細胞學檢查,分析肺泡細胞死亡比例及類型。結果(一)氣管導管內注入胎糞混懸液2ml/kg的MAS組出現(xiàn)了相應的肺損傷。(二)1.A549細胞經(jīng)過不同濃度的胎糞刺激后,細胞死亡比例明顯增加,呈濃度依賴性,胎糞混懸液濃度越高,A549細胞死亡比例越高。2.MAS與C組和NS組比較,MAS組動物肺組織FXR及RIPK3蛋白表達水平有增加趨勢。3.不同濃度胎糞刺激A549細胞后,FXR、RIPK3的蛋白表達水平增加。(三)1.肺源性氣管插管組細胞死亡平均數(shù)為25%(凋亡21%)左右,非肺源性插管患者細胞死亡平均數(shù)為5%左右。2.肺源性氣管插管組血液中IL-6的平均值為533,明顯高于非肺源性插管患者。結論1.通過氣管導管內注入胎糞混懸液2ml/kg可成功制作胎糞吸入綜合征模型,為研究胎糞吸入綜合征及其相關疾病提供更好的動物模型。2.胎糞可誘導A549細胞死亡比例增加,呈濃度依賴性,20mg/ml組刺激24小時為最適濃度組,細胞死亡比例在39%左右。隨著胎糞混懸液濃度的增加,FXR、RIPK3的蛋白表達水平明顯增加。3.肺源性氣管插管組患者中,細胞死亡比例明顯大于非肺源性氣管插管患者,且死亡方式以凋亡為主。
[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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R722.1

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本文編號：2019013