人中性粒细胞多肽介导肺损伤的体外研究

发布时间：2018-05-11 13:36

本文选题：人中性粒细胞多肽 + P2Y6　；参考：《广州医科大学》2014年硕士论文

【摘要】：背景急性呼吸窘迫综合征（Acuterespiratorydistresssyndrome,ARDS）是由心源性以外的各种肺内、外致病因素导致的急性、进行性呼吸衰竭，由于缺乏特异有效的治疗方法，其病死率仍然高达36%－44%。保护性机械辅助通气是ARDS目前的主要支持治疗手段，也是目前唯一能降低ARDS死亡率的治疗手段，有效的药物治疗手段仍然缺乏，而即使是保护性机械通气也无法避免机械通气相关性肺损伤的发生，ARDS的死亡率仍居高不下。肺泡上皮及微血管内皮通透构成呼吸膜结构，任何因素导致的肺泡上皮及肺微血管内皮的损伤都将导致肺泡结构的破坏，造成肺气体交换功能障碍。ARDS的发病机制研究对探索有效的药物治疗手段降低ARDS死亡率至关重要。肺泡上皮和肺微血管内皮通透性增高及肺泡内大量中性粒浸润是ARDS急性期的特征性病理表现。活化的中性粒细胞在肺泡浸润并释放大量炎症介质和蛋白酶类进一步加重肺损伤，并趋化巨噬细胞细胞及其他炎症细胞的浸润，造成持续的肺泡损伤。人中性粒细胞多肽(HNPs)是中性粒含量最多的蛋白，占中性粒细胞总蛋白5%以上，初级颗粒蛋白含量的50%以上。研究表明高浓度的HNPs对肺部损伤的诱导及进展具有重要作用。嘌呤受体P2Y家族是一类促代谢型受体，由细胞外核苷酸激活并通过G-蛋白偶联受体生成细胞内第二信使。P2Y6是P2Y家族成员之一，可介导HNPs诱导IL-8生成，在炎症诱导及免疫调节中具有重要作用。本研究以HNPs刺激原代小气道上皮细胞(SAECs)及肺微血管内皮细胞(HPMVECs)，探讨HNPs介导SAECs及HPMVECs损伤的相关机制及P2Y6在其中的作用。目的研究HNPs对人肺小气道上皮细胞及肺微血管内皮细胞通透性的影响。方法体外培养人原代小气道上皮细胞及肺微血管内皮细胞，以不同浓度的HNPs对细胞进行刺激，研究细胞通透性改变。结果 1.20μg/ml及50μg/ml HNPs8h刺激可诱导HPMVECs通透性升高，50μg/ml8h刺激可诱导SAECs通透性升高 2. HNPs诱导的HPMVECs及SAECs通透性增加由P2Y6介导，可被MRS2578部分阻断。 3. HPMVECs对HNPs敏感性高于SAECs，可能与P2Y6在HPMVCs表达明显高于SAECs所致。结论 HNPs可通过P2Y6介导SAECs及HPMVECs通透性增加。背景脓毒血症是感染造成的全身性损伤性炎症反应，脓毒血症病因复杂，以肺部感染最为常见，，其中又以革兰阴性菌为主，由于病原菌阳性诊断率低造成脓毒血症早期诊断及治疗困难。另一方面，脓毒症患者病情异质性大，老年患者居多，病人常有不同的基础疾病，进一步加重脓毒症治疗困难。脓毒血症依病情轻重可分为脓毒血症，重症脓毒血症及脓毒症休克。目前，脓毒症发病率仍以每年5~10%的速度上升，随病情进展患者的死亡率明显升高，脓毒症患者整体死亡率为30~50%，是目前ICU面临的难题。感染诱导全身性炎症反应并造成一系列不同程度的组织器官损伤，如器官功能障碍，凝血与抗凝失衡，全身组织灌注不足与缺血缺氧等，患者最终往往不是死于感染，而是死于感染诱导的全身性损伤性炎症。感染及其控制程度，全身性炎症及其导致的组织器官功能障碍，基础疾病，并发疾病，疾病进展等都不同程度影响患者预后。衡量患者基础病情，针对疾病的本质监测病情进展，筛选脓毒血症预后早期敏感因素，并及时有效纠正预后影响对脓毒血症患者具重要意义。目的对重症脓毒血症患者的预后进行多因素分析，并分析相对最敏感因素的早期预测指标，为脓毒血症早期干预提供理论依据。方法收集广州医科大学附属第一医院重症医学科2012-10-17至2013-8-8脓毒血症患者临床资料，纳入脓毒血症患者53例，根据患者ICU住院期间是否存在凝血功能障碍将病人分为A,B两组，根据脓毒血症严重程度将病人分为脓毒血症，重症脓毒血症，脓毒血症休克，根据APACHEII评分将病人分为低危(APACHEII＜10)，中危(APACHEII≥10)，高危(APACHEII≥20)。入组后记录患者年龄，性别，等基本临床资料，并进行APACHEII评分及。24h内采取血液标本，血浆IL-6，IL-8；血乳酸，血清降钙素原(PCT)为临床检查项目。根据临床检查及表现分析ICU住院期间是否存在凝血功能障碍及凝血功能障碍是否得到纠正与重症脓毒血症患者的28天预后及各影响因素的敏感性分析，并分析患者预后相对最敏感因素的早期评估。结果年龄、性别是重症脓毒血症患者28天预后的混杂因素；脓毒血症分级，APACHEII评分，ICU住院期间是否存在凝血功能障碍及凝血功能障碍是否得到纠正，患者ICU住院首次血乳酸，血清PCT，血浆IL-6，IL-8与患者预后明显相关；是否存在凝血功能障碍及凝血功能障碍是否得到纠正，血乳酸水平是脓毒血症患者预后的独立影响因素；是否存在凝血功能障碍及凝血功能障碍是否得到纠正是重症脓毒血症患者的最敏感预后影响因素；血浆IL-6，IL-8浓度是脓毒血症患者是否出现凝血功能障碍的早期预测因子。结论脓毒血症患者病情异质性大，预后影响因素复杂，血乳酸、是否出现凝血功能障碍及凝血功能障碍是否纠正是脓毒血症患者预后的独立影响因素。IL-6，IL-8水平与是否出现凝血功能障碍密切相关，可能成为凝血功能早期检测指标。本研究以脓毒血症患者ICU住院期间是否出现凝血功能障碍及28天预后研究观察目标，以患者入组第一天各项指标为监测指标，未对各项指标进行动态观察，IL-6，IL-8水平与患者从入组至出现凝血功能障碍的时间是否存在关系尚待进一步研究。
[Abstract]:background
Acuterespiratorydistresssyndrome (ARDS) is an acute, progressive respiratory failure caused by a variety of pulmonary and external pathogenic factors other than cardiogenic. Due to the lack of specific and effective treatment, the mortality rate is still up to 36% - 44%. protective mechanical ventilation is the main support for the treatment of ARDS. It is also the only treatment that can reduce ARDS mortality, and effective drug treatment is still lacking, and even protective mechanical ventilation can not avoid the occurrence of mechanical ventilation related lung injury. The mortality of ARDS is still high. Alveolar epithelium and microvascular endothelium constitute the respiratory membrane structure, and any factor caused by the lung. The damage of alveolar epithelium and pulmonary microvascular endothelium will result in the destruction of the alveolar structure. The pathogenesis of.ARDS, which causes pulmonary gas exchange dysfunction, is very important for the exploration of effective drug treatment to reduce the mortality of ARDS. The increased permeability of alveolar epithelium and pulmonary microvascular endothelium and a large number of neutrophils in the alveoli are acute phase of ARDS. Characteristic pathological manifestations. Activated neutrophils infiltrate in the alveoli and release a large number of inflammatory mediators and proteases to further aggravate the lung injury, and chemotaxis the infiltration of macrophage cells and other inflammatory cells, causing persistent alveolar damage. Human neutrophil polypeptide (HNPs) is the most neutrophilic protein, which accounts for neutrophils. More than 5% of the total protein and more than 50% of the primary particle protein content. The study showed that the high concentration of HNPs was important for the induction and progression of lung injury. The purine receptor P2Y family is a class of metabolic receptor, activated by extracellular nucleotides and second messenger of.P2Y6 by the G- protein coupling receptor, one of the members of the P2Y family, This study can mediate HNPs induced IL-8 formation, which plays an important role in inflammatory induction and immunomodulation. This study used HNPs to stimulate primary small airway epithelial cells (SAECs) and pulmonary microvascular endothelial cells (HPMVECs) to explore the mechanism of HNPs mediated SAECs and HPMVECs damage and the effect of P2Y6 in it.
objective
Objective to study the effects of HNPs on the permeability of human pulmonary small airway epithelial cells and pulmonary microvascular endothelial cells.
Method
Human primary small airway epithelial cells and pulmonary microvascular endothelial cells were cultured in vitro and stimulated with different concentrations of HNPs to study cell permeability changes.
Result
1.20 mu g/ml and 50 g/ml HNPs8h stimulation can induce HPMVECs permeability increase, and 50 g/ml8h stimulation can induce SAECs permeability increase.
2. the permeability of HPMVECs and SAECs induced by HNPs was mediated by P2Y6 and partially blocked by MRS2578.
3. the sensitivity of HPMVECs to HNPs is higher than that of SAECs, which is probably higher than that of P2Y6 in HPMVCs expression.
conclusion
HNPs can increase the permeability of SAECs and HPMVECs through P2Y6.
background
Sepsis is a systemic inflammatory response caused by infection. The etiology of sepsis is complex and the most common in the lung infection, which is mainly Gram-negative bacteria. Because of the low diagnostic rate of pathogenic bacteria, the early diagnosis and treatment of sepsis are difficult. On the other hand, the patients with sepsis are with large heterogeneity and many elderly patients. There are often different basic diseases that further aggravate the difficulty of the treatment of sepsis. Sepsis can be divided into sepsis, severe sepsis and septic shock according to the severity of the disease. At present, the incidence of sepsis is still rising at the rate of 5~10% every year. The mortality of patients with progressing is significantly higher, and the overall mortality rate of sepsis is 30~50%, ICU is a difficult problem at present. Infection induces systemic inflammatory response and causes a series of different degrees of tissue and organ damage, such as organ dysfunction, coagulation and anticoagulant imbalance, deficiency of whole body tissue perfusion and ischemia and hypoxia, and the patients are often not dying from infection, but die from infection induced systemic inflammatory inflammation. Infection and The degree of control, systemic inflammation and its resulting tissue and organ dysfunction, basic disease, disease, and disease progression all affect the patient's prognosis in varying degrees. Measure the patient's basic condition, monitor the disease progression, screen the early sensitivity factors of the prognosis of sepsis, and correct the prognosis effect on sepsis in time and effectively. The patient is of great significance.
objective
The prognosis of patients with severe sepsis was analyzed by multifactor analysis, and the early prediction indexes of the relative most sensitive factors were analyzed to provide the theoretical basis for the early intervention of sepsis.
Method
The clinical data of patients with sepsis from 2012-10-17 to 2013-8-8, the First Affiliated Hospital of Guangzhou Medical University, were collected and included in 53 patients with sepsis. The patients were divided into A and B two according to the presence of coagulation dysfunction during the hospitalization of ICU, and the patients were divided into sepsis and severe sepsis according to the severity of sepsis. Septic shock was divided into low risk (APACHEII < 10), middle risk (APACHEII > 10), and high risk (APACHEII > 20). The age, sex, and other basic clinical data were recorded in the group, and the APACHEII score and the blood samples in.24h, the plasma IL-6, IL-8, blood lactic acid, and serum calcitonin (PCT) were the clinical examination items. Objective. On the basis of clinical examination and performance analysis, an analysis of the 28 day prognosis of patients with severe sepsis and the sensitivity analysis of the 28 day prognosis of patients with severe sepsis and the early evaluation of the relative most sensitive factors in patients with severe sepsis were analyzed.
Result
Age, sex was a confounding factor in the 28 day prognosis of severe sepsis; sepsis grading, APACHEII score, whether coagulation dysfunction and coagulation dysfunction were corrected during ICU hospitalization; patients with ICU first blood lactic acid, serum PCT, plasma IL-6, IL-8 were significantly related to the prognosis of patients; and the presence of coagulation function was found. Whether obstruction and coagulation dysfunction are corrected, blood lactic acid level is an independent prognostic factor in patients with sepsis; whether or not clotting dysfunction and coagulation dysfunction are corrected is the most sensitive prognostic factor in patients with severe sepsis; plasma IL-6, IL-8 concentration is a coagulant in patients with sepsis. Early predictors of blood dysfunction.
conclusion
Patients with sepsis have large heterogeneity and complex prognostic factors. Blood lactic acid, coagulation dysfunction and coagulation dysfunction are independent factors that affect the prognosis of patients with sepsis.IL-6. The level of IL-8 is closely related to the appearance of coagulation dysfunction. This study may be an early detection index of coagulation function. In patients with sepsis, whether coagulation dysfunction and 28 days of prognosis were observed during the hospitalization of patients with sepsis, the indexes of the first day of the patients were monitored as the monitoring indexes and no dynamic observation on the indexes was carried out. The relationship between the level of IL-6, IL-8 and the time of the patients from entering the group to the obstruction of coagulation function remains to be further studied.

【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R563.8

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