脂肪干细胞对脂多糖诱导急性肺损伤大鼠炎性因子表达的影响

发布时间：2018-05-20 07:51

本文选题：急性肺损伤 + 脂肪干细胞　；参考：《郑州大学》2017年硕士论文

【摘要】：研究背景:急性肺损伤(acute lung injury,ALI)/急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)继发于严重感染、创伤、误吸、缺血再灌注、酸中毒等原因,失控的炎症反应二次打击后肺往往为最早受损的器官,是ICU中常见的危重病之一,进一步发展可出现严重的呼吸衰竭而危及生命。尽管ALl/ARDS病因不同,但有共同的特征是难治性低氧血症和进行性呼吸窘迫,其确切的发病机制目前仍未完全阐明。治疗也因原发病不同变得复杂,目前临床中主要采用小潮气量肺保护性通气策略、严格的液体控制管理以及CRRT、ECMO等高端仪器的应用,疗效有限。虽然在过去的二十年里治疗方法及ICU病房的使用对疾病预后有所改善,但较高的发病率和死亡率,治疗困难以及高昂的医疗成本等问题一直困扰着医疗界,成为全球性重要的公共卫生问题。因此迫切寻求新的治疗方法来改善现状,近年来研究发现具有多向分化潜能和抗炎作用的干细胞,有望改善这些症状。脂肪干细胞(adipose-derived stem cells,ADSCs)是近年来新发现的一种成体干细胞,具有来源丰富、取材方便,而且增殖能力强、免疫功能低等优点引起人们的广泛关注。目前关于ADSCs在脂多糖(lipopolysaccharide,LPS)诱导的ALI方面的研究较少,本研究进一步探讨ADSCs对LPS诱导急性肺损伤大鼠炎性因子表达的影响及其抗炎作用机制。目的:建立用LPS诱导的大鼠ALI模型,EdU标记ADSCs后观察在肺部定植情况,观察ADSCs对LPS诱导的ALI大鼠的治疗作用及炎性因子的影响,进一步探讨抗炎作用机制。方法:雄性SD大鼠70只,按随机数字表法分为对照组(10只)、LPS组(30只)、LPS+ADSCs组(30只)。ADSCs培养至第4代时加入EdU培育24h标记干细胞。腹腔注射LPS8mg/kg制备大鼠ALI模型,LPS+ADSCs组于制模30min后尾静脉注射300μL ADSCs进行干预;对照组无处理直接取标本,其余两组在6h、24h、72 h取标本。股动脉取血检测动脉血氧分压(PaO2)及乳酸水平;左心室取血,用酶联免疫吸附试验(ELISA)检测血清髓过氧化物酶(MPO)和白细胞介素-10(IL-10)水平;开胸取肺组织,检测肺湿/干质量(W/D)比值,光镜下观察肺组织病理学改变,荧光显微镜下观察肺组织EdU标记的干细胞定植情况,用蛋白质免疫印迹试验(Western Blot)检测肺组织核转录因子-κB(NF-κB)表达。结果:用EdU标记的ADSCs通过大鼠尾静脉注射后6h、24h、72h均有不同程度的在ALI肺部定植。与对照组比较,LPS组6h起肺组织损伤较重,肺W/D比值、血乳酸及血清MPO、IL-10水平和肺组织NF-κB表达均显著增加,PaO2显著下降;与LPS组比较,LPS+ADSCs组6h起肺组织损伤程度明显减轻,肺W/D比值、血乳酸及血清MPO水平和肺组织NF-κB表达均明显降低,PaO2显著增加,72 h已接近正常对照组水平[肺W/D比值:6 h为5.33±0.29比5.77±0.42,72 h为5.14±0.46比5.43±0.38;血乳酸(mmol/L):6 h为3.6±1.0比5.7±1.1,72 h为3.1±1.0比3.8±1.2;血MPO(μg/L):6 h为1.50±0.90比2.70±1.85,72 h为0.46±0.30比0.71±0.22;血NF-κB(灰度值):6 h为0.40±0.11比0.50±0.09,72 h为0.24±0.03比0.33±0.06;PaO2(mmHg,1 mmHg=0.133 kPa):6 h为78.0±4.1比74.5±3.2,72 h为89.3±9.4比81.9±3.4;均P0.05],IL-10水平仅24 h显著高于LPS模型组(ng/L:27.75±15.49比17.52±6.56,P0.05)。结论:ADSCs移植可在ALI大鼠肺部定植,能改善氧合升高PaO2及降低血清乳酸水平,减轻血清中炎症因子及肺部炎症反应,可能与抑制NF-κB活性有关。
[Abstract]:Background: acute lung injury (acute lung injury, ALI) / acute respiratory distress syndrome (acute respiratory distress syndrome, ARDS) is secondary to severe infection, trauma, mistaken aspiration, ischemia reperfusion, acidosis and other reasons, and the lung is often the earliest damaged organ after two hits, which is one of the most common critical diseases in ICU. In one step, severe respiratory failure can occur. Although the causes of ALl/ARDS are different, the common characteristics are refractory hypoxemia and progressive respiratory distress. The exact pathogenesis is still not fully elucidated. The treatment is also complicated because of the different primary diseases. Gas strategy, strict liquid control management, and the application of CRRT, ECMO and other high-end instruments have limited curative effect. Although the treatment methods and the use of ICU in the past twenty years have improved the prognosis of the disease, the higher incidence and mortality, the difficulty of treatment and the high cost of medical treatment have plagued the medical community. As an important public health problem, it is urgent to seek new treatments to improve the status. In recent years, studies have discovered that stem cells with multiple differentiation potential and anti-inflammatory effects are expected to improve these symptoms. Adipose-derived stem cells (ADSCs) is a newly discovered adult stem cell in recent years, with a rich source of origin. The advantages of ADSCs in lipopolysaccharide (LPS) induced ALI are less. The effect of ADSCs on the expression of inflammatory factors in rats with acute lung injury induced by LPS and the mechanism of its anti-inflammatory mechanism are further explored. The rat ALI model induced by LPS was established and the lung colonization was observed by EdU marker ADSCs. The effect of ADSCs on LPS induced ALI rats and the effect of inflammatory factors were observed. The mechanism of anti-inflammatory action was further explored. Methods: 70 male SD rats were divided into group (10), LPS group (30), LPS+ADSCs group (30).ADSCs. 24h labeled stem cells were cultured with EdU in the fourth generation. ALI model of rats was prepared by intraperitoneal injection of LPS8mg/kg, and LPS+ADSCs group was injected with 300 mu L ADSCs for the injection of LPS+ADSCs. The control group had no treatment directly and the other two groups were collected in 6h, 24h, 72 h. The serum levels of myeloperoxidase (MPO) and interleukin -10 (IL-10) were detected by enzyme linked immunosorbent assay (ELISA); lung tissue was detected in the lungs and the ratio of lung wet / dry mass (W/D) was detected. The pathological changes of lung tissue were observed under light microscope. The colonization of EdU labeled stem cells in lung tissue was observed under fluorescence microscope, and protein immuno printing was used. The expression of nuclear factor kappa B (NF- kappa B) in lung tissue was detected by trace test (Western Blot). Results: EdU marked ADSCs was injected in the tail vein of the rat through the rat tail vein, 6h, 24h, 72h all were in the ALI lung colonization. Compared with group LPS, the damage degree of lung tissue in LPS+ADSCs group was significantly reduced, the ratio of lung W/D in LPS+ADSCs group, the ratio of W/D, serum MPO and NF- kappa B in lung tissue were significantly decreased, PaO2 increased significantly, and the 72 h was close to the normal control group [lung W/D ratio: 6 h was 5.33 + 0.29, 5.77 + and 5.14 + 0.46 ratio 5. Blood lactic acid (mmol/L): 6 h is 3.6 + 1, 5.7 + 1.1,72 h is 3.1 + 1 and 3.8 + 1.2, and blood MPO (g/L): 6 h is 1.50 + 0.90 than 2.70 + 1.85,72 h, and NF- kappa B (gray value). It was 89.3 + 9.4 ratio 81.9 + 3.4, P0.05], IL-10 level was only 24 h significantly higher than that of LPS model group (ng/L:27.75 15.49 than 17.52 + 6.56, P0.05). Conclusion: ADSCs transplantation can be used to colonize the lungs of ALI rats. It can improve the increase of oxygenation and decrease the level of serum lactic acid, reduce the inflammatory factors in the serum and the inflammatory reaction in the lungs, and may be related to the inhibition of NF- kappa B activity.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563.8

【参考文献】