重组人Elafin对慢性高氧致新生小鼠支气管肺发育不良的保护作用

发布时间：2018-05-14 14:43

本文选题：支气管肺发育不良 + 人重组Elafin　；参考：《重庆医科大学》2016年硕士论文

【摘要】：目的:探讨人重组Elafin对慢性高氧致新生小鼠支气管肺发育不良(bronchopulmonary dysplasia,BPD)的保护作用及作用机制。方法:新生24h内的C57BL/6J小鼠随机分3组:空气对照组,高氧+L/R组(L/R:lactated-ringer solution,乳酸钠林格液,为安慰剂),高氧+Elafin组。高氧+L/R组、高氧+Elafin组予以85%慢性高氧暴露,空气组置于同一室内空气中。高氧+Elafin组高氧暴露前30min给予Elafin 8ng/μl,5μl/g腹腔注射,每日1次,高氧+L/R组给予等量的L/R。于第1、3、7、14、21d采集肺组织,收集BALF行蛋白浓度测定和细胞分类计数评估气道炎症,运用HE染色、肺泡平均截距(mean linear intercept,MLI)、放射状肺泡计数(radical alveolar counts,RAC)观察肺组织形态学改变,弹性纤维染色观察弹性纤维表达情况,DQ-弹性蛋白底物法检测弹性蛋白酶(neutrophil elastase,NE)活性,免疫组化(IHC)测定弹性蛋白(elastin)表达,分别用实时荧光定量PCR(RT-PCR)及Western Blot检测NE、IL-1β、Tropoelastin、Fibulin-5的mRNA及蛋白表达变化,透射电镜观察弹性蛋白微观结构。结果:与空气组比较,高氧+L/R组可见炎性浸润、间质水肿,次级脊3d时开始减少,14d、21d减少显著(P0.01),MLI增加,RAC减少,肺泡发育受阻,BALF蛋白浓度增高,细胞总数及中性粒细胞计数增高,肺组织NE活性1d时即升高,后续各时间点均高于空气组,且1d、3d升高最为显著(P0.01),Elastin和Fibulin-5表达增多,无序沉积于肺泡间隔,NE、IL-1β、Tropoelastin、Fibulin-5 mRNA及蛋白表达在14d、21d时较空气组不同程度增加。与高氧+L/R组相比,Elafin干预组肺组织炎性浸润及间质水肿减轻,次级脊逐渐恢复,14d时肺泡发育明显改善,BALF蛋白浓度、细胞总数及中性粒细胞计数减少,肺组织NE活性受到抑制,14d时NE活性减少显著(P0.05);Elastin和Fibulin-5表达减弱,且主要集中于次级脊处;NE、IL-1β、Tropoelastin、Fibulin-5 mRNA的mRNA及蛋白表达均不同程度减少。结论:BPD的发生与NE活性增高有关,人重组Elafin对慢性高氧致支气管肺发育不良具有保护作用,其作用可能通过减少炎症浸润抑制NE活性、减少Elastin及其相关蛋白过度沉积,阻断弹性蛋白平衡紊乱体系而实现。
[Abstract]:Objective: to investigate the protective effect and mechanism of recombinant human Elafin on bronchopulmonary dysplasia (BPDs) in neonatal mice induced by chronic hyperoxia. Methods: C57BL/6J mice within 24 hours were randomly divided into three groups: air control group, hyperoxic L / R group, L / R% lactated-ringer solution, sodium lactate Ringer solution, placebo and hyperoxic Elafin group. Hyperoxia L / R group and hyperoxia Elafin group were exposed to 85% chronic hyperoxia, and the air group was placed in the same indoor air. Before hyperoxia exposure, 30min was injected intraperitoneally with Elafin 8ng/ 渭 l 5 渭 l / g, once a day, and the same amount of L / R was given to hyperoxia L / R group before hyperoxia exposure. Lung tissues were collected on day 1, 1, 3, 7, 7, 14, 21, and BALF were collected to determine the concentration of histopathologic proteins and to evaluate airway inflammation. The morphological changes of lung tissue were observed by HE staining, mean alveolar mean linear interceptance MLI, radial alveolar count and radial alveolar count. The expression of elastotrophil elastase was detected by DQ- elastoprotein substrate method, and the expression of elastin was detected by immunohistochemistry (IHC). The expression of mRNA and protein were detected by real-time fluorescence quantitative PCR RT-PCR and Western Blot, respectively. The microstructure of elastin was observed by transmission electron microscope. Results: compared with the air group, inflammatory infiltration and interstitial edema were observed in the hyperoxia L / R group. The number of cells and the neutrophil count in the hyperoxia L / R group were increased, the number of cells and neutrophils were increased, the increase of RAC, the concentration of BALF protein and the number of neutrophils began to decrease at 14 days and 21 days after the onset of hyperoxia in the L / R group. The activity of NE in lung tissue increased on day 1, and was higher than that in air group at the following time points, and the expression of Elastin and Fibulin-5 in lung tissue increased most significantly on day 1 and day 3, and the expression of mRNA and protein increased in different degrees at day 21 compared with that in group of air at day 14 and day 21 after the deposition of NEIL-1 尾 and Trobulin-5 in alveolar septum. Compared with the hyperoxia L / R group, the inflammatory infiltration and interstitial edema of lung tissue in the Elafin intervention group decreased, and the alveolar development significantly improved the BALF protein concentration, the total number of cells and the neutrophil count at 14 days after the restoration of the secondary ridge. After 14 days of inhibition of NE activity in lung tissue, NE activity decreased significantly (P 0.05) and the expression of Elastin and Fibulin-5 decreased significantly, and the expression of mRNA and protein mainly concentrated in the secondary ridge of nebulin 1 尾 tropoelastin 5 mRNA. Conclusion the occurrence of BPD is related to the increase of NE activity. Human recombinant Elafin has a protective effect on chronic hyperoxia-induced bronchopulmonary dysplasia, which may inhibit NE activity and reduce the excessive deposition of Elastin and its related proteins by reducing inflammatory infiltration. Blockage of elastin homeostasis system is achieved.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R722.1

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