弹性蛋白系统在高氧致新生鼠支气管肺发育不良中的作用及干预研究

发布时间：2018-07-12 15:10

本文选题：支气管肺发育不良 + 弹性蛋白　；参考：《重庆医科大学》2014年博士论文

【摘要】：目的观察弹性蛋白系统在支气管肺发育不良(bronchopulmonary dysplasia, BPD)小鼠肺组织的动态表达情况,并通过对关键环节之一中性粒细胞弹性蛋白酶(neutrophil elastase inhibitor, NE)进行干预,以初步揭示弹性蛋白系统在BPD发生及发展过程中的作用以及可能机制,为寻求有效的BPD防治药物和治疗策略,提高治疗效果提供实验支持和理论依据。方法1.弹性蛋白系统在高氧致新生鼠BPD的动态表达规律利用新生小鼠肺发育的生物学规律与人类肺发育规律相似这一特点,通过将新生12 h C57BL/6小鼠持续吸入高氧(85%)构建BPD动物模型,应用组织病理学、免疫荧光双标法(dual immunofluorescence,DIF)和分子生物学技术等,检测出生后1-21 d正常小鼠和BPD小鼠中弹性蛋白系统的动态变化规律,确定高氧对肺弹性蛋白数量和分布的影响,判断与肺弹性蛋白组装、分解相关蛋白的变化及功能差异,分析与BPD发生的关系。2.弹性蛋白酶抑制剂Elafin对高氧致新生鼠BPD的保护作用建立高氧致新生小鼠BPD模型,通过气道滴注中性粒细胞弹性蛋白酶抑制剂(neutrophil elastase inhibitor, NEI) Elafin干预弹性蛋白系统。将孕19～21 d自然分娩的C57母鼠和新生小鼠,在生后12h内(记为日龄0 d),随机分为3组：(1)空气对照组(Fi02=0.21,Air)；(2)模型对照组(Fi02=0.85,02)。在小鼠日龄3 d、6 d、10 d时,气道滴注10μl/g(小鼠体重)乳酸林格溶液(lactated-ringer solution, L/R);(3)模型给药组(Fi02=0.85,02),在小鼠日龄3d、6d、10d时,将按照10 g1/g(小鼠体重),Elafin 40 ng/g通过气道滴注入小鼠肺组织内。应用组织病理学观察肺组织形态变化、酶联免疫吸附试验(enzyme-linked immunoabsorbent Assay,ELISA)检测肺组织NE活性和尿液锁链素含量；透射电镜(transmission electron microscope, TEM)检测弹性纤维超微结构。3. Elafin对高氧致新生鼠BPD肺TGF-β/Smad2通路的影响建立高氧致新生小鼠BPD模型,给予NEI Elafin(40 ng/g), ELIS A检测肺组织中活化型和总转化生长因子β1(transforming growth factor-β, TGF-β1)的含量；免疫组织化学法(immunohistochemistry,IHC)检测磷酸化Smad2 (phospho-Smad2, pSmad2)在肺组织细胞内的数量与分布；实时荧光定量聚合酶链反应(quantitative real-time polymerase chain reaction, Q-RT-PCR)检测小鼠肺组织中的弹性蛋白原(tropoelastin)、TGF-β1、TTF-1和HGF-3β基因的表达水平；蛋白印迹法(western Blot, WB)测定pSmad2表达,以及pSmad2占总Smad2的比例。结果1.新生12 h以内的C57小鼠持续85%02暴露21 d,可见弹性蛋白过度无序表达于受损肺泡,次级脊与肺泡数量显著减少,肺泡腔扩大,肺泡结构简单化,肺泡再分化过程受阻。结果提示,高氧致新生小鼠BPD模型建立成功。高氧暴露促进合成、分泌弹性蛋白原的肌成纤维细胞(myofibroblast)显著分化增殖,弹性蛋白原合成、分泌显著增加。促进弹性蛋白原脱氨基转化成熟的赖氨酰氧化酶(lysyl oxidase, Lox)与赖氨酰氧化酶样1(lysyl oxidase-like 1, Loxl-1) mRNA表达上调；ECM蛋白结合受体整合素αv与弹性蛋白共表达增强；以弹性蛋白为最适底物的NE表达增加(P0.05)。2. Elafin给予,小鼠肺泡数量增加,肺泡腔明显变小；高氧模型组(7.17±2.25)的RAC比Air组(13.75±1.81)显著降低(P0.01),而Elafin处理组比模型显著增高(10.05±3.12)(P0.01)；与高氧组相比,Elafin处理组弹性蛋白异常增生量显著减少(P0.01),次级脊数量明显增加,弹性蛋白在肺泡间隔中沉积减少,而主要在次级脊顶端分布；透射电镜检测显示,与高氧组相比,Elafin处理组小鼠弹性蛋白排列更加规整,与之交联形成弹性纤维的微纤维也排列成正常的条索状。3.高氧会提高小鼠肺活化TGF-β1的含量,而对总TGF-β1的含量无影响, Elafin能够抑制活化TGF-β1比例的升高,而不影响TGF-β1总蛋白的表达；模型组pSmad2表达以及pSmad2占总Smad2蛋白的比例均较空气对照组显著增加(P0.05),给予Elafin后,两者均显著下降；与空气对照组相比,模型组的tropoelastin,TTF-1以及HGF一3p表达显著增加(P0.01),而Elafin组tropoelastin,TTF-1以及HGF-3β表达显著较模型降低(P0.05)。结论1.高氧暴露后,弹性蛋白的合成、组装、沉积和降解等环节间的不协调导致了弹性蛋白表达过度且沉积无序,扰乱了弹性蛋白在初级囊泡壁上的正确沉积从而阻碍次级分隔级的形成,进而阻碍高氧暴露后的肺发育进程。早产儿不完善的弹性蛋白系统参与了BPD的发生发展,并在其中起着非常重要的作用。2.高氧致新生小鼠BPD发生的机制可能与NE表达与活性增加,过度激活TGF-β1-Smad2信号通路有关。3.Elafin给予后能明显改善BPD小鼠肺组织的形态学、弹性蛋白表达分布和超微结构改变,促进肺泡分隔数量的增加,对高氧致肺发育阻滞有明显保护作用。4.抑制NE表达与活性,进而重塑TGF-β1-Smad2信号通路平衡,可能是Elafin对高氧致肺发育阻滞产生保护作用的重要机制之一
[Abstract]:Objective To observe the dynamic expression of elastin system in the lung tissues of bronchopulmonary dysplasia (BPD) mice and to interfere with the neutrophil elastase inhibitor (NE), one of the key links, in order to preliminarily reveal the development and development of the elastin system in BPD. The role and possible mechanisms provide experimental support and theoretical basis for seeking effective BPD control drugs and treatment strategies and improving the therapeutic effect. Method 1. the dynamic expression of BPD in neonatal rats induced by high oxygen protein system is similar to that of the lung development of newborn mice, which is similar to that of human lung development. The new 12 h C57BL/6 mice inhaled hyperoxia (85%) to construct the BPD animal model. Histopathology, immunofluorescence double standard (dual immunofluorescence, DIF) and molecular biology techniques were used to detect the dynamic changes of elastin system in 1-21 D normal mice and BPD mice after birth, and determine the number and score of hyperoxia to the lung elastin. The influence of cloth, determination of the changes in protein assembly, changes of protein and function difference, analysis of the relationship between BPD and.2. elastase inhibitor Elafin on the protection of hyperoxia induced neonatal rat BPD, the BPD model of hyperoxia induced neonatal mice was established, and the neutrophil elastase inhibitor (neutrophil elastas) was injected through the airway. E inhibitor, NEI) Elafin intervention elastin system. The C57 female and newborn mice of natural birth 19~21 D were randomly divided into 3 groups: (1) air control group (Fi02=0.21, Air), and (2) model control group (Fi02=0.85,02). At the age of 3 D, 6, and 10, the airway infusion of 10 micron (mice weight) lactate forest Lattice solution (lactated-Ringer solution, L/R); (3) model administration group (Fi02=0.85,02), when mice were 3D, 6D, 10d, the mice were injected into the lung tissue according to the 10 g1/g (mice weight) and Elafin 40 ng/g through the airway drops. The morphological changes of lung tissue were observed by histopathology, enzyme linked immunosorbent assay (enzyme-linked immunoabsorbent) ISA) detection of lung tissue NE activity and urine lock chain content; transmission electron microscopy (transmission electron microscope, TEM) detection of the ultrastructure of elastic fiber.3. Elafin on the BPD lung TGF- beta /Smad2 pathway in hyperoxia induced neonatal rats. The content of total transforming growth factor beta 1 (transforming growth factor- beta, TGF- beta 1); the quantitative and distribution of Smad2 (phospho-Smad2, pSmad2) phosphorylated in the lung tissue by immunohistochemistry (immunohistochemistry, IHC); real-time fluorescent quantitative polymerase chain reaction (quantitative real-time polymerase) T-PCR) detected the elastin (tropoelastin), the expression level of TGF- beta 1, TTF-1 and HGF-3 beta in the lung tissue of mice, the expression of pSmad2 and pSmad2 in Western Blot (WB), and the ratio of pSmad2 to Smad2. The results showed that 21 of the mice within the newborn 12 h were exposed to 21, which showed that the elastin was overexpressed in the disorder. The alveoli, the number of secondary ridges and alveoli decreased significantly, the alveoli enlarged, the alveolar structure was simplified, and the redifferentiation of the alveoli was blocked. The results suggest that the BPD model of hyperoxia induced neonatal mice is successful. The hyperoxic exposure promotes synthesis, and the myofibroblast (myofibroblast) that secretes elastin is significantly differentiated and proliferated, and the elastin is synthesized and secreted. The expression of lysine oxidase (lysyl oxidase, Lox) and lysine like oxidase like 1 (lysyl oxidase-like 1, Loxl-1) mRNA was up-regulated, and the co expression of ECM protein binding receptor integrin alpha v and elastin was enhanced, and NE expression with elastin as the most suitable substrate for ECM protein increased (P0.05).2. The number of alveoli in mice increased and the alveoli of the alveolus were significantly smaller, and the RAC in the hyperoxic model group (7.17 + 2.25) was significantly lower than that in the Air group (13.75 + 1.81) (P0.01), while the Elafin treatment group was significantly higher than the model (10.05 + 3.12) (P0.01). Compared with the hyperoxia group, the abnormal proliferation of elastin in the Elafin treatment group was significantly decreased (P0.01) and the number of secondary ridges increased significantly. Adding, the deposition of elastin in the alveolar septum was reduced and mainly distributed at the top of the secondary ridge. Transmission electron microscopy showed that the elastin in the Elafin treatment group were more orderly than those in the hyperoxia group, and the microfibril linked with the elastic fibers formed into normal.3. hyperoxia could increase the content of TGF- beta 1 in mice lung activation. There was no effect on the content of total TGF- beta 1. Elafin could inhibit the increase in the ratio of activated TGF- beta 1, but did not affect the expression of total protein of TGF- beta 1. The pSmad2 expression of the model group and the proportion of pSmad2 to total Smad2 protein increased significantly (P0.05) in the control group, and both of the two were significantly decreased after giving Elafin; compared with the air control group, the model was compared with the air control group. The expression of tropoelastin, TTF-1 and HGF 3P increased significantly (P0.01), and the expression of tropoelastin, TTF-1 and HGF-3 beta in the Elafin group was significantly lower than that of the model (P0.05). Conclusion after 1. hyperoxia exposure, the incongruity of the synthesis, assembly, deposition and degradation of elastin resulted in the excessive and disorderly deposition of elastin and disturbed elasticity. The correct deposition of protein on the wall of primary vesicles hinders the formation of secondary separation level and hinders the development of lung development after hyperoxia exposure. The imperfect elastin system in premature infants participates in the development of BPD and plays a very important role in the pathogenesis of BPD in.2. hyperoxic mice, which may be associated with the expression and activity of NE. In addition, over activation of the TGF- beta 1-Smad2 signaling pathway related to.3.Elafin can obviously improve the morphology of lung tissue in BPD mice, the distribution of elastin expression and ultrastructure, promote the increase of the number of alveolar septum, and have a clear protective effect on hyperoxic lung development block,.4. inhibits the expression and activity of NE, and then reshape the TGF- beta 1-Smad2 letter. The balance of pathway may be one of the important mechanisms by which Elafin can protect lung from hyperoxia induced lung development.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R722.6

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