發(fā)布時間：2018-07-12 15:10

  本文選題：支氣管肺發(fā)育不良 + 彈性蛋白　； 參考：《重慶醫(yī)科大學》2014年博士論文

【摘要】：目的觀察彈性蛋白系統(tǒng)在支氣管肺發(fā)育不良(bronchopulmonary dysplasia, BPD)小鼠肺組織的動態(tài)表達情況,并通過對關(guān)鍵環(huán)節(jié)之一中性粒細胞彈性蛋白酶(neutrophil elastase inhibitor, NE)進行干預(yù),以初步揭示彈性蛋白系統(tǒng)在BPD發(fā)生及發(fā)展過程中的作用以及可能機制,為尋求有效的BPD防治藥物和治療策略,提高治療效果提供實驗支持和理論依據(jù)。方法1.彈性蛋白系統(tǒng)在高氧致新生鼠BPD的動態(tài)表達規(guī)律利用新生小鼠肺發(fā)育的生物學規(guī)律與人類肺發(fā)育規(guī)律相似這一特點,通過將新生12 h C57BL/6小鼠持續(xù)吸入高氧(85%)構(gòu)建BPD動物模型,應(yīng)用組織病理學、免疫熒光雙標法(dual immunofluorescence,DIF)和分子生物學技術(shù)等,檢測出生后1-21 d正常小鼠和BPD小鼠中彈性蛋白系統(tǒng)的動態(tài)變化規(guī)律,確定高氧對肺彈性蛋白數(shù)量和分布的影響,判斷與肺彈性蛋白組裝、分解相關(guān)蛋白的變化及功能差異,分析與BPD發(fā)生的關(guān)系。2.彈性蛋白酶抑制劑Elafin對高氧致新生鼠BPD的保護作用建立高氧致新生小鼠BPD模型,通過氣道滴注中性粒細胞彈性蛋白酶抑制劑(neutrophil elastase inhibitor, NEI) Elafin干預(yù)彈性蛋白系統(tǒng)。將孕19～21 d自然分娩的C57母鼠和新生小鼠,在生后12h內(nèi)(記為日齡0 d),隨機分為3組：(1)空氣對照組(Fi02=0.21,Air)；(2)模型對照組(Fi02=0.85,02)。在小鼠日齡3 d、6 d、10 d時,氣道滴注10μl/g(小鼠體重)乳酸林格溶液(lactated-ringer solution, L/R);(3)模型給藥組(Fi02=0.85,02),在小鼠日齡3d、6d、10d時,將按照10 g1/g(小鼠體重),Elafin 40 ng/g通過氣道滴注入小鼠肺組織內(nèi)。應(yīng)用組織病理學觀察肺組織形態(tài)變化、酶聯(lián)免疫吸附試驗(enzyme-linked immunoabsorbent Assay,ELISA)檢測肺組織NE活性和尿液鎖鏈素含量；透射電鏡(transmission electron microscope, TEM)檢測彈性纖維超微結(jié)構(gòu)。3. Elafin對高氧致新生鼠BPD肺TGF-β/Smad2通路的影響建立高氧致新生小鼠BPD模型,給予NEI Elafin(40 ng/g), ELIS A檢測肺組織中活化型和總轉(zhuǎn)化生長因子β1(transforming growth factor-β, TGF-β1)的含量；免疫組織化學法(immunohistochemistry,IHC)檢測磷酸化Smad2 (phospho-Smad2, pSmad2)在肺組織細胞內(nèi)的數(shù)量與分布；實時熒光定量聚合酶鏈反應(yīng)(quantitative real-time polymerase chain reaction, Q-RT-PCR)檢測小鼠肺組織中的彈性蛋白原(tropoelastin)、TGF-β1、TTF-1和HGF-3β基因的表達水平；蛋白印跡法(western Blot, WB)測定pSmad2表達,以及pSmad2占總Smad2的比例。結(jié)果1.新生12 h以內(nèi)的C57小鼠持續(xù)85%02暴露21 d,可見彈性蛋白過度無序表達于受損肺泡,次級脊與肺泡數(shù)量顯著減少,肺泡腔擴大,肺泡結(jié)構(gòu)簡單化,肺泡再分化過程受阻。結(jié)果提示,高氧致新生小鼠BPD模型建立成功。高氧暴露促進合成、分泌彈性蛋白原的肌成纖維細胞(myofibroblast)顯著分化增殖,彈性蛋白原合成、分泌顯著增加。促進彈性蛋白原脫氨基轉(zhuǎn)化成熟的賴氨酰氧化酶(lysyl oxidase, Lox)與賴氨酰氧化酶樣1(lysyl oxidase-like 1, Loxl-1) mRNA表達上調(diào)；ECM蛋白結(jié)合受體整合素αv與彈性蛋白共表達增強；以彈性蛋白為最適底物的NE表達增加(P0.05)。2. Elafin給予,小鼠肺泡數(shù)量增加,肺泡腔明顯變�。桓哐跄Ｐ徒M(7.17±2.25)的RAC比Air組(13.75±1.81)顯著降低(P0.01),而Elafin處理組比模型顯著增高(10.05±3.12)(P0.01)；與高氧組相比,Elafin處理組彈性蛋白異常增生量顯著減少(P0.01),次級脊數(shù)量明顯增加,彈性蛋白在肺泡間隔中沉積減少,而主要在次級脊頂端分布；透射電鏡檢測顯示,與高氧組相比,Elafin處理組小鼠彈性蛋白排列更加規(guī)整,與之交聯(lián)形成彈性纖維的微纖維也排列成正常的條索狀。3.高氧會提高小鼠肺活化TGF-β1的含量,而對總TGF-β1的含量無影響, Elafin能夠抑制活化TGF-β1比例的升高,而不影響TGF-β1總蛋白的表達；模型組pSmad2表達以及pSmad2占總Smad2蛋白的比例均較空氣對照組顯著增加(P0.05),給予Elafin后,兩者均顯著下降；與空氣對照組相比,模型組的tropoelastin,TTF-1以及HGF一3p表達顯著增加(P0.01),而Elafin組tropoelastin,TTF-1以及HGF-3β表達顯著較模型降低(P0.05)。結(jié)論1.高氧暴露后,彈性蛋白的合成、組裝、沉積和降解等環(huán)節(jié)間的不協(xié)調(diào)導(dǎo)致了彈性蛋白表達過度且沉積無序,擾亂了彈性蛋白在初級囊泡壁上的正確沉積從而阻礙次級分隔級的形成,進而阻礙高氧暴露后的肺發(fā)育進程。早產(chǎn)兒不完善的彈性蛋白系統(tǒng)參與了BPD的發(fā)生發(fā)展,并在其中起著非常重要的作用。2.高氧致新生小鼠BPD發(fā)生的機制可能與NE表達與活性增加,過度激活TGF-β1-Smad2信號通路有關(guān)。3.Elafin給予后能明顯改善BPD小鼠肺組織的形態(tài)學、彈性蛋白表達分布和超微結(jié)構(gòu)改變,促進肺泡分隔數(shù)量的增加,對高氧致肺發(fā)育阻滯有明顯保護作用。4.抑制NE表達與活性,進而重塑TGF-β1-Smad2信號通路平衡,可能是Elafin對高氧致肺發(fā)育阻滯產(chǎn)生保護作用的重要機制之一
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R722.6

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