【作者】 金贞爱；

【导师】 鲁继荣；

【作者基本信息】 吉林大学 ， 内科学， 2006， 博士

【摘要】 本研究利用新生大鼠吸入高浓度氧的方法,建立支气管肺发育不良的动物模型,从组织病理学观察吸入高氧后新生大鼠不同时程肺组织结构改变,并应用TUNEL染色方法检测肺细胞凋亡、免疫组织化学染色和Western blot方法观察高氧性肺损伤大鼠不同时程肺组织中的Clara细胞分泌蛋白、肺表面活性物质相关蛋白-A以及转化生长因子-β受体1、2的表达变化,以了解高氧对肺损伤和肺发育阻滞的影响及其机制。并应用人重组胰岛素样生长因子-1和地塞米松进行干预后,再观察肺发育程度和肺内Clara细胞分泌蛋白、肺表面活性物质相关蛋白-A表达变化以及肺细胞凋亡程度,探讨重组人胰岛素样生长因子-1是否对高氧性肺损伤有保护作用。为今后进一步临床应用重组人胰岛素样生长因子-1预防和治疗支气管肺发育不良提供有力的理论和实验依据。另外,将来利用基因重组的方法制作Clara细胞分泌蛋白,用于支气管肺发育不良的防治研究提供可能性。更多还原

【Abstract】 Introduction The development of neonatology and the appearance of pulmonarysurfac-tant have been increasingly effective in reducing the mortality of very lowbirth weight infants at the expense of an increasing number of survivors withbronchopulmonary dysplasia (BPD) caused by lung immaturity. BPD is acommon syndrome in newborns, especially in preterm infants, when treated withhyperxia and mechanical ventilation. Unfortunately, there have been no effectivemedicines applied in clinic for the prevention and treatment of BPD limited by itsunclear pathogenesis. Newborn infants with severe health problems are most prone to suffer fromhypoxemia, which may be treated with oxygen therapy to save their lives. Butmore and more cases indicate that prolonged inhalation of high concentration ofoxygen may cause acute or chronic pulmonary injury for not only lung is liable tobe attacked due to its characterized structure and position but also oxygen is akind of gas medicine with harmful side effect. At present, new-type of BPD with the restricted lung development isconsidered most important in neonatal intensive care. BPD has been suggested toinfluence lung maturation by causing progressive and sustained lung inflammation,which would result in the depression of the development of alveoli, smallrespiratory tubes and small blood vessels. Therefore, the lung injury occurredduring the formation stage of respiratory system may result in retardation of lungdevelopment.Apoptosis, together with the characteristic death of endothelial and alveolarepithelial cells are reported to participate in the pathogenic process of lung injuryinduced by high concentration of oxygen. When lung injury occurs, thedenaturalization and destruction of type II alveolar epithelium will lead to thedecreased secretion of pulmonary surfactant protein (SP), disfunction ofpulmonary surfactants and finally to inhibit the maturation of pulmonary alveoli.Transforming growth factor-β (TGF-β) is one of the important growth regulatingfactors, which restrains the development of endothelial and epithelial cells andparticipates in the abnormal repair process after hyperoxia-induced lung injury.Clara cell secretory protein (CCSP), the major protein secreted by remotebronchiole Clara cells, is an inhibitor of secretary phospholipase A2, which takespart in the pathophysiological processes such as inflammation and tissuereconstruction.Insulin-like growth factor-1 (IGF-1), an important growth factor, plays acrucial regulatory role on cell multiplication, differentiation, apoptosis anddevelopment of organisms. It is reported that IGF-1 is related to thecompartmentation and maturation of alveoli. For example, IGF-1 expressiondecreases while exposed to hyperoxia at the alveoli stage of lung development.In this study, we are intent to establish BPD model induced by hyperoxiaexposure to observe pulmonary apoptosis, to investigate expression of CCSP,SP-A, TGF-βR1, TGF-βR2 and process of alveoli stage, and finally to discuss themechanism of hyperoxia-induced lung injury with or without external supplementof IGF-1 so that we can provide theoretical and experimental evidences forclinical prevention and treatment of BPD.ObjectiveExperimental neonatal rat BPD model, induced by prolonged exposure tohyperoxia, was used to investigate the histomorphology, different gene expression,such as CCSP, SP-A, TGF-βR1, TGF-βR2 and cell apoptosis process, and todiscuss the influence and mechanism of hyperoxia on lung development andinjury. Meanwhile, rh-IGF-1 and Dexamethasone were delivered to the model tocompare their different effects to provide powerful theoretical and experimentalevidences of rh-IGF-1 for prevention and treatment of BPD.MethodsMechanism of hyperoxia-exposed lung injury120 full term neonatal Wistar rats and 40 full term neonatal BAL B/c rats,under the same condition were divided randomly into two groups: air controlgroup and hyperoxia group the second day after birth. Pups were allowed foodand water ad libitum and maintained on a 12h dark-light cycle. Pups in hyperoxiagroup were kept into a Plexiglas chamber and exposed to over 85% oxygen,which was monitored daily by an oxygen sensor, while pups in control group wasunder the same raising condition except for the exposure to room air. Chamberwas opened 1 hour everyday for change of water, food, bedding and exchange oflactation rats to avoid their decreasing care ability caused by oxygen toxicity.The number of dead pups was recorded and mortality was calculated on 4d,7d, 10d and14d after birth of both groups. In addition, lung tissues were obtainedat same time points following induction of anesthesia to perform hematoxylin-and eosin-stained (HE), immunohistochemistry, TUNEL, Western blot analysisand to measure wet to dry weight ratio (W/D) to examine pneumonedema.HE assay is used to evaluate the lung tissue histological changes andexamine radical alveolar counts (RAC);Immunohistochemistry is to observeexpression of CCSP, SP-A and TGF-βR1, TGF-βR2 at different time;Apoptoticcell number and index of lung tissue were calculated on day 7 and day 14 ofhyperoxia exposure by the method of TUNEL;And expression content ofTGF-βR1, TGF-βR2 after 14d of hyperoxia exposure were detected by Westernblot analysis, besides, semiquantitative analysis was performed using the Bcionimage Software.Influence of rh-IGF-1 to hyperoxia-exposed lung injury160 full term neonatal Wistar rats in the second day after birth were assignedrandomly into four groups: Ⅰair + normal sodium control group, Ⅱhyperoxia +normal sodium group, Ⅲ hyperoxia + Dexamethasone group, and Ⅳ hyperoxia +rh-IGF-1 group. BPD model were established as described before. Except forcontrol group, chambers of the other three groups were opened 1 hour everydayfor change of bedding and lactation rats. Extra control groups for Ⅲ、Ⅳ weredesigned only for change the lactation rats.Pups in group Ⅲ were administrated everyday from the third day ofhyperoxia with intraperitoneal injection of Dexamethasone (0.25mg/Kg) whilepups in group Ⅳ with rh-IGF-1 (1ug/Kg), pups in the other two groups weregiven the same dosage of normal sodium.Lung tissues of neonatal rats in every group were obtained after 7d and 14dof hyperoxia exposure to perform HE, immunohistochemistry, TUNEL analysis.HE is used to evaluate the lung tissue histological changes and examine radicalalveolar counts (RAC). Immunohistochemistry is used to observe expression ofCCSP, SP-A at different time. Apoptotic cell number and index of lung tissuewere calculated after 7d and 14d of hyperoxia exposure by the method of TUNEL.ResultsMechanism of hyperoxia lung injury1. Changes of general condition and mortality of rats exposed tohyperoxiaNeonatal rats in air group were active, sensitive, with abundant adipose layer,high appetite and were in a healthy development status while rats in the othergroup were lazy, clumsy, emaciated with bad appetite and hypoevolutism. Withprolonged hyperoxia exposure, the mortalities of both groups were increased.Compared with air group, the mortality of hyperoxia group increased sharply after7 days of exposure, and there was significant difference between them (P<0.01).2. Pneumonedema after hyperoxia exposureThe wet to dry weight ratio of both groups had no difference at day 4 and day14 of exposure (P>0.05);but it showed a significantly increase at day 7 and day10 compared to the air control group (P<0.05).3. Lung morphology with hyperoxia(1) changes of lung histopathologyFrom birth until day 4, the terminal bronchioli of lung tissue in both groupsbranched into smooth-walled channels that ended in sacculi, the wall of whichcontained a lot of interstitial cells. The septae of the newborn lungs appeared andwere much thicker. And there were very little number of alveoli in this lungdevelopment stage. On day 7 and day 10, the number of alveoli was increased inthe air group compared to the hyperoxia group. By day 14, alveoli number of airgroup continued to multiplication resulting in a homogenous alveolar, and its wallwas thick with many interstitial cells. But for the rats of hyperoxia group, sacculicontinued to dominate, the number of small diameter alveoli decreased sharplyand distal air space enlarged obviously. On day 7 and 10 of hyperoxia exposure,edema was observed in enlarged alveoli with red blood cells and inflammatorycells in it.(2) Radical alveolar counts (RAC)The results of RAC demonstrated that there was no difference after 4 days ofhyperoixa (P>0.05) and on the other three days, RAC reduced significantlycompared to the control group (P<0.05).4. Expression of CCSP and SP-A of lung tissues with hyperoxiaexposureWith immunohistochemistry, it was found a focus of positive staining inClara cells of bronchiole epithelium. The percentage of Clara cells in distal andrespiratory bronchioles epithelium were decreased with hyperoixa compared tothat in air group (P<0.01). And statistically significant relationships were found inpositive intensity of Clara cells between hyperoxia and air group (P<0.01). Withprolonged hyperoxia, the positive ratio of Clara cells and the expression of CCSPreduced, and difference considered statistically significant (P<0.01 and P<0.05).The express of SP-A decreased on day 14 after exposure, and differenceconsidered statistically significant (P<0.01). And on day 7, the express of SP-A intwo groups has no difference (P>0.05).5. Cell apoptosis of lung tissues under hyperoxiaWith the assay of TUNEL, it showed that most apoptotic cells were alveoliand bronchi epithelial cells. The results revealed on day 7 and day 14 afterexposure, the apoptotic index were significantly higher than that in air group (P<0.01). Although cell apoptosis aggregated with the prolonged exposure time,there was no statistically difference between the two groups at every time point (P>0.05).6. Expression of TGF-βR1, TGF-βR2 of lung tissues under hyperoxiaThe results of immunohistochemistry told that on day 14 of exposure, theexpression intensity of TGF-βR1, TGF-βR2 were both higher than that in air group(P<0.01). And the positive staining was turned to mainly distribute in bronchi andalveoli epithelium and interstitial cells.It was observed with the assay of Western blot and analyzed that expressioncontent of TGF-βR1, TGF-βR2 after hyperoxia exposure were significantly morethan that of air group (P<0.05).Effect of rh-IGF-1 on lung injury induced by hyperoxia exposure1. RAC and morphological quantitative analysisCompared to air group, RAC of hyperoxia group reduced and Alveolar arealratio increased;and compared to hyperoxia and Dexamethasone groups, RAC ofrh-IGF-1 group increased and Alveolar areal ratio decreased obviously, and alldifferences were considered statistically significant (P<0.01). There was nostatistically difference between Dexamethasone and hyperoixa group (P>0.05).2. Effect of rh-IGF-1 on the expression of CCSP、SP-A after hyperoxiaexposureIt was observed from the assay of immunohistochemistry that positivestaining of CCSP distributed mainly in distal and in respiratory bronchioles. Thepositive ratio of Clara cells and the expression of CCSP with hyperoxiasignificantly decreased when compared with air and rh-IGF-1 group respectively(P<0.01). In addition, differences were statistically significant betweenDexamethasone and hyperoxia groups (P<0.05).On day 7 of hyperoxia, there were no differences in the expression of SP-Abetween hyperoxia and other groups (P>0.05);but on day 14, The positive stainswith hyperoxia significantly decreased when compared with air and rh-IGF-1group respectively (P<0.05). Meanwhile, no statistically differences were foundbetween Dexamethasone and hyperoxia group (P>0.05).3. Effect of rh-IGF-1 on the pneumocyte apoptosis after hyperoxiaexposureWith the assay of TUNEL, it showed that most apoptotic cells were alveolarand bronchi epithelial cells. The apoptotic index were increased to a significantlygreater extent for the hyperoxia group compared to the air and rh-IGF-1grouprespectively (P<0.01).Conclusions1. Lung injury and developmental arrest of neonatal rats can be induced by85% hyperoxia exposure.2. Hyperoxia exposure can promote the pneumocyte apoptosis, inhabit theexpression of CCSP and SP-A, increase TGF-βR1, TGF-βR2 to suppress theformation of lung alveoli to aggregate lung injury of neonatal rats.3. Rh-IGF-1 can remove the block of the formation of lung alveoli,increase the secretion of CCSP and SP-A to alleviate lung injury.Creation pointBronchopulmonary dysplasia has become a hot issue of neonatology with theincreased survival rates of premature infants. But its pathogenesis is still unclearand no medicine has been applied clinically with definite therapeutic effect. Andso far, no studies on the relationship of CCSP, hyperoxia and BPD, as well as theinfluence of rh-IGF-1 on lung injury induced by hyperoxia are reported indomestic. Therefore the results of this study provide a theoretic and experimentalevidence for clinic application of rh-IGF-1 to the prevention and treatment ofBPD. Furthermore, rh-CCSP obtained from gene recombination will greatlypromote the possibility of treatment of BPD.更多还原