| BackgroundsBronchopulmonary dysplasia (BPD) is the most familiar complications during the treatment of serious heart or pulmonary diseases in low-birth-weight premature infants by using long term oxygen and mechanical ventilation, which has been the most important disease threating preterm infants'health and life. More recently, with the introduction of antenatal steroids, exogenous surfactant therapy and non-invasive modes of ventilation, the survival rate of very low birth weight infants is raised quickly and the incidence of BPD is also to be growing year by year. Simultaneously, BPD has been one of the most troublesome problems in neonatal intensive care unit. According to the present report, BPD is diagnosed in about 20% of 60,000 infants who are born at 30 weeks gestation and weigh 1,500 g. and in 50% of infants less than 750 g, Infants develop BPD in about 1.5% of all newborn births. The survivors' lung function and gas exchange are usually abnormal,who need long term trearment of oxygen and mechanical ventilation and bring grave economics and psychic burden. to families and the society, affecting the patients'life quality seriously in future. therefore, to investigate how to prevent BPD and reduce the incidence is the important topic attecting the economic development of the society,which can protect our limit medic resources.BPD is classified two types based on the pathological feature:classic or "old BPD"and "new BPD". The "old BPD"was first described by Northway and Colleagues in 1967. Classic BPD is characterized by lung inflammation, parenchyma fibrosis due to hyperinflation and pulmonary atelectasis; New BPD is a milder form and be much more frequent today. The essential features are both lung microvascular and alveolarization dysplasia or abnormal, lung inflammation and parenchyma fibrosis are usually milder. New BPD almost always occurs in premature infants who are delivered at a gestational age of less than 26weeks and who have a birth weight of less than 1000g. They are born with mild lung disease or no pumalnary disease and need to inhale low concentrate oxygen or even no oxygen.However,these infants are gradually requiring supplemental oxygen by 36 weeks correct gestational age. The pathogenesy of new BPD is extremely complex,which are involved preterm, oxidative stress, prenatal infection,postpartum Inflammary and pulmonary dysplasia etc.However,the pathogenesy of BPD is not definite. The reports about the new BPD'pathophysiology were mainly on pulmonary alveoli dysplasia,which achieved no advancement in the past. As the Abnormality of pulmonary vascular is essential to lung dysplasia, genes and proteins which could control lung angiogenesis and vasculogenesis are investigated recentantly. Abnormal lung microvascular can directly induce BPD. If the expressions of these genes are illegitimate, the normal development of lung vascular and alveolization will be interfered.It has important effect on the pathophysiology of new BPD.Epidermal growth factor-like domain7 is a protein discovered recently that is exclusively secreted by endothelial cells.EGFL7 is one of epidermal growth factor-like domain growth factors family,which contains two epidermal growth factor-like domain and be evolutively conservative.indicating it may participate in important vital activity of interaction of proterns in different species.The expression of EGFL7 gene is special, which mainly exist in lung,heart,kidney and placenta. EGFL7 is secreted by endothelial cells to extracellular matrix and then may promote to angiogenesis by autocrining.Importantly,EGFL7 is highly expressed in process of growth in lungs of embryons and newborns. The distinct pattern of expression reveals that EGFL7 may be an important factor controlling lung angiogenesis and vasculogenesis. Parker and her colleagues found that EGFL7 was essential in the formation of tube in angiogenesis,which could promote endothelial cell to immigration and adhesion. Accordingly, EGFL7 is regarded as an important factor in lung angiogenesis and vasculogenesis. Whether EGFL7 is participated in development of new BPD and the relation of EGFL7 and pathogenesy of new BPD is unknown.Objectives1. As the sequence and the timing of postnatal lung development in rats is similar to human's.In the study, we prepared animal model of new BPD using newborn rats by persistantly breathing 600ml.L-1 oxygen and observed the effect of hyperxia on rats's general state. Using pathological section,immunohistochemistry,western blotting and fluorescent quantitation real time polymerase chain reaction to observe the rule of the lung tissue pathology in neonate rats and measure the EGFL7 protein and molecular level2. To study the passible contribution of them in development of new BPD. Finally, we hope to provide more information for pathophysiologicai mechanism and treatment of new BPD in future.Methods1. We prepared animal model of new BPD using newborn rats induced by persistantly breathing 600ml.L-1 oxygen.full-term newborn Sprague-Dawley rats were randomly divided into experimental group and control group according to the fraction of inspired oxygen. The rats of experimental group began to breathe 600ml.L-1 oxygen after 12 hours of postnatal age and rats in control group were exposed on room air. They were observed for general state at 4,7,10,14-day respectively. The pathological change was observed by hematoxylin-eosin stain2. The platelet endothelial cell adhesion molecule-1 (PECAM-1) protein was detected by immunohistochemistry (IHC) metheod. we quantified capillary density by measuring the area of PEC AM-1 immunostaining relative to the total area of pa-renchymal cells in order to identify the damage of lung microvascular induced by hyperxia.3. The mRNA and protein expression of EGFL7 were determined by fluorescent quantitation real time-PCR and Western blotting.4. Data presented by X±S, statistics done with SPSS13.0. EGFL7 mRNA etc compared using factorial analysis, number between the two groups were compared using t test and One-way ANOVA were used to compare the number among groups, if homogeneity of variance, welch method if heterogeneity of variance, use pearson correlation analysis between EGFL7 and capillary density. P<0.05 presents statistics different.Results1. Effect of 600ml.L-1 oxygen on general status of neonate rats:the animal's actions were fine, the reaction was sensitive, the pelage was tramosericeous the skin colour was florid and the growth of rats was increased rapidly.When inhaling oxygen at 10 days,some of them had slightly dyspneic respiration and cyanosis.After 14 days of breathing oxygen,the general status of neonate rats was worse than the control group.the weight of them were lighter. As separate them from oxygen,almost all of the rats were dyspnea at different extent.the heads of them wavered and the whole body were cyanosis.When they breathing oxygen again, the symptoms were relieved at different degree.2. The lung morphology induced by 600ml.L-1 oxygen:the alveolar structures of newborns in control group at days 4 was regular,the size of alveolar was uniformity.the septa became thinner; As times went on, the alveoli were regular uniformly distributed. In the hyperoxia treated group:there were a few red blood cells in some air space, interstitial tissue were infiltrated by some inflammatory cells at days 4.After 7 th day, the air space became larger gradually and the alveolar numbers started to become fewer. the septa became thinner and the interstitial cells of lung tissue were increased;When inhaling oxygen at days 10,the alveolar space became even much larger,some of them were fused. the numbers of pulmonary alveoli were decreased and interstitial cells were increased rapidly.On days 14, the architecture of air sac was interrupted,the sizes of alveoli were un-uniformity.most of them were fused.there were fewer pulmonary alveolis and more interstitial cells.3. The dynamic changes of PECAM-1 protein and microvessel density in lung tissues of neonatal rats exposed to 600ml.L-1 oxygen:1) The expression of PECAM-1 protein in lung tissues of neonatal rats exposed to 600ml.L-1 oxygen:PECAM-1 protein expressed predominantely in cytoplasm of pulmonary microvascular endothelial cells. there were statistical significance between the control group and treated group. The expression of PECAM-1 protein was lower than that in control group. Differences caused by hyperoxia in pulmonary microvascular changed over time.2) The dynamic changes of microvessel density in lung tissues of neonatal rats exposed to 600ml.L-1 oxygen:the microvessel density of lung tissues presented adscendent tendency and got highest at days 14 in control group; At postnatal 4,7,10,14days, the microvessel density of lung tissues in neonatal rats decreased in treated group, there were statistical significance between the control group and treated group at different times.4. The dynamic changes of EGFL7 mRNA and protein level in lung tissues of neonatal rats exposed to 600ml.L-1 oxygen:1) the mRNA expression of EGFL7 in lung tissues:EGFL7mRNA expression was high in control group and got the highest at 10 days; Compared with the control group,the mRNA expression of EGFL7 in lung tissues of neonatal rats were significantly decreased at postnatal 4,7,10,14days. the differences had statistical significance.2) the protein expression of EGFL7 in lung tissues:EGFL7 protein expression was high in control group. Compared with the control group, the protein expression of EGFL7 in lung tissues of neonatal rats were significantly decreased at postnatal 4,7, 10,14days. there were statistical significance between the control group and treated group at different times.5. The protein expression of EGFL7 and capillary density in lung tissues has a relationship. Correlation analysis showed that EGFL7 protein and capillary density (r=0.511,P<0.01) have a positive correlation.Conclusions1.14 days after exposed to 600ml.L-loxygen, similar pathological changes occurred in the structure of lung of the rats compared to that of the'new'BPD.the feature was that pulmonary alveolus was dysplasia,the normal structure of air sac disappeared,the air space was obviously large,the numbers of lung alveolus significantly decreased. The alveolar septum was much thinner and considerable lung interstitial cells were hyperplasia.2.600ml.L-loxygen could lead to the decreased expression of PECAM-1 and lung capillary, revealing that hyperxia which amaged vascular endothelial cell and interfered vasculogenesis could result in pulmonary hypoplasia.3. The mRNA and protein expression of EGFL7 were significantly decreased in lung tissues of newborn rats after exposed to 600ml.L-loxygen. There were elementary expressions in normal lung tissues,however,when exposed to 600ml.L-loxygen, both EGFL7 mRNA and protein expression significantly decreased, Presumeing that EGFL7 is involved in the whole process of'new' BPD4. Vasculogenesis is vital for normal pulmonary development. EGFL7 may mediate pulmonary microvascular dysplasia induced by 60% oxygen. |
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