| prefaceChronic lung disease (CLD) is a kind of syndrome caused by long time oxygen inhalation with high concentration or mechanical ventilation with high piercer. In recent years, with the conduct of assisted reproductive technology and increasingly sophisticated management techniques of premature infants, the rate of low weight infants, especially low or very low weight infants is increasing year by year, However, CLD incidence rate is also increasing, which seriously affects the quality of premature infants life. It is currently one of the major diseases leading to neonatal disability. Although the occurrence of hyperoxia-induced lung injury mechanism is unclear, the pathological changes and its pathological process has been basically clear, that is the early pulmonary edema and late pulmonary interstitial fibrosis. Most of the current study attempts to antagonize the perspective of pulmonary fibrosis to achieve their goals, but the effect is not so obvious. So if we explore the possible mechanism of its occurrence from the early stages of hyperoxia-induced lung injury to pulmonary edema, it will provide the experimental and theoretical basis for prevention and treatment of hyperoxia-induced lung injury.Hyperoxia-induced lung injury is a kind of diffuse pulmonary inflammation, injury repair and reconstruction of complex pathologic and physiological process. The early typical change of lung injury is pulmonary edema. At present, the early pulmonary edema of hyperoxia-induced lung injury is related to abnormal expression of inflammatory factors. After using its antagonist, the symptom of pulmonary edema is not significantly reduced. Oxidative stress reaction is also considered associated with pulmonary edema, but it is also not effective by application of antioxidants in the treatment and prevention.What is the the mechanism of early pulmonary edema by hyperoxia-induced lung injury?Aquaporins (AQP) are recently discovered cell membrane transporters related to water permeability.Currently AQP1, AQP3 AQP4, AQP5 are closed with the lung tissue. AQP1 expression in fetal lung tissues slowly rises, but increases significantly near the birth. In the initial period afer birth it changes timely day by day, which prompts that AQP1 is closely related with the rapid transportation of pulmonary fluid.ENaC is a trans-membrane protein and an important transport channel in pulmonary tissue, which is composed by three homologous subunitαβγThe expression of the airway epithelial ENaC in premature infants is much lower than that in normal infants. The low level expression is closely related with respiratory distress.The study is on lung injury of premature rat models exposed to hyperoxia by detection of the expression of AQP 1,α-ENaC gene and protein in lung tissue. The aim of this research is to clarify the dynamic AQP1, a-ENaC expression in hyperoxia-induced lung injury.Materials and Methods1. Animal experiment(1) Subgroup and animal model①Object of study:premature Wistar rats②he establishment of animal models:use of previously established research methodsEighty premature Wistar rats were randomly divided into two groups based on oxygen concentration level:experimental group (FiO20.90) and the control group (FiO20.21).Each group contained 40 rats.In the experimental group,the premature rats were placed in Plexiglas chamber where oxygen was continuously delivered to achieve a constant level of 90% oxygen concentration.CO2 was absorbed by sodalime to keep CO2 level below 0.5%.Temperature and humidity were maintained at 25℃-27℃and 50%-70% respectively.Chamber was opened for 0.5h daily to change water, add food and clean dirty cages.Nursing mothers were rotated between oxygen exposed and room air litters everyday to avoid oxygen toxicity.The control group was placed in air conditions (21% oxygen concentration).Methods and control factors were same with the experimental group.(2) Example collection and methodsRats from each group were killed on 24h,48h,72h,5d,7d. Thoracic cavity was opened after anesthesia with 10% chloral hydrate.Lungs were removed, and the left lungs were placed in 4% paraformaldehyde for hematoxylin-eosin stain an immunohistochemistry.2.Experimental methods and test indicators(1) histomorphologyObserve lung histomorphology using light microscope to invest the pathology of hyperoxia-induced lung injury.(2) The immunohistochemistry of lung (AQP1) and the immunofluorescence technology of lung (a-ENaC)10 slices were selected randomly at different time points and five fields of every slice was selected randomly using light microscope.Windows were fixed if buffy grains were founded in endochylema and the cell was positive. American universal imaging porppration system was used to analyse the quantitative determination while Meta Morph software was used to accumulate the mean density value.(3) AQP1mRNA in lung tissue detecting using RT-PCR technologyThe RNA of destinated gene(AQP1 gene and a-ENaC gene)and house-keeping gene(GAPDH) was constructed first while standard curve was also made. Destinated gene and house-keeping gene were quantitated by standard curve respectively. The relative expression of AQP1 gene and a-ENaC gene was detected by the rectification of house-keeping gene in cells of all groups. The expression of AQP1 mRNA=AQP1 gene copy/GAPDH gene copy. The expression of a-ENaC mRNA=a-ENaC gene copy /GAPDH gene copy.The results were corrected by the control group with physiologic saline. 3.atistical analysisSPSS 14.0 was used to perform statistical analysis,with all data expressed as (x±S), according to the test of homogeneity.F-test or f-test was used in two groups and spearman was to analyse the correlation.ANOVA was used to analyse multigroups and it had significance when P<0.05.Results1.The histmorphology findings of lung tissue24 hours of the experiment,it was observed in both room air and hyperoxia group that the alveolar was thick and the alveolar structure was irregular.On day2,3, the alveolar septum was thinner and the alveolar of the air group was more regular meanwhile in hyperoxia group there was inflammatory response,more RBC in room..In hyperoxia group on day 5-7,it was observed that there was inflammatory response,more interstitial cells, lung septum degarded and lung edema meanwhile in air group alveolar septum much thinner and the alveolar structure was graduatelly irregular and there was inflammatory response and so on.2.The expression of AQP1 protein in the lungsIn the experiment, on day land 2,there was no significant deviation in the expression strength of AQP1 protein between two groups(P>0.05).On day 3,the expression was9.01±8.95 in experiment group and 13.97±6.78 in air group.on day 5..5.42±2.60 in experiment group,14.08±6.84 in air group, On day 7,6.69±8.53 in experiment group and13.93±8.28 in air group (there was light recovery.There was significant deviation between two groups (P<0.05)3.a-ENaC in lung tissue detecting using Immunofluorescence technologyIn the experiment, on day land 2,there was no significant deviation in the expression strength of a-ENaC protein between two groups.On day 3,5and 7,the expression strength of a-ENaC protein was gradually decreased in experiment group4. AQP1mRNA,α-ENaCmRNA in lung tissue detecting using RT-PCR technologyThe expression of the lung tissue AQP1mRNA was 8.20±5.14,11.76±8.66, 9.01±8.95,5.42±2.60,6.69±8.53 in the experiment group and 8.63±4.14 14.09±13.54 13.97±6.78 14.08±6.84 13.93±8.28 in the air group on 24h,48h,72h,5d,7dThe expression of the lung tissue a-ENaC mRNA was 0.89±0.16,0.83±0.14, 0.69±0.17,0.63±0.17,0.66±0.15 in the experiment group and 0.91±0.30, 0.88±0.15,0.89±0.21,0.90±0.21,0.88±0.20 in the air group on 24h,48h,72h,5d,7dConclusion1.The expression of AQP1 and mRNA in lung tissue of rats exposed to hyperoxia was lower on day3 and much lower on day5. On day7, it slightly recovered.There was significant deviation on day 3,5,7.2.The expression ofα-ENaC mRNA in lung tissue of rats exposed to hyperoxia was gradually decreased; there was significant deviation on day5,7 and it had statistically significant. |
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