Effects Of Different State Of Oxidative Stress For The Premature Rat Alveolar Type II Epithelial Cells And ROS Induced The Changes Of WNT Signaling Pathway Expression

BackgroundOxygen therapy is an important clinical treatment of hypoxemia. But long time exposured in high concentration oxygen can cause oxygen free radicals and their derivatives produced in the lung tissue.Once over the compensation limit of body ,hyperoxia lung injury would be occurred .Lots of studies have shown that the repair of injuries lung entirely depends on the proliferation and differentiation of AECâ…¡.The proliferation and differentiation of AECâ…¡not only can fix the normal alveolar struture,but even function also can be repaired more effectively. AECâ…¡can be seen an the stem cells of alveolar.Because of the lung injury caused by over high volume fraction oxygen 0.5 (50%O₂) has been confirmes by many experiments, in the paper, we chose high volume fraction oxygen 0.4 which always used on clinic to exposed on the premature rat alveolar typeΙΙepithelial cells in vitro,and compared with high volume fraction oxygen>0.95 (95%O2) and room air group, to study the growth and apoptosis of AECâ…¡.Objective(1) To culture the premature rat alveolar typeΙΙepithelial cells in vitro and prepare oxidative damage cell model.(2) To observe the morphological changes of cells were separately exposed in room air,oxygen volume fraction 0.4 and oxygen volume fraction>0.95 for 12H,24H,48H and evaluate cell model.(3) To research the survival rate and apoptosis rate of AECâ…¡were exposed separately in room air,oxygen volume fraction 0.4 and oxygen volume fraction >0.95 for 12H,24H,48H.Methods(1) To culture the premature rat AECâ…¡and were identified by electron microscopy ,and then placed in 40% and 95% oxygen concentration in a oxygen chamber and the concentration of oxygen was continuously monitored. Exposure time was determined according to requirements of experiments.(2) The morphologic changes of cells in each group in each time had been observed under inverted phase contrast microscope. (3) The survival rates was measured by MTT. (4) The apoptosis rates were measured by flow cytometry.ResultsThe morphology,survival rates and apoptosis rates of cells in groups of oxygen volume fraction 0.4 were all better than them in groups oxygen volume fraction >0.95 .But with prolonged exposure time, then oxidative damamge of oxygen volume fraction 0.4 group was increased, after 48H appeared statistically significant contrasted with room air group.ConclusionsUse premature rat AECâ…¡cultured in vitro to be exposed in different volume oxygen as the cells in vitro model of oxidative stress. With oxidative stress time prolonged, even if the stimulation from low concentrations oxygen (40%), corresponding oxidative damage will also be appeared after 48H.BackgroundIn recent years, in the embryo and postnatal rat lungs various Wnt signaling pathways were found, and these studies suggested that Wnt signaling pathway in lung played an important role during the development of lung. Studies have shown that Wnt5a gene was thought to related to separate brabch and vascular development of embryonic lung.β-catenin protein participated the formation process of lung distal airway .There were all expressingβ-catenin in primitive undifferentiated epithelial cells and differentiated alveolar epithelium and the adjacent interstitial tissue during development of embryonic lung.Classic WNT /β-catenin signaling pathway plays an important role in embryonic development and wound repair process. Under the condition of a long time oxidative stress, the body produces excess ROS can serve as an exogenous signal molecules, by changing the normal expression of WNT signaling pathway to interfere with cell proliferation and lung development and differentiation, which led to the occurrence of BPD.In this study, premature rat alveolar typeâ…¡cells (alveolar typeâ…¡epithelial cell, AECâ…¡) as the object , contrast after high volume fraction oxygen> 0.95 (95%) and air group (21%) exposed, ROS affect AECâ…¡And WNT signaling pathway.Objective(1) To culture premature rat AECâ…¡in vitro and prepare oxidative injury model of cells.(2) To observe different concentration of ROS induced the changes of WNT5a-mRNA andβ-catenin protein expression in the AECâ…¡.Methods(1) To culture the premature rat AECâ…¡and were identified by electron microscopy ,and then placed in 40% and 95% oxygen concentration in a oxygen chamber and the concentration of oxygen was continuously monitored. Exposure time was determined according to requirements of experiments.(2) ROS was detected by flow cytometry.(3) RT-PCR detecte oxidative stress influence WNT5a-mRNA expression in the AECâ…¡.(4) Western-Blot detected oxidative stress influenceβ-catenin protein expression in the AECâ…¡.Results(1) At 12H, ROS of volume fraction oxygen> 0.95 group was significantly higher than the one of air group, and at 24H continue to rise (P<0.05).(2) WNT5a and nuclearβ-catenin protein expression of volume fraction oxygen> 0.95 groups were the highest at 12H and then reduced, those of room air group would be highest in the 48H.ConclusionAs oxidative stress time prolonged, even if the stimulation of low concentrations oxygen (40%) would also appear oxidative damage after more than 48H; WNT signaling pathway as a early regulatory factor involved in hyperoxia lung injury , ROS can prematurely active it.