| Acute lung injury (ALI) is an acute progressive hypoxia respiratory dysfunction induced by various reasons except cardiogenic factors, and its serious stage is acute respiratory distress syndrome (ARDS). It is the common complication and death cause after clinical infection,external injury, operation ,et al. Up to now, there is no satisfactory measure to cure it.,and the fatality always reach to 50% or higher. Endotoxin, which was produced by G" bacillus is the most important etiological factor, and LPS (lipopolysaccharide) is the effective component of it. During the progress of ALI, the pulmonary vascular endothelial cells and alveolar epithelium were injuried extensively. The intergrated structure, reactivity and permeability of pulmonary vascular got changed, and a series pathology alterations come into being as a result. Out of these alterations, the pulmonary artery endothelial cells' alteration often took place earliest, also the most obviously. Injury is the principal influence of LPS on PAEC , resulting in the cellular swelling, necrosis and shedding. So, to study the injury mechanism of LPS on PAEC is important for the protection of PAECs and integrated structure of pulmonary vessels, then it will result in the prevention of ALI.NHE is an late discovered ion exchanger locating in cell membrane. It mediates the extracellular Na+ and intracellular H+ exchange at a ratio of 1:1. Until now, there are altogether 7 isoforms of NHE have been found. NHE1 is scattered most widely, and it is expressed in almost all kinds of hisocytes. NHE1 has special function compared to the other isoforms. It belongs to house-keeping gene, major take part in the bellowing functions: a. adjustment of intracellular pH; b. control of intracellular osmotic pressure and cellular volume; c. influence on cells' proliferation split and death. NHE1 is expressed in the vascular endothelial cells. But there is still no report on whether NHE1 take part in the procession of endothelial cells injury as a result of LPS presence. So, this question causes our attention. In this study, the relation between NHE1 and endothelial cells death caused by LPS will be investigated at cell level.METHOD: 1) bovine pulmonary endothelial cells (BPAECs) were cultured for study, NHE1 gene express of BPAECs after the addition of LPS were investigated by RT-PCR and DNA electrophoresis. 2) The effect of LPS on the proliferation of BPAECs were investigated by MTT method, and LDH in the serum of cultured BPAECs were also measured. 3) The effective ofLPS on the intracellular Ca2+ were measured by laser scanning confocal microscopy (LSCM) . 4) After staining by BECEF-AM fluorescent probe, the changes of intracellular pH were observed, and the ability of NHE1 were measured by detecting recovery rate of intracellular pH. The main results are following:1. The effect of LPS on cell proliferation of cultured BPAECsAfter 48h by the presence of LPS, the OD value of MTT is obviously below the contrast group in a dose-dependent manner, which showed LPS could inhibit the proliferation of BPAECs and increse the cell death.2. The effect of LPS on the ability of LDH in the serum of cultured BPAECsAfter 24h by the presence of LPS, the OD value of LDH increased rapidly in a dose-dependent manner, which showed LPS could cause cell injury in BPAECs.3. The effect of LPS on NHE1 gene expression of cultured BPAECs.By RT-PCR, the e/c (the densitometric comparison of NHE1 and P-actin ) in each experiment group is higher than contrast group in both does-dependent and time-dependent manner , which showed that the gene expression of NHE1 in BPAECs could increased as a result of LPS presence. 4.The effect of DMA on BPAECs injury caused by LPS.With the presence of DMA (an inhibitor of NHE1), the OD value of MTT and LDH both increased compared with LPS group, which showed that DMA could attenuate the cell injury caused by LPS.5. The effect of LPS on intracellular free Ca2+ in cultured BPAECsThe LSCM detection result showed that intracellular... |
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