A Preliminary Research On Sialin, A Nitrate Transporter In Anoxia Reoxygeneration Model Of Human Umbilical Vein Endothelial Cells

Objective: To establish human endothelial anoxia/reoxygenation cell model, simulating vascular ischemia-reperfusion injury. To study the relationship between time and cell damage degree of Anoxia/reoxygenation, exploring the nitrate transporter Sialin expression level of anoxia reoxygenation model. To explore the role of nitrate to the cells of anoxia reoxygenation preliminarily, and provide new theoretical basis for clinical prevention and treatment of tissue flap ischemia-reperfusion injury.Material and Methods: 1. To establish a stable anoxia/reoxygenation model of human umbilical vein endothelial cells(HUVEC)with an anaero-pack system, simulating vascular ischemia-reperfusion injury. HUVEC is randomly divided into four groups according to different anoxia time: Control group, cultured as normal; 3h/ 2h group, anoxia for 3h followed by 2h reoxygenation; 6h/ 2h group, anoxia for 6h followed by 2h reoxygenation; 9h/ 2h group, anoxia for 9h followed by 2h reoxygenation;. Cells are cultured in anaero-pack system. Before the process of anoxia, we change cell culture medium with low sugar serum-free DMEM medium. After anoxia/reoxygenation, several items are detected to determine cells state and to choose the optimal group of anoxia/ reoxygenation: observating cell morphology under the microscopic; determinating cell activity with Cell Counting Kit-8(CCK-8);Flow Cytometry with Annexin V-FITC Apoptosis Detection Kit detecting apoptosis ratio. 2. Studying Sialin expression in HUVEC anoxia/reoxygenation model. Observing Sialin on HUVEC cell expression under the Immunofluorescence staining microscope; Real-time PCR and Western Blot techniques testing Sialin expression in gene and protein level. 3. Loading concentration of 30mmol/L nitrate extracellular, flow cytometry and Cell Counting Kit results showed that under normal oxygen condition and after anoxia/ reoxygenation, compared to control group, the experimental group cell activity decreased obviously(P<0.05), the ratio of early and late apoptosis cells increased significantly(P<0.05). Relatively, cells under the condition of anoxia/reoxygenation, the experimental group and control group have no differences. In addition, after anoxia, observating the experimental group loading nitrate under the microscope, we found that,cell morphological changed from polygon to long spindle and a kind of lumen sample structure formed. But, after 2 hours of reoxygenation, cell restores normal morphology.Results: 1.The results showed that compared to Control group,with anoxia time extending, dead cells number increased, viable cells reduced obviously.The flow cytometry results showed that early and late apoptosis increased with anoxia injury aggravated. 2.The experiment of RT-PCR, Western Blot and Immunofluorescence showed that on HUVEC cell membrane and cytoplasm contain high expression level of Sialin. Realtime-PCR and Western Blot showed that Sialin expression reduces with the anoxia time increasing. 3. Loading concentration of 30mmol/L nitrate extracellular, flow cytometry and Cell Counting Kit results showed that: under normal oxygen condition, compared to control group, the experimental group cell activity decreased obviously and early and late apoptosis increased. Relatively, cells under the condition of anoxia/ reoxygenation, the experimental group and control group have no differences.Besides, after anoxia, observating the experimental group loading nitrate under the microscope, we found that, cell morphological changed from polygon to long spindle and a kind of lumen sample structure formed. But, after 2 hours of reoxygenation, cell restores normal morphology.Conclusion: 1. Established the anoxia reoxygenation injury model of human umbilical vein endothelial cells successful;and proved that with the anoxia time prolonged,cell viatility reduce and cell apoptosis increase. 2.The experiment proved Sialin had a higher expression in HUVEC cell membrane and cytoplasm; And with anoxia damage aggravating, Sialin expression decreased obviously. 3. Loading concentration of 30mmol/L nitrate extracellular,under normal oxygen condition, high concentration of nitrate had obvious cytotoxicity; but when cells experience anoxia/reoxygenation, nitrate cytotoxic effect decreased; Prompt that anoxia changes nitrate effect on cell.