Haemodialysis Patients Sera Induce Angiogenesis And Apoptosis Of Endothelial Cells In Vitro

Part One: Haemodialysis patients sera induce angiogenesis in vitroObjective To explore the angiogenesis induced by uraemic sera medium in vitro.Method HUVECs are respectively cultured in 10% haemodialysis patients sera medium(trial group) and 10% healthy persons sera medium(control group).Tube formation are observed under inverted microscope,cell proliferation are examinded by MTT,cell migration are measured in transwell chamber,p-p38 are evaluated by western-blot and ROS are investigated by colorimetric method.Results There are tube formation and more cell proliferation and migration after cells are exposured to uraemic sera medium for 30 hours in trial group,the average tube area is1438±112um2,mean optical density value is 1.073±0.046 in MTT method,average number of migration cells are 63.5±6.2. The expression of p-p38 and the production of ROS are higher increased than cells exposed to healthy persons sera respectively for 10 hours and 30 hours,there is correlation between ROS and cell migration and between p-p38 and cell proliferation.Conclusion Haemodialysis patients sera can induce angiogenesis in vitro,and the higher expression of p-p38 and ROS may be the mechanism.Part Two: Haemodialysis patients sera induce angiogenesis and apoptosis of endothelial cells in vitroObjective The angiogenesis and the apoptosis of endothelial cells induced by haemodialysis patients sera in vitro will be validated. Methed HUVECs are exposed to culture medium containing 10% of pooled sera obtained from 11 different patients with chronic renal failure undergoing haemodialysis(trial group) and 12 healthy volunteers(control group).The effect of haemodialysis patients sera on vascular structure formation are observed under inverted microscope,cell proliferation are evaluated by MTT, cell migration is measured by transwell chamber.Apoptosic cells in vascular structure are evaluated by TUNEL.To investigate the expression of p-p38,caspase-3 and ROS, cell lysates are evaluated by western-blot and spectrophotometry. Result After HUVECs are exposed to pooled sera medium 30 hours, vascular structure formation can be observed,average area per field is 1438±112um2,cell pliferation and migration increase in trial group.There are apoptosic cells in vascular structure after 40 hours in trial group,average positive cells number are 13 per field under light-microscope(magnified 400 time),differences are significant compared with contol group ,and there is also significant differences beteewn vascular structure cells and non vascular structure cells in trial group. Before vascular structure formation,the cells in trial group have a high expression of p-p38,afer vascular structure formation,the expression of caspase-3 is higher .The production of ROS increase after vascular structure formation. There is rorrelation between ROS and cell apoptosis. Conclusion Haemodialysis patients sera can induce angiogenesis and apoptosis of endothelial cells in vitro. Higher expression of p-p38,caspase-3 and more production of ROS may be the mechanism.Part Three: the comparation of angiogenesis and apoptosis of endothelial cells induced by sera medium between long-term haemodialysis and short-term haemodialysis patients.Objective To compare angiogenesis and apoptosis of endothelial cells induced by sera medium between long-term haemodialysis and short-term haemodialysis patients.Method HUVECs are respectively cultured in 10% long-term haemodialysis patients sera medium and 10% short-term haemodialysis patients sera medium.Tube formation are observed under inverted microscope,cell proliferation are examinded by MTT,cell migration are measured in transwell,and ROS are investigated by colorimetric method. Apoptosic cells in vascular structure are evaluated by TUNEL.To investigate the expression of p-p38 and caspase-3, cell lysates are evaluated by western-blot. Result There are tube formation(average area per field 1438±112um2 )and more cell proliferation and migration after cells are exposured to long-term haemodialysis patients sera for 30 hours. There are apoptosic cells in vascular structure after 40 hours in long-term haemodialysis patients group,average positive cells number are 13 per field under light-microscope(magnified 400 time),differences are significant compared with short-term haemodialysis patients group,and there is also significant differences beteewn vascular structure cells and non vascular structure cells in long-term haemodialysis patients group. Before vascular structure formation,the cells in long-term haemodialysis patients group have a high expression of p-p38,afer vascular structure formation,the expression of caspase-3 is higher. The production of ROS are higher increased in the cells exposed to long-term haemodialysis patients sera medium than short-term haemodialysis patients.There is correlation between ROS and cell migration,apoptosis and between p-p38 and cell proliferation. Conclusion Compared with short-term haemodialysis patients, long-term haemodialysis patients sera medium can induce angiogenesis and the apoptosis of endothelial cells and the more production of ROS and the higher expression of p-p38 and caspase-3 may be the mechanism.