| Objective: Endothelial progenitor cells(EPCs)are the precursor cells of endothelial cells, have the functions of promoting the new born of blood vessels and repairing damaged blood vessels.Currently, diabetes vascular lesions have become an undoubtful threaten to Human health,plenty of studies of the pathomechanism on diabetes vascular lesions have indicated that the damaging of EPCs functions plays a key role.Cell apoptosis is a kind of autonomos and procedural process of cells death mediated by self-gene,when different kinds of reasons caused the level of Calcium and Phosphonium in EPCs rapidly increased,it will lead cell apoptosis and the formation of apoptotic bodies.Advanced glycation end products(AGEs) is a kind of covalent complex formed by the combination of proteoin lipid nucleic acid and glucose under the condition of high glucose,it is the majior composition induces the cell apoptosis under the condition of high glucose. AGEs hardly expressed in normal physiological conditions,but the expression increased significantly in the patients with diabetes. Many signal pathways involved in this progress of which the Mitogen-activated Protein Kinases(MAPK) is the most important one.Studies have reported that the apoptosis of EPCs induced by AGEs can be inhibited while the inhibition of oxidative stress.In this experiment we are trying to explore wheather AGEs could induce the apoptosis of EPCs of SD-Rat through oxidative stress by activating the MAPK signal pathways and the mechanisms of this progress.Methods : SD rats are executed by cutting off the heads, to be put in 75% alcohol for 15 min.Then place the disinfected rats in clean workbench, separating the femur and tibia with animal anatomy instruments ?After the separation, wash the bones with PBS which contains 1% heparin steile until the marrow cavity become colorless and collect bone-marrow, centrifuge the bone-marow with high speed centrifugal, then collect cells in the bottom of the tubes. Using Ficoll density gradient centrifugation to separate the mononuclear cells from rat bone marrow cells; After the separation, the Monocytes are inoculated in 6 well-plate, Change cell medium for the first time after three days inoculation, since then, Changing cell medium for every 3 to 4 days, using inverted microscope to observe the morphological changes of the cells and EPCs were identified by the method of double swallowing of Di I-ac-LDL/ FITC-UEA-1.EPCs were treated with the medium without FBS for 24 h,then EPCs were treated with different concentrations of AGEs(50ug/l?100ug/l?200ug/l) while using the same concentration of AGEs(200ug/ml) treating for different time(6h?12h?24h?48h),then flow cytometry instrument detecting EPCs apoptosis and Western blot test cells promoting apoptosis related protein Bax and resistance to apoptosis related protein Bcl-2,we also used Western blot to detect the activation of phosphory-lated products of EPCs JNK and p38 MAPK. Molecular probe(DCFH- DA) was used to detect the changes of reactive oxygen species(ROS)content of EPCs in treating with different time(3h?6h?12h?24h) under the 200ug/ml concentration of AGEs.Then Molecular probe(DCFH- DA) was used again to detect the changes of reactive oxygen species(ROS)content of EPCs after treated with antioxidants(NAC).To continue to verify p38 MAPK and jnk pathway,respectly introduced JNK inhibitor SP600125 and p38 MAPK inhibitor SB203580 intervention cells,again using Western blot detecting Bcl-2?Bax?protein and the activation of phosphory-lated products of EPCs JNK and p38 MAPK.Results: After using the EGM-2 culture medium three days, Most EPCs were roundness, began to adhere, Seven days later, cell grows rapidly, visible under the microscope to assume the colony growth, after fourteen days, "paving stone" cells can be found. By laser confocal microscopy, cells after dealing with the Di I-ac-LDL/ FITC-UEA-1, double staining positive cells were considered to be endothelial progenitor cells.Stimulating EPCs with different concentrations(0ug/ml?50ug/ml?100ug/ml?200ug/ml)of AGEs for 24 h,cells apoptosis increased concentration-dependent,apoptosis rate are 5.94%,9.83% and 24.81%,while stimulating EPCs with the 200ug/ml concentration of AGEs for different time(6h?12h?24h?48h),cells apoptosis increased time-dependent,the apoptostis rate are 15.83%,22.75%,27.86% and 23.24%,the peak point is 24 h.The expression of promoting apoptosis related protein Bax increased time and concentration independent, however the resistance to apoptosis related protein Bcl-2 decreased time and concentration independent. As concentration of AGEs and stimulating time increased, he activation of phosphory-lated products of EPCs JNK and p38 MAPK also increased(P-P38 MAPK,0.28±0.01?0.33±0.01?0.41±0.02,vs Medium ?,P<0.01,n=3;P-JNK1.08±0.02?1.36±0.01?1.75±0.01,vs Medium ?,P<0.01,n=3),but the peak point of P-JNK is 12 h while the peak point of P-p38 MAPK is 24 h. Stimulating EPCs with the 200ug/ml concentration of AGEs for different time(3h?6h?12h?24h),the generation of ROS also increased from 3h to 12 h but degan to decrease at the time of 24 h. When pretreated with antioxidant NAC(20um), the generation of ROS and expression of promoting apoptosis protein Bax respectly decreased compared with the group of Medium, satistically significant(P < 0.01).After pretreating with SP600125(inhibitor of JNK,20um) and SB203580(inhibitor of p38 MAPK,20um),the expression of Bax and the activation of phosphory-lated products of EPCs JNK and p38 MAPK. decreased while the expression of Bcl-2 increased, all compared with the group of Medium,statistically significant(P<0.01).Conclusion:1.AGEs induces EPCs apoptosis through generation of Reactive oxygen species.2.AGEs induces EPCs apoptosis by activating p38MAPK/JNK signal pathways. |
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