Effects Of Stromalcell-derived-factor-1on Endothelial Progenitor Cells Of Diabetes From Peripheral Blood And PI3K/AKT Signal Transduction Mechanism

Objective：1. To observe the effects of stromalcell-derived-factor-1(SDF-1) on theendothelial progenitor cells’（EPCs）function of diabetes from peripheral blood.2. To discuss the effects of SDF-1on the EPCs whether or not be related toPI3K/AKT signaling pathway.Methods：1.10healthy people were included in healthy control group(HC group),20diabetes patients were included in diabetes mellitus group(DM group).Collect each20ml peripheral venous blood on an empty stomach,and be anticoagulated withheparin.To obtain peripheral blood mononuclear cell by means of the density gradientcentrifugation.After four days with EGM-2MV culture medium(containing5%FBS)cultivating,abandon the absorption solution,digest the EPCs with0.25%trypsin,identify the EPCs by means of DiI-ac LDL and FITC-UEA-1which arepositive with inverted fluorescence microscope,and identify the EPCs furtherly byflow cytometry instrument which can detect its surface marker(CD34,CD133andKDR). HC2group and DM2group continue to develop as above,HC1group andDM1group be cultivated by100ng/ml SDF-1until the seventh day.After sevendays,collect the EPCs. The Boyden chamber and In vitro angiogenesis kit wereanalyzed retrospectively.2. Collect20ml peripheral venous blood on an empty stomach from1healthypeople and1diabetes patient.After four days with EGM-2MV culture mediumcultivating,6holes in proper sequence are blank control、1ng/ml SDF-1、10ng/mlSDF-1、100ng/ml SDF-1、 pure AMD3100and100ng/mlSDF-1withAMD3100.The seventh day,abandon the absorption solution,wash twice withPBS,blow the adherent cells.After precipitating and centrifugating,join the cell lysissolution,collect the supernatant fluid and centrifugating1minute in high speed.Thatyou can go on with SDS-PAGE electrophoresis,and finally go on with proteinimprinting analysis. Results：1. DM2group of EPCs chemotactic migration and angiogenesis ability waslower than that in group HC2（respectively15.50±2.224vs22.20±1.304and113.87±15.198vs181.80±9.202）,the difference was statistically significant (p＜0.001).Compared with HC2group,EPCs’chemotactic migration ability（24.60±1.517vs22.20±1.304）and angiogenesis ability（197.98±10.855vs181.80±9.202）of HC1group were higer,the difference was statistically significant (p＜0.05).Compared withDM2group,EPCs chemotactic migration ability（19.30±1.337vs15.50±2.224）andangiogenesis ability（140.52±10.622vs113.87±15.198）of DM1group were enhancedobviously,the difference was statistically significant (p＜0.001).2. To detect the EPCs of AKT protein expression level in two groupsrespectively using Western blot method.For the HC group,AKT protein expression inthe blank control group and1ng/ml SDF-1group（0.83±0.008vs0.84±0.004）had nostatistically significant difference（p＞0.05）;Compared with the blank controlgroup,10ng/ml SDF-1group of AKT protein expression（0.87±0.005vs0.83±0.008）was higher,the difference was statistically significant（p＜0.05）;100ng/ml SDF-1group of AKT protein expression （0.90±0.009vs0.83±0.008） was highersignificantly,the difference was statistically significant（p＜0.001）;pure AMD3100group of AKT protein expression （0.75±0.014vs0.83±0.008） was lower,thedifference was statistically significant（p＜0.001）; Compared with100ng/mlSDF-1group,100ng/mlSDF-1with AMD3100group of AKT protein expression（0.64±0.023vs0.90±0.009）was lower especially, the difference was statisticallysignificant（p＜0.001）.For the DM group,AKT protein expression in the blankcontrol group、1ng/mlSDF-1group、10ng/mlSDF-1group and100ng/ml SDF-1group(0.71±0.011vs0.74±0.012vs0.79±0.007vs0.84±0.012) was increased bydegrees, Compared with the blank control group,1ng/ml SDF-1group of AKTprotein expression was higher,the difference was statistically significan（tp＜0.05）;10ng/ml SDF-1group and100ng/ml SDF-1group were increased obviously,thedifference was statistically significant（p＜0.001）;pure AMD3100group of AKTprotein expression （0.63±0.008vs0.71±0.011） was lower,the difference wasstatistically significant （p＜0.001）; Compared with100ng/mlSDF-1group, 100ng/mlSDF-1with AMD3100group of AKT protein expression（0.52±0.020vs0.84±0.012）was lower especially,the difference was statistically significant（p＜0.001）.Conclusions：SDF-1can increase the chemotactic migration and angiogenesis ability of theEPCs from peripheral blood, especially for the diabetes，and the effects of SDF-1onthe EPCs are related to PI3K/AKT signaling pathway.