| Abstract:Objective:To investigate the protective effects of quercetin (Que) on the cultured endothelial progenitor cells (EPCs) following oxidative injury induced by oxygen free radical, and tried to elucidate their possible mechanism.Methods:(1) the mononuclear cells separation:60 ml human umbilical cord blood was collected from placenta of healthy women with full term pregnancy,common heparin anticoagulation (by 20 IU per 1ml), total mononuclear cells were isolated by gelatin precipitation combination density gradient centrifugation, the calculation of cell viability (dead cells were blue, living cells were not colored),the number of viable cells counted for more than 98% could be vaccinated,and finished above in four hours after blood samples being collected.(2) induction of differentiation:The cells were suspended in 5 ml M199 culture medium with 4×106/cm2 density of cells and plated on fibronectin-coated 25 cm2 culture flask(by 4μg per 1cm2),adding 10% fetal bovine serum, 1% mycillin (by 10,000 units per 1 ml),VEGF 50ng/ml,b-FGF 1ng/ml.The cells were cultivated in 5% CO2,saturated humidity,37℃incubation box. Because the fibroblasts were attached less than 24 hours,the floating cells after 24 hours were continued to cultivate to the fourth day,then washed out non- adherent cells with phosphate buffer,and changed the culture medium to went on cultivating until the seventh day,further washed out the non- adherent cells with phosphate buffer,after all above,the attached cells could be used for experiments.(3)EPCs identification:using immunofluores-cence assay to detect cell epitope of anti-CD133;using specific antibodies of EPCs Dil-ac-LDL and FITC-UEA-1 to detect adherent cells by immunofluor-escence assay.(4) groups and intervention:after being cultured 7days later,the collection of adherent cells having digested into cell suspension by 0.25% trypsin were counted and randomly divided into normal control group;Que (60μmol/L,90μmol/L,120μmol/L)group;H2O2(500μmol/L)group;Que(60μmol/L ,90μmol/L,120μmol/L)+H2O2(500μmol/L)group.cultured in 96-well or 6-well plates for 24 hours,the proliferation and apoptosis of EPCs were asssyed.(5) EPCs proliferation observation:each group had six parallel holes and then the cell suspension had been vaccinated in the 96-well plates,about 2000 cells/well,WST-1 assay was used to detect the effect of Que on the 24 hours and 48 hours proliferation ability of EPCs exposed to H2O2 or not. (6) EPCs apoptosis observation:the use of fluorescein isothiocyanate-labeled Annexin-v, propidium iodide(PI)double staining immunofluorescence method observed EPCs apoptosis, Flow cytometry was used to evaluate the apoptotic rate of EPCs induced by H2O2.(7) the research of apoptosis mechanism:the p-JNK expression of control group,H2O2 group and Que+H2O2 group were detected by immunocytochemical method,then Flow cytometry was used again to detect the apoptotic rate of EPCs pretreating with JNK agonist Anisomycin 10μmol/L in each group. Results:(1) compared with the control group,Que at 60-90μmol/L could stimulate EPCs proliferation,but Que at 120μmol/L could inhibit the proliferation of EPCs, the difference had statistical significance (P<0.05), compared with the 24 hours,the EPCs had lower proliferation ability at the 48 hours,the difference had statistical significance(P<0.05). (2) compared with the control group,H2O2 group significantly impaired the proliferation of EPCs (P<0.01), pretreating with Que at 60-120μmol/L,the proliferation ability of EPCs induced by H2O2 had been improved dramatically, and it was time-dependent and dose-dependent in experiment range,contrast to the H2O2 group, the data had obviously statistical significance(P<0.01).(3) while the concentra-tion of Que gradually increased in the range of 60-120μmol/L, the apoptotic rate EPCs induced by H2O2 showed a tendency to decline,when the concentration of Que at 60μmol/L,90μmol/L,120μmol/L,the apoptosis rate was respectively(27.43±0.58)%, (23.12±0.37)%, (22.43±0.16)%, compared with H2O2 group ((36.58±0.45)%),the apoptotic rate of Que (60μmol/L 90μmol/L,120μmol/L)+H2O2 group had apparent statistical significance (P<0.01); compared with 90μmol/LQue+H2O2 group, the apoptotic rate of 120μmol/L Que+H2O2 group had no significant difference(P>0.05).(4) the p-JNK positive expression showed cytoplasm and nucleus contained yellow particles, with the increase of concentration of Que, p-JNK positive expression products were gradually decreased and the coloration became lighter.(5) Que could inhibited the increase of apoptotic rate of EPCs after pretreating with the JNK agonist, the apoptotic rate of Anisomycin+Que(60μmol/L,90μmol/L, 120μmol/L)+H2O2 group was respectively (42.36+0.53)%, (38.32±0.30) %, (35.78±0.54)%, in contrast to H2O2 group((47.35±0.64)%), the difference had statistical significance(P<0.05). Conclusion:(1) on the condition of physiology, low concentration Que could promote the EPCs proliferation,but high concentration Que could inhibit the EPCs proliferation; on the condition of oxidative stress, Que improve the ability of proliferation of EPCs in a dose-dependent manner. (2) oxidative stress increased the apoptosis of EPCs, Que can inhibit the the apoptosis of EPCs induced by H2O2.(3) the mechanism of Que inhibits apoptosis of EPCs induced by H2O2 has relation with the inhibition of JNK signal pathways. |
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