Estrogen Promotes Proliferation And Migration Of Endothelial Progenitor Cells Via α-receptor Subtypes

bjective:Aim is to study the effect of estrogen(E2) and estrogen receptor(ER) agonists PPT(selective ERa agonist) and DPN(selective ERβagonist) on proliferation and migration of endothelial progenitor cells(EPCs).Ivestigate the role of E2 and the relationship to its receptor subtypesMethods:(1)The mononuclear cells separation:60 ml cord bloods were collected from placenta of healthy women with full term pregnancy,common heparin anticoagulation (by 20IU per 1ml), Total mononuclear cells (MNCs) were isolated by gelatin precipitation combination density gradient centrifugation, the calculation of cell viability (dead cells were the blue,live cells were not colored),the number of viable cells counted for more than 98% can be vaccinated,finished above in four hours after blood samples were 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 1 cm2,adding 10% fetal bovine serum,1% mycillin(by 10,000 units per 1 ml),VEGF 50 ng/ml,b-FGF 1ng/ml.The cells were cultivated in 5% CO2,saturated humidity,37℃incubation box.Because the fibroblasts were less than 24 hours adherent,the non-adherent cells after 24 hours continued to cultivate to the fourth days,w-ashed out non-adherent cells with phosphate buffer,changed the culture mediu-m to continue to cultivate to the seventh day,further washed out the non-adher ent cells with phosphate buffer, adherent cells for experimental use.(3)EPCs id entification:7d attached cells were detached and stained with DIL-ac-LDL,FIT C-UEA-1,CD133,and observed under fluromicroscope.(4)Investigate the effect of different concentrations of E2,PPT and DPN on proliferation and migration of EPCs. EPCs were seeded onto the the upper of Coster Transwell (membrane diameter of 6.5mm, aperture 8um)and 600ul culture medium with different concentrations of E2,PPT,DPN were added into lower layer.(experiment located a pairs of hole). Manual counting calucated the number of EPCs migrated toward lower surface under high magnification and investgated the effect of different concentrations of E2,PPT and DPN on migration of EPCs.Results:(1) Attached cells were observed after 4 days and cluster growth were noticed after 7days, it can be seen more blood island-like cell clusters after 9days. More than 90% attached cells were double positive fluorescenceand CD 133 green positive fluorescence according immu-neofluorescence.(2)WST showed that E2 and PPT can promote the proliferation of EPCs presenting concentration-dependent,DPN had no effect.The absorption values at 450 nm of EPCs in responsed to E2 of the concentration of 1×10-10mol/L,1×10-9mol/L,1×10-8mol/L,1×10-7 mol/L were 0.3466±0.0132,0.5083±0.016, 0.5397±0.020,0.4785±0.0189,under the same concentration PPT were 0.3265±0.1290,0.4503±0.0210,0.5040±0.0162,0.4272±0.0220, which had significantly statistically significant (P<0.05) compared with the control group 0.2961±0.0106;At the same concentration DPN were 0.2963±0.0141,0.3129±0.0483,0.2951±0.013,0.3016±0.0241,which had no significantly statistically significant (P>0.05) compared with the control group; at the same concentration E2 and PPT had significantly statistically significant (P<0.05) compared with DPN and E2 had significantly statistically significant (P<0.05) compared with PPT.(3)Manual counting test showed the cell numbers were increased in response to E2 and PPT of 1×10-8mol/l,the cell number were (2.83±0.29)×105,(2.56±0.11)×105,which had signifycantly statistically significant (P<0.05) compared with the control group (2.21±0.09)×105.At the same concentration DPN was (2.21±0.99)×105 which had no significantly statistically significant (P>0.05) compared with the control group; at the same concentration E2 and PPT had significantly statistically significant (P<0.05) compared with DPN and E2 had significantly statistically significant (P< 0.05) compared with PPT.(4).Cell cycle analysis disclosed that S phase EPCs increased in response to E2 and PPT,the percentage were (17.26±3.89)%,(12.1±1.84)%, which had significa ntly statistically significant (P<0.05) compared with the control group (2.93±0.15)%; DPN was (3.37±0.64) %,which had no significantly statistically significantly (P>0.05) compared with the control group;the percentage of G1 phase EPCs in response to E2 is (72.5333±6.2645)%,which had significantly statistically significant (P<0.05) compared with control group (87.2667±7.9758)% and DPN(87.6±0.7104) %; E2 had significantly statistically significant (P<0.05) compared with PPT (77.3±5.2602)%.(5)Transwell assay revealed that E2 and PPT enhanced EPCs migration and the peak impact occurred with the concentration 1×10-8mol/l, the cell numbers migrated toward lower layer in responsed to E2 of 1×10-10mo 1/L,1×10-9mo 1/L,1×10-8mol/L,1×10-7mol/L of were 35.64±4.95,44.26±3.93,50.10±6.22,39.81±4.73,at the same concentration PPT were 29.00±3.61,40.06±4.68,43.47±4.78,35.18±2.71,which had significantly statistically significant (P< 0.05) compared with the control group 18.78±1.35;DPN were 18.97±2.01,22.13±4.14,24.72±3.43,20.48±4.35, which had no significantly statistically significant (P>0.05) compared with the control group; at the same concentration E2 and PPT had significantly statistically significant (P<0.05) compared with DPN and E2 had significantly statistically significant (P<0.05) compared with PPT.Conclusion:E2 and PPT can enhance the proliferation and migration and increase the numbers of EPCs in S phase, DPN has no such effect.E2 promotes proliferation and migration of endothelial progenitor cells via a-receptor subtypes.