Estrogen Promotes Proliferation And Migration Of Endothelial Progenitor Cell Via MAPK Signal Pathway

Objective：Aim is to study the effect of mitogen-activatied protein kinase（MAPK）, extracellular-signal regulated protein kinase （ERK），c-JunN-terminal kinase（JNK）/Stress-activated protein kinase（SAPK）andP38on proliferation and migration of endothelial progenitor cell（EPCs）stimulated by Estrogen.Method：（1）The mononuclear cells separation：50ml cord blood werecollected with an empty needle wetted by2ml heparin（50IU/ml），precipitated with16ml gelatin(cord blood: gelatin=3:1)， totalmononuclear cells were isolated by combination density gradientcentrifugation after added with human lymphocyte separation，mediumcalculated the cell viability，the number of viable cells countedfor more than98%could be vaccinated.（2）induction of differentiation：Because the fibroblasts were attached less than24hours，so washed outnon-adherent cells and suspended in1ml M199culture mediun with4×10~6/cm~2density of cells and plated on fibonectin-coated24wellculture plate，adding10%fetal bovine serum，1%mycillin（by10,000units/ml），VEGF50ng/ml，b-FGF1ng/ml.The cells were cultivated in5%CO2，saturated humidity，37℃incubation box，changed the culturemedium to continue to cultivate to the seventh day，adherent cells for experimental use.（3）EPCs identification：attached cells were detachedand stained with CD133，DIL-ac-LDL，FITC-UEA-1，then observedunder inverted phase contrast microscope and immuno-fluoroscope.（4）Experimental Classification：The experiment groups are designed as①EPCs②EPCs+E2③EPCs+PD98,059+E2④EPCs+SP600125+E2(5)ObservationIndicator：①viable cell count of every group were determined by enzymelinked immunosorbent assay②Selected specific concentrations and Cellcycle changes of every group were analysed by fluorescence activatedcall sorting (FACS) analysis③cell suspension were seeded onto theupper of Coster Transwell and chemokines were added into lower layer.Manual counting culucated the number of EPCs migrated toward lowersurface.Results：（1）mononuclear cells manifested as spherical and radiolucentcells under inverted phase contrast microscope，the percent of viable cellswere proved to be95%by Trypan Blue Staining.（2）More than95%attached cells were CD133green positive fluorescence accordingimmu-neofluorescence after cultured7days. After cultured9days，endothelial cells specific antibodies were manifested red withDil-ac-LDL and green with FITC-UAE-1，double manifested yellow withDil-ac-LDL and FITC-UAE-1.(3) CCK-8test: the absorbance at450nmof E2group was0.6940±0.0143,which had marked statistical significance(P<0.05) compared with control group0.4163±0.0503; theabsorbance at450nm of PD98,059of concentration of5umol/L、10umol/L、20umol/L、40umol/L、80umol/L were0.7406±0.0376、0.6177±0.1043、0.5211±0.0793、0.5032±0.0975、0.4522±0.0406.Compared with E2group, the absorbance of PD98,059of concentrationof20umol/L、40umol/L、80umol/L showed marked statisticalsignificance(P<0.05); the absorbance at450nm of SP600125ofconcentration of1umol/L、25umol/L、50umol/L、75umol/L、100umol/Lwere0.6152±0.0406、0.4255±0.0395、0.3882±0.0185、0.4090±0.0761、0.4168±0.1012. Compared with E2group, the absorbance of SP600125of concentration of25umol/L、50umol/L、75umol/L、100umol/L hadmarked statistical significance(P<0.05); the absorbance at450nm ofSB203580of concentration of5umol/L、10umol/L、15umol/L、20umol/L、25umol/L were0.6670±0.6223、0.6350±0.8311、0.6312±0.6437、0.6285±0.6462、0.6500±0.6036. Compared with E2group, the absorbance of SB203580of concentration of all above had nostatistical significance(P>0.05).(4) cell cycle analysis: S phase percentageof DNA in cell shows the ability of proliferation. The S phase percentageof E2group was （33.15±0.212）%, which had marked statisticalsignificance(P<0.05) compared with control group （11.55±0.212）%.The S phase percentage of PD98,059group was（16.25±6.434）%, whichhad marked statistical significance(P<0.05) compared with E2group. The S phase percentage of SP600125was （12.55±1.202）%, which hadmarked statistical significance(P<0.05) compared with E2group. The Sphase percentage of SB203580was（24.80±6.223）%, which had nostatistical significance(P>0.05) compared with E2group.(5) Transwellassay: the cell numbers of lower layer shows the ability of immigration.The cell numbers of lower layer of E2group was65.20±12.617, whichhad marked statistical significance(P<0.05) compared with control group22.80±8.643. The cell numbers of lower layer of PD98,059ofconcentration of5umol/L、10umol/L、20umol/L、40umol/L、80umol/Lwere为54.00±11.576、42.40±7.057、42.00±7.416、29.40±5.640、22.80±5.263, and compared with E2group the concentration of20umol/L、40umol/L、80umol/L had marked statistical significance(P<0.05). The cell numbers of lower layer of SP600125ofconcentration of1umol/L、25umol/L、50umol/L、75umol/L、100umol/Lwere56.00±12.390、44.00±9.027、50.40±14.484、51.80±17.513、68.40±5.320, and compared with E2group the concentration of25umol/L、50umol/L、75umol/L had marked statistical significance(P<0.05). The cell numbers of lower layer of SB203580of concentrationof5umol/L、10umol/L、15umol/L、20umol/L、25umol/L were62.40±7.830、59.40±11.330、55.80±7.050、61.20±9.038、60.40±7.128,and compared with E2group the concentration of all above had nostatistical significance (P>0.05). Conclusion:(1) Estrogen can enhance the ability of proliferation andimmigration of endothelial progenitor cell.(2) The effect of estrogen onendothelial progenitor cell may related to extracellular-signal regulatedprotein kinase and c-Jun N-terminal kinase; P38can not interrupt theeffect of estrogen on normal endothelial progenitor cell.