The Establishment Of SKOV3Co-culture With EPCs And VEGF-antibody’s Influence On EPCs In Tube Formation

Background：Ovarian cancer is one of common malignant tumor of female reproductive organs,themain treatment is surgery,but the5-year survival rate of only25%-30%.The main reason ofhigh mortality rate is the metastatis and the recurrence of ovarian cancer,the key factor of itis the tumor angiogenesis.Currently besides the accepted VEGF(vascular endothelialgrowth factor) is the main factor to promote tumor angiogenesis,Endothelial cells fromvarious sources (such as vascular endothelial cells from tumor,endothelial cells incirculation,etc.) are also important factors to promote tumor angiogenesis.EPCs(endothelialprogenitor cells) is the precursor cells of mature endothelial cells,can differentiate intoendothelial cells and participate in angiogenesis,so EPCs is also a important factor of tumorangiogenesis,so the studies of EPCs have important significance to tumor angiogenesis andinhibition.As the features of EPCs’ surface markers and differentiation,it is possible to canreduce tumor metastasis and recurrence,improve cancer patients’ quality of life and survivalrate by inhibit EPCs participating in tumor angiogenesis.The experiment observed how theovarian cancer cell lines SKOV3affect EPCs’ tube formation and it’s related proteinexpression by blocking VEGF act on EPCs,in order to provide some experiment basis forthe study of EPCs angiogenesis.Objective:Probing the methods of EPCs’ separation from umbilical cord blood,cultivation andidentification,lay the foundation of EPCs’ study.Observing influence on EPCs’ tubeformation and MMP-2/MMP-9protein expression by adding different quantities of VEGFantibody into the co-culture system of SKOV3and EPCs,deeply realize the relationshipbetween VEGF antibody and EPCs angiogenesis and provide some experiment basis for thefurther vivo study of EPCs. Materials and methods:1. EPCs’ separation from umbilical cord blood,cultivation and identificationGeted umbilical cord blood from puerpera of natural labour and cesarean who hadpermitted,then blended the HES at the ratio6:1,Separating mononuclear cells by densitygradient centrifugation,cultivated and observed cells growth in six orifice plate;Changedmedium every2days, photographed cells morphology every7days.Used the method of thedouble swallow of FITC-UEA-1/DiI-ac-LDL and FITC-CD34,FITC-CD133,PE-VEGFR2antibodies and flow cytometry detecting surface markers to identify EPCs.2.The non-contact co-culture system of SKOV3and EPCsPut0.4μm bore diameter’s Transwell on six orifice plate and inoculated SKOV3in upperchamber.Added different quantities of VEGF antibodies and divided experiment into5teams:EPCs team,EPCs+SKOV3co-culture team,EPCs+SKOV3+25μgVEGF antibody co-cultureteam,EPCs+SKOV3+50μgVEGF antibody co-culture team,EPCs+SKOV3+100μgVEGFantibody co-culture team.Removed the co-culture system after2days and used every team’sEPCs for the next experiment.3.EPCs’ tube formation and MMP-2/MMP-9protein expressionInoculated every team’s EPCs in Matrigel,cultured at37℃and calculate the EPCs’tube formation by inverted microscope after24h.Observed the EPCs’ MMP-2/MMP-9protein expression by Western-blot and RT-qPCR.Results:1. EPCs’ separation from umbilical cord blood,cultivation and identificationA small amount of cells began to deform,most of cells were drifting after inoculationthe first day.Part of cells began to deform,enlarge in fusiformis shape in the third day.Mostof cells turned round as “paving stone” shape,only just some fusiformis cells in theforteenth day.Double swallow of FITC-UEA-1/DiI-ac-LDL appeared green andred,FITC-CD34,FITC-CD133,PE-VEGFR2antibodies detecting surface markers appearedgreen,green and red; Positive rate was (2.65±0.48%),(19.40±6.35%),(64.01±1.07%).2.The non-contact co-culture system of SKOV3and EPCsSKOV3growthed in good condition,appeared fusiformis after cells recovery.When cells fused at80%-90%,passaged cells.We can observed two kinds cells growthed in goodcondition without blending after establishing the non-contact co-culture system.3.EPCs’ tube formation and MMP-2/MMP-9protein expressionThe tube number of EPC team was less than EPCs+SKOV3co-culture team(P<0.05).The tube number of EPCs+SKOV3+25μg,EPCs+SKOV3+50μg,EPCs+SKOV3+100μgVEGFantibody co-culture team were less than EPCs+SKOV3co-culture team(P<0.05),the moreantibody,the less tube number.The result of RT-qPCR and Western blot display:Protein andmRNA expression of MMP-2/MMP-9of EPC team were less than EPCs+SKOV3co-culture team(P<0.05),protein and mRNA expression of MMP-2of EPCs+SKOV3+50μg,EPCs+SKOV3+100μgVEGF antibody co-culture team were less thanEPCs+SKOV3co-culture team(P<0.05), protein and mRNA expression of MMP-9ofEPCs+SKOV3+100μgVEGF antibody co-culture team were less than EPCs+SKOV3co-culture team(P<0.05).Conclusion:The results indicated that density gradient centrifugation could separation EPCs fromumbilical cord blood, identification results demonstrate the cells were EPCs,cells functionand tube formation maintain good status.The separation method provided technique andaccumulated some experience.The experiment established the non-contact co-culture system of SKOV3andEPCs,observed VEGF antibody could inhibit EPCs’ tube formation and MMP-2/MMP-9expression by compare with conrol group.The results indicated that SKOV3’s secretionVEGF might act on EPCs and promote MMP-2/MMP-9expression,then enhanced EPCs’tube formation,it provied experiment basis for the further study of EPCs promoting tumorangiogenesis.