Autologous Platelet-Rich Plasma(PRP) Promotes Functional Expression Of Endothelial Progenitor Cells (EPCs)

Objective:To study the effects of platelet-rich plasma on proliferation,migration,adhesion and differentiation of endothelial progenitor cells,5o as to establish a safe and effective EPC amplification system.Methods:Platelet-rich plasma is obtained by manual centrifugation,and PRP quality is evaluated by calculating the platelet recovery and eruichment rate by the ammonium oxalate method.Endothelial progenitor cells are obtained by centrifugation in vitro and cultured in vitro.Immunological identification was performed using CD31 immunofluorescence staining,DiI-AC-LDL phagocytosis assay and FITC-UEA-1 binding assay.The experiment was basically divided into ECM,ECM+supporting growth factor,The experiment was basically divided into group ECM,ECM+supporting growth factor,ECM+PRP(10 ul/ml).ECM group only contained ECM basal medium+5%fetal bovine serum+1%Penicillin-Streptomycin Solution,without Endothelial Cell Growth Supplement(ECGS).The ECM+supporting growth factor group is the ECM complete medium.The ECM+PRP group is the ECM group with PRP extract(10 ul/ml).Cell viability was measured by MTT assay,and cell migration and adhesion were detected by Transwell chamber and cell adhesion assay.The expression of CD34 and CD45 in cells was detected by RT-PCR to analysis the effect of PRP on the differentiation of EPCs.In the RT-PCR experiment,the primitive cultured cells for 7-9 days in the complete medium are chosen as the Primary Cell group.Results:The platelet concentration in whole blood is about 164.43±14.69(×109)(N=12),the concentration of platelet concentrate is about 1045.24±99.36(×109)(N=12),the platelet recovery rate is about 79%,and the enrichment rate is about 6.36(x102).The results of CD31 immunofluorescence staining?DiI-AC-LDL phagocytosis test and FITC-UEA-1 binding assay are positive.The detected cells are confirmed as EPCs.The results of MTT assay:On the first day,the ECM group(0.18±0.139),the ECM+ECGS(0.222±0.020),and the ECM+PRP(10ul/ml)(0.22±0.198)group are statistically significant on the data(P<0.01)(N=12).There is no statistically significant between group ECM+PRP(10ul/ml)and ECM+ECGS.The group ECM+PRP(10ul/ml)and ECM+ECGS are statistically significant from group ECM.(P<0.01).On Day 7?14,group ECMM+PRP(10ul/ml),ECM+ECGS?ECM?ECM+PRP(10ul/ml)are statistically significant on the data(P<0.01).There is also statistically significance between any two of them.The adhesion assay:Group ECM±PRP(10ul/ml)(27.67±2.11),ECM+ECGS(27.37± 1.30)and ECM(18.67± 1.45)(N=12)are statistically significant on the data(P<0.01).There is statistieal significance between group ECM+PRP(10ul/ml)?ECM+ECGS and ECM(P<0.01).There is no statistical significance between group ECM+PRP(10ul/ml)and ECM+ECGS.The migration assay:Group ECM+PRP(10ul/ml)(22.13±0.93)?ECMECGS(17.43±0.53)and ECM(9.87±0.46)(N=12)are statistically significant on the data(P<0.01).It is suggested that PRP can improve the migration ability of EPC cells.RT-PCR results:In the CD34 expression,there is statistical significance between the four experimental groups(P<0.01).On the data,there is statistical significance between the First group,the ECM group and the ECM±ECGS(P<0.01).There is statistical significance between group ECM+PRP and ECM+ECGS(P<0.01).There is no statistical significance between the Primary Cell and ECM groups.In the expression of CD45,there is statistical significance between the four groups(P<0.01).There is statistical significance between the ECM+ECGS?the Primary Cell group and the ECM group(P<0.01).There is statistical significance between the ECM+PRP group and the ECM+ECGS(P<0.01).Suggesting that PRP can promote the differentiation of EPCs.Conclusion:The results of statistical analysis suggest that the platelet concentrate obtained by the Aghaloo method can reach the PRP standard.As a substitute for growth factors,PRP has a stronger ability to promote the proliferation,migration and differentiation of EPCs than ECGS,but there is no statistical significance in the effect of adhesion between PRP and ECGS to EPCs.PRP is hopeful in the culture of EPCs in vitro,as a potential alternative to ECGS.