Effect Of Acid Fibroblast Growth Factor On HUCB-derived Endothelial Progenitor Cells

【Objective】To investigate whether acid fibroblast growth factor(aFGF) can augument the number of endothelial progenitor cells(EPCs),enhance their biological function and inhibit their apoptosis.To further explore the mechanisms of aFGF on the role of EPCs,and find the possible induction agent,which raise the amount of EPCs in vitro.【Methods】Mononuclearcells(MNCs) from human umbilical cord blood were isolated by density gradient centrifugation,and cultured in the human fibronectin-coated culture plates in vitro. The cells were cultured by the method of adherent culture.M199 culture medium cultered it for 4 days,which adding VEGF and bFGF. PBS washed the non-adherent cells .After being cultured for 7 days, adherent cells were digested and made preparations for the experiment. The morphology of EPCs were characterized as adherent cells double positive DiI-LDL uptake and lectin binding by direct flurescent staining under a laser scanning confocal microscope,and methods immunohistochemical and double immunofluorescence techniques were used to detectⅧfactor in the study ,the cells were documented by demonstrating the expression of CD34,VEGFR-2,CD133 with flow cytometry.After being cultured for 7 days,the attached cells were stimulated with aFGF(final concentration: 0μg/L,2μg/L,5μg/L and 10μg/L ) for the respective time points(6h,12h,24h and 48h),the group of 0μg/L aFGF as control group. Then its counted under the inverted microscope. EPCs proliferation was assayed with CCK-8 kit assay.EPCs migration was assayed by modified Boyden chamber assay. EPCs adhesion assay was performed by replating cells on fibronectin-coated dishes,and then adherent cells were counted. Flow cytometry was uesd to detect cell apoptosis.The expression of Bcl-2 mRNA was detected respectively by RT-PCR at 24h.Accordingly, to explore the relationship between time and effect of the groups,respectively,and further explore the mechanism of aFGF inhibiting apotosis of EPCs.【Results】After MNCs were extracted by density gradient centrifugation, adherent cells were cultured by the method of adherent culture successfully,the attached cells were conformed to be EPCs.Compared with control group,aFGF can argument the number of EPCs and enhance the biological function of proliferation,migration,adhesion.In addition,aFGF can inhibit apoptosis of EPCs(P<0.05).In our research,5μg/L aFGF roled in EPCs for 24h can significant augment the number,improve the biological functions and inhibit apoptosis of EPCs from human umbilical cord(P<0.05). When aFGF roled in EPCs for 24h,we found to compare with control group,the different concentrations of aFGF could make the expression effect of Bcl-2 mRNA that from cord blood EPCs significantly increased(P<0.05).The most significant effect group is the group of 5μg/L aFGF.【Conclusions】1.It is feasible to isolate and culture endothelial progenitor cells from human cord blood mononuclear cells by density gradient centrifugation and adherent culture ,the attached cell was identified to be EPCs.2.aFGF can increase the number of human umbilical cord blood EPCs and improve the biological function of EPCs proliferation, migration and adhesion and inhibit their apoptosis.3.In the study, 5μg/L aFGF act on EPCs for 24h,that most biological functions and inhibit apoptosis is most notable, adhesion function of EPCs which in the aFGF 10μg/L for 12h was most obviously improvemented.4.aFGF inhibition mechanism of EPCs apoptosis may be relate to enhance the Bcl-2 genes expression.