Effect Of A23187 On Proteins Expression Of Endothelial Progenitor Cells In Umbilical Cord Blood Using SELDI-TOF-MS

Objective Part one:To establish a culture system of Endothelial progenitor cells from umbilical cord blood. Part two:To identify the effect of A23187 with different concentration on EPCs and use SELDI protein chip for screening protein expressions of cell protein samples of EPC after intervention of A23187.Methods Part one:using density gradient centrifugation to obtain Mononuclear cells,and then culture them with our labs'culture system. In this study, immunocytochemistry and flow cytometric analysis of CD144, CD146, vWF, CD34 and CD133 were performed. Part two:Culture the cells of 7+ld in the medium in gradient concentration of A23187(1μM,3μM,5μM) for 24h. Flow cytometry analysed the percentage of cells expressing CD 146. Obtain the cells, proteins were extracted from cell samples by cell disruption buffer established in our lab, respectively. The concentrations of proteins were detected by BCA method, then protein samples were applied directly to the ProteinChips arrays using the chip of WCX2.Results Part one:The first five days were latent period, cells became adherent, but the number of cells did not increase obviously. At the 6th day, the number of cells came up to 287±45(the average number under 10X10 visual field),and the 9th day up to 282+46 (P>0.05, compared with 6th day).After cultured for 12 days, the cells came into logarithmic phase,and the number of cells were 805.33±66.61 (P<0.05, compared with 6th day),and continued to incease at day 19, the number of cells were up to11115±182 (P<0.05, compared with 6th day).Apoptosis took place at day 23, the number of cells decreased to 265±615 (P<0.05, compared with 6th day). Immunocytochemistric analysis indicated that the cells were weakly positive for vWF, CD 144 and CD 146, and the percentage of CD34 in cobblestone-shaped cells were 88.98%±5.15%(P<0.05), and that of CD133 were 1.20%±1.44%(P<0.05) with flow cytometric analysis. Part two:SELDI analysis indicated that there were 12 protein or peptide peaks overexpressed and 8 downexpressed in A23187 treated EPCs compared with the control. A23187 also reduced the percentage of cells expressing CD146, and A23187 induced EPCs'apoptosis when it's concentration came up to 10μM.Conclusions:Part one:The method established by our lab for EPCs culture is effective. The first five days is a latent period, cells become adherent, but do not increase in number obviously; and from day 12, the cells come into logarithmic phase. The number of EPCs is increasing at day 19, and then the cells undergo apoptosis at day 23. Part two:A23187 induces EPCs'apoptosis at the concentration of 10μM, and it also inhibited the differentiation of EPCs to mature endothelial cells at low concentration, and A23187 induced EPCs'apoptosis when it's concentration came uo to 10μM.The work to be caried out:To identify the proteins expressed differentially in SELDI using 2-DE and mass spectrum.