EP4受体激动剂可通过激活Wnt/β-catenin信号途径促进人CD34+细胞体外增殖

摘要

目的:探讨前列腺素E2特异性受体激动剂4(EP4A)促进人CD34+细胞体外增殖的可能信号途径.方法:收集中山一院血液科20例健康供者经G-CSF动员后的外周血造血干细胞采集物.应用免疫磁珠法分选出人CD34+细胞,应用CCK8法明确EP4A促进人CD34+细胞体外增殖的最佳条件(最佳浓度和最佳作用时间);在此条件下EP4A作用于人CD34+细胞后,用实时荧光定量PCR(qRT-PCR)检测β-catenin mRNA水平的变化、Westernblot检测β-catenin和P-GSK-3β蛋白表达情况.结果:EP4A 10 μmol/L、在培养72 h能明显促进人CD34+细胞体外增殖,增殖率为对照组的1.36倍(P =0.002).最佳条件EP4A作用于人CD34+细胞后,β-catenin mRNA及其蛋白的表达增高,GSK-3β蛋白的磷酸化增加,而这些作用均可被相应的抑制剂(EP4AA)所拮抗.结论:EP4A可通过激活Wnt/β-catenin信号途径促进人CD34+细胞体外增殖.%Objective:To investigate the potential signaling pathway that regulates the proliferation of human CD34 + cells stimulated by prostaglandin E2 receptor 4 agonist (EP4A) in vitro.Methods:Twenty samples of peripheral blood containing stem cells were collected from the G-CSF mobilized healthy donors in our department of hematology.Human CD34 + cells were isolated by magnetic activated cell sorting (MACS) microbeads kit.The Cell Counting Kit-8 (CCK8) assay was used to determine the optimal concentration and time of EP4A to promote human CD34 + cell proliferation in vitro.Under the optimal condition,quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect mRNA level of β-catenin,and Western blot was used to assay protein expression of β-catenin and P-GSK-3β in human CD34 + cells treated with EP4A.Results:Culturing with 10 μmol/L EP4A for 72 h,it was found that EP4A promoted human CD34 + cell proliferation significantly,and the proliferation rate of human CD34 + cells was 1.36 times higher than that of the control(P =0.002).Under the optimal condition,it was also found that EP4A enhanced the β-catenin expression at both mRNA and protein levels,and up-regulated phosphorylation of GSK-3β in human CD34+ cells,but these effects could be inhibited by the EP4A antagonist EP4AA.Conclusion:EP4A can enhance human CD34 + cell proliferation in vitro by activating Wnt/β-catenin signaling pathway.