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

Prostaglandin E2 Receptor 4 Agonist Promotes Human CD34^+ Cell Proliferation in vitro by Activating Wnt/β-Catenin Signaling Pathway

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、Western blot检测β-catenin和P-GSK-3β蛋白表达情况。结果:EP4A 10μmol/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 ＋ ceils 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 prolif- eration in vitro. Under the optimal condition, quantitative real-time polymerase chain reaction （qRT-PCR） was used to detect mRNA level of 13-catenin, and Western blot was used to assay protein expression of 13-catenin and P-GSK-313 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 f3- catenin expression at both mRNA and protein levels, and up-regulated phosphorylation of GSK-313 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/μ-catenin signaling pathway.