【摘要】 【目的】明确介导前列腺素E2(PGE2)促进人CD34~+细胞增殖的具体受体亚型。【方法】收集2013年6月至2014年10月期间18例健康志愿者经G-CSF动员的外周血,采用免疫磁珠法分选人CD34~+细胞,并用流式细胞术鉴定其纯度;用q RT-PCR检测经PGE2作用后的人CD34~+细胞中PGE2四个亚受体(EP1~EP4)m RNA表达情况;根据处理方法不同,将人CD34~+细胞分为对照组、前列腺素E2(PGE2)组、前列腺素E2受体EP2激动剂(EP2A)组、EP2A~+前列腺素E2受体EP2拮抗剂(EP2AA)组、前列腺素E2受体EP4激动剂(EP4A)组及EP4A~+前列腺素E2受体EP4拮抗剂(EP4AA)组,每组3例,分别接受上述组药物处理,采用集落形成实验检测红系集落形成单位CFU-E、红系爆式集落形成单位BFU-E、粒系集落形成单位CFUG、单核系集落形成单位CFU-M、粒-单核系集落形成单位CFU-GM及粒、红、单核、巨核系集落形成单位CFU-GEMM集落形成情况;用流式细胞术检测细胞周期分布;用Real time-PCR检测CD34~+细胞内survivin-m RNA水平、Western blot检测胞浆内β-catenin和survivin蛋白表达情况。【结果】人CD34~+细胞可表达PGE2的四个亚受体,但主要表达受体EP2和EP4;PGE2作用后,EP2和EP4m RNA表达量较对照组明显升高,而EP1和EP3的m RNA表达变化不明显;EP4A可促进人CD34~+细胞各系造血集落形成;EP4A可促进人CD34~+细胞进入细胞周期,使G2/M期比例增加;EP4A可促进凋亡抑制蛋白survivin的m RNA和蛋白表达、β-catenin蛋白表达。【结论】前列腺素E2体外主要通过受体EP4促进人CD34~+细胞增殖。更多还原

【Abstract】 【Objective】 To identify the receptors involved in PGE2 promoting human CD34~+cells proliferation. 【Methods】 18 samples of peripheral blood mobilized by G-CSF from 2013.6~2014.10 was collected. Human CD34~+cells were isolated by magnetic activated cell sorting(MACS) microbeads kits, and the purity of CD34~+cells was identified by flow cytometry. Four PGE2receptors(EP1~ EP4) expressed on human CD34~+cells which were treated with dm PGE2 were detected by q RT-PCR. The human CD34~+cells were divided into 6 groups. Each group cells were treated with control, PGE2, EP2agonist(EP2A), EP2A~+EP2antagonist(EP2AA),EP4 agonist(EP4A) and EP4A~+EP4antagonist(EP4AA), respectively. Twenty four hours later, burst forming unit-Erythrocyte(BFUE) number, colony forming unit-Erythrocyte(CFU-E)number, colony forming unit-Granulocyte(CFU-G) number, colony forming unit-Monocyte(CFU-M) number, colony forming unit-Granulocyte monocyte(CFU-GM) number and colony forming unit-Granulocyte,Erythrocyte, Monocyte and Megakaryocyte(CFU-GEMM) were evaluated by colony-forming assay. Furthermore, cell cycledistribution was analyzed by flow cytometry, and the expression of survivin m RNA,β-catenin and survivin protein of human CD34~+cells was detected by q RT-PCR and Western blot,respectively. 【Results】 Human CD34~+cells could express four PGE2 receptors.While the relative expression of EP2 and EP4m RNA were significantly higher than EP1 and EP3m RNA. The EP2 and EP4m RNA relative expression in human CD34~+cells which were treated with dm PGE2 were significantly up-regulated compared with control groups, while the EP1 and EP3m RNA relative expression had no obviously changed. After exposured to PGE2, EP2 A, EP2A~+EP2AA,EP4 A and EP4A~+EP4AA, we observed that EP4 A stimulates CFU-E, BFU-E, CFU-GEMM, CFU-G, CFU-M and CFU-GM formation,while EP4 AA plays a negative effects on those colony formations. We also found that EP4 A induces human CD34~+cells entering into cell cycle(G2/ M phase) from rest period(G0/ G1phase). In addition, we found that EP4 A elevates survivin m RNA, survivin and β-catenin protein expression by real time-PCR and Western blot analysis. 【Conclusion】 PGE2 enhancing human CD34~+cells proliferation could mainly mediate by EP4 prostanoid receptor.更多还原