S-亚硝基谷胱甘肽诱导脐带血CD34^+细胞来源的巨核细胞产生血小板

Formation of platelets from cord blood CD34^+ cells-derived megakaryocytes induced by S-nitrosoglutathione

为了探讨一氧化氮供体S-亚硝基谷胱甘肽(S-nitrosoglutathione,GSNO)对脐带血CD34+细胞分化来源的巨核细胞产生血小板的可能作用,我们采用免疫磁珠法从8例健康产妇足月顺产的胎儿脐带血中分选CD34+细胞,并在含血小板生成素(thrombopoietin,TPO,50ng/ml)、白细胞介素-3(IL-3,10ng/ml)、干细胞因子(stemcellfactor,SCF,50ng/ml)和重组人粒-巨噬细胞刺激因子(rHuGM-CSF,20ng/ml)的无血清培养基中培养14d。随后,用免疫磁珠法分选CD61+细胞。CD61+细胞在含有(实验组)和缺乏(对照组)GSNO(20mg/ml)的无血清培养基[含TPO(50ng/ml)、IL-3(10ng/ml)、SCF(50ng/ml)]中培养不同时间(30min、2h)。采用流式细胞仪检测培养体系中的血小板数;电子显微镜观察巨核细胞的形态学;倒置显微镜和流式细胞仪观察凝血酶诱导的血小板凝集;ELISA方法检测巨核细胞中cGMP的含量。结果显示,与对照组比较,实验组血小板数量明显增加(P<0.05);电子显微镜下可见巨核细胞有明显伪足形成和突出;凝血酶诱导后在倒置显微镜和流式细胞仪上均可观察到血小板凝集现象;GSNO作用后巨核细胞中的cGMP明显升高(P<0.05)。这些结果提示,GSNO可以促进脐带血CD34+细胞来源的巨核细胞产生具有一定功能的血小板,其产生的机制可能部分与cGMP途径有关。

To investigate the effect of S-nitrosoglutathione （GSNO）, a nitric oxide donor, on platelet production from megakaryocytes differentiated from cord blood CD34^＋ cells in vitro, the CD34^＋ cells from eight fresh umbilical cord blood samples by a high-gradient magnetic cell sorting （MACS） system were cultured in serum-free medium for 14 d with thrombopoietin （TPO） 50 ng/ml, IL-3 10 ng/ml, stem cell factor （SCF） 50 ng/ml and rHuGM-CSF 20 ng/ml. Then, CD61^＋ cells were purified by MACS system from these CD34^＋ cells, and were cultured in serum-free medium supplemented with TPO 50 ng/ml, IL-3 10 ng/ml and SCF 50 ng/ml in the presence （treatment group） and absence （control group） of GSNO for 30 min or 2 h. Platelet-sized particles were counted by flow cytometry; megakaryocyte structure was detected by scanning electron microscope. Aggregation of the thrombin-induced platelet particle was observed under inversion microscope, cGMP was assessed by commercial ELISA kit. The results showed that, compared with the control group, the number of platelet-sized particles significantly increased （P〈0.05） in the treatment group, in which megakaryocytes presented significant pseudopod formation and extensive membrane blebbing. The platelet particle aggregation could be observed under microscope after thrombin induction, cGMP activity was significantly increased after treatment with GSNO （P〈0.05）. These results propose that GSNO can facilitate platelet production from megakaryocyte, and it may be partly through cGMP pathway.