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目的:探讨正常人外周血造血干细胞(PB-HSC)分泌的微泡(microvesicle,MV)的免疫调节功能及对造血集落形成的影响。方法:采用密度梯度离心法分离PB-HSC并培养,收集第48 h上清液,使用超速离心法分离提取MV。通过电子显微镜观察MV形态;采用BCA法定量检测MV蛋白分泌量;采用流式细胞术检测其表面标志物。将MV与正常人外周血单个核细胞(PB-MNC)共培养,共培养12 h后通过共聚焦扫描电子显微镜观察MV与PBMNC的作用方式;共培养48 h后采用酶联免疫吸附试验法(ELISA)法检测上清中IL-2,IL-6,IL-8,IL-10,IFN-γ及TNF-α的分泌量;采用流式细胞仪检测T细胞亚群、T细胞激活变化及不同亚群细胞胞内细胞因子染色情况。用甲基纤维素半固体培养基检测MV及MV与PB-MNC共培养48 h后上清液对外周血造血干细胞集落形成的影响。结果:电子显微镜观察分离得到的MV为卵圆形膜性小囊泡,BCA法检测MV蛋白分泌量为29-110μg。流式细胞术测得MV带有混合标志,其中高表达特异性标志CD63(85.86%)与干细胞标志CD34(33.52%)。共培养12 h后共聚焦扫描电子显微镜显示,MV与PB-MNC二者相融合。共培养48 h后,ELISA法检测结果表明,MV可促进PB-MNC分泌IL-6,IL-8,IL-10与TNF-α,而IL-2和IFN-γ水平均无变化明显。流式细胞术结果显示,T细胞亚群及T细胞的激活无明显改变。胞内因子染色结果表明,CD11c+细胞内IL-8因子显著增多。集落培养实验表明,MV及MV与PB-MNC共培养48 h后上清液可促进造血集落形成。结论:PB-HSC来源的MV具有免疫调节及促进造血集落形成的作用。
Objective: To investigate the immunomodulatory function of microvesicle (MV) secreted by normal human peripheral blood hematopoietic stem cells (PB-HSC) and its effect on hematopoietic colony formation. Methods: PB-HSCs were isolated by density gradient centrifugation and cultured for 48 h. The supernatant was collected and the MVs were isolated by ultracentrifugation. MV morphology was observed by electron microscopy; MV protein secretion was quantitatively determined by BCA method; and surface markers were detected by flow cytometry. The MVs were co-cultured with normal PBMCs and co-cultured for 12 h, then the mode of action of MV and PBMNC was observed by confocal scanning electron microscopy. After co-cultured for 48 h, enzyme-linked immunosorbent assay The levels of IL-2, IL-6, IL-8, IL-10, IFN-γ and TNF-α in the supernatant were measured by ELISA. The changes of T cell subsets and T cell activation were detected by flow cytometry Intracellular cytokine staining in different subpopulations. The effect of supernatant on the colony formation of peripheral blood hematopoietic stem cells was examined with methylcellulose semi-solid medium 48 hours after co-culture of MV, MV and PB-MNC. Results: The MV obtained by electron microscopy was oval membranous vesicles. The amount of MV protein secreted by BCA was 29-110μg. Flow cytometry measured MV with mixed markers, including high expression of specific markers CD63 (85.86%) and the stem cell marker CD34 (33.52%). Confocal scanning electron microscopy 12 h after co-culture showed that both MV and PB-MNC were fused. After co-cultured for 48 h, the results of ELISA showed that MV could promote the secretion of IL-6, IL-8, IL-10 and TNF-α by PB-MNC, while the levels of IL-2 and IFN- Flow cytometry showed no significant changes in T cell subsets and T cell activation. The results of intracellular factor staining showed that IL-8 was significantly increased in CD11c + cells. Colony culture experiments showed that the supernatant could promote hematopoietic colony formation after MV and MV and PB-MNC co-cultured for 48 h. Conclusion: PB-HSC-derived MVs play an immunomodulatory role in promoting hematopoietic colony formation.