外周血造血干细胞来源微泡的生物学特性

Biological Characteristics of Microvesicles Secreted by Human Peripheral Blood Hematopoietic Stem Cells

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 effects of microvesicles （MV） isolated from human peripheral blood hematopoietic stem ceils （PB-HSC） on immune regulation and hematopoiesis. Methods： PB-HSCs were separated by density-gradient centrifugation and cultrued. The supernatants of PB-HSC at 48 h were harvested for isolation and purification of MV by using ultracentrifugation. The electron microscopy was used to observe the morphology of MV. The protein level in MV was quantified through bicinchoninic acid （BCA） protein assay. Flow cytometry was used to detect the immunophenotype of MV. Human peripheral blood mononuclear cells（PB-MNC） were isolated from healthy donor and treated with isolated MV. After being co-cultured for 12 h, confocal microscopy was used to observe the action mode of MV on PB-MNC. After being co-cultured for 48 h, the levels of IL-2, IL-6, IL-8, IL-10, IFN-7 and TNF-et were detected by ELISA. Flow cytometry was used to detect the changes of T cell subsets and the activation of T cell subsets as well as intracellular cytokine staining after co-culture for 48 h. The methylcellulose was used to assess the hematopoiesis-supportive function of MV as well as co-cultured supernatants. Results： The eletron microscopy revealed that MV were elliptical membrane vesicles. The protein amount in MV ranges from 29 to 110 p.g. Flow cytometry showed that MV expressed mix markers on the surface, especially highly expressed MV specific marker CD63 （85.86%） and hematopoietic stem cell marker CD34（ 33.52% ）. After being co-cultured for 12 h, confocal microscopyshowed that MV were merged with PB-MNC. After being co-cultured for 48 h, ELISA showed that the secretion of cytokines IL-6 ,IL-8 ,IL-10 as well as TNF-a was increased while the level of IL-2 and IFN-a was not changed much. The results of flow cytometry showed that there was no significant change in T cell subsets and T cell activation. Staining of intracellular factor showed that IL-8 was increased significantly in CDI 1 a cells. The colony-forming experiments revealed that MV and the co-cultured supernatants could facilitate the colony formation. Conclusion： MV isolated from PB-HSC have immune -reulatery function and can promote hematopoiesis.