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人脐带血浆细胞样树突状细胞亚群的体外诱导及鉴定
In vitro culture and characterization of human umbilical cord blood-derived plasmacytoid dendritic cell subsets
文章发布日期:2015年11月19日  来源:  作者:彭建平,孙克伟,银丝丝,等  点击次数:1102次  下载次数:259次

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【摘要】:目的建立浆细胞样树突状细胞(pDC)的体外培养方法。 方法采集2014年6月-2015年2月于湖南中医药大学第一附属医院就诊的健康产妇脐带血40 ml,分离脐带血单个核细胞(CBMC),用含重组人FMS样酪氨酸激酶3配体(Flt3-L)100 ng/ml、白细胞介素(IL)3 10 ng/ml 的RPMI1640完全培养基连续培养7 d,每隔1天半换液。第8天加入2 μg/ml的CpG ODN,24 h后收集细胞行流式检测,同时检测pDC培养上清干扰素(IFN)α水平。在培养细胞的第1、3、5、7、8天,观察培养孔中的pDC形态特征变化。 结果CBMC培养2 h后可见圆形扁平细胞布满视野,24 h后细胞开始贴壁,细胞质伸展开,体积有所增大,圆形,透亮,并可见散在的小集落形成;培养第3~4天,细胞体积较前继续增大,多数为圆形,部分细胞表面可见小的突起,并可见少量梭形、蝌蚪状、星形或其他不规则形的细胞,培养液中集落较前明显增多、增大;培养第5~8天,集落数量及集落内细胞数逐渐减少,而培养液中圆形或具有小突起的悬浮细胞逐渐增多。流式细胞仪检测发现CD123、BDCA-2、BDCA-4均表达阳性的细胞,该细胞为pDC。在培养过程中,pDC比例不断增加,其在培养开始时仅占CBMC总数的1.08%,第 4天升至5.32%,第8天时达到高峰,为19.8%。培养第8天时,pDC培养上清IFNα水平达(11 302.61±1745.31)pg/ml。 结论以人CBMC为培养初始细胞,利用Flt3-L与IL-3联合,可成功体外诱导出pDC。
【Abstract】:ObjectiveTo establish a method for in vitro culture of plasmacytoid dendritic cell (pDC). MethodsUmbilical cord blood (40 ml) was collected from healthy parturients in the First Affiliated Hospital of Hunan University of Chinese Medicine, and cord blood mononuclear cell (CBMC) were isolated. The CBMC were cultured for 7 days with RPMI 1640 complete medium containing rh Flt3-ligand (Flt3-L) (100 ng/ml) and rh interleukin (IL)-3 (10 ng/ml), and the medium was half changed every 2 days. On the eighth day, CpG ODN (2 μg/ml) was added to the cells, and the attached cells and supernatant were collected 24 h later for flow cytometry and interferon (IFN)α measurement, respectively. On days 1, 3, 5, 7, and 8 of cell culture, the morphological changes of pDC were observed. Results After 2 h of culture, the CBMC showed circular, flat morphology. Twenty-four hours later, the cells began to adhere to the wall, with extended cytoplasm and increased volumes, and they became round and translucent, with scattered small colonies. On days 3-4 of culture, the cell volume continued increasing; most cells were round, and some had small protrusions; few cells were spindle-, tadpole-, star- or irregularly shaped; the number and volumes of colonies increased substantially. On days 5-8 of culture, the number of colonies and the number of cells in colonies gradually decreased, and suspended cells that were round or had small protrusions gradually increased in the medium. The cells expressing CD123, BDCA-2, and BDCA-4, which were considered pDC, were detected by flow cytometry. Flow cytometry revealed that the proportion of pDC in CBMC increased during the culture: increasing from 1.08% at the beginning of culture to 5.32% on day 4, and finally reaching a peak of 19.8% on day 8. On day 8, the level of IFNα in pDC culture supernatant was(11 302.61±1745.31) pg/ml. ConclusionpDC can be successfully induced in vitro by rh Flt3-L combined with IL-3 from human umbilical CBMC.
【关键字】:胎血;树突细胞;细胞培养技术
【Key words】:fetal blood; dendritic cells; cell culture techniques
【引证本文】:彭建平, 孙克伟, 银丝丝, 等. 人脐带血浆细胞样树突状细胞亚群的体外诱导及鉴定[J]. 临床肝胆病杂志, 2015, 31(11): 1862-1866.

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