人脐带血浆细胞样树突状细胞亚群的体外诱导及鉴定

发布时间：2018-01-25 08:00

本文关键词： 胎血树突细胞细胞培养技术　出处：《临床肝胆病杂志》2015年11期 　论文类型：期刊论文

【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)100ng/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2μg/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]:Objective to establish a plasmacyte-like dendritic cell (PDC). Methods 40 ml umbilical cord blood was collected from the first affiliated hospital of Hunan University of traditional Chinese Medicine from June 2014 to February 2015. Umbilical cord blood mononuclear cells (CBMC) were isolated and 100ng / ml with recombinant human FMS like tyrosine kinase 3 ligand Flt3-Ln / ml. The complete RPMI1640 medium of IL-310 ng/ml was cultured continuously for 7 days, and the solution was changed every 1 and a half days. On the 8th day, 2 渭 g / ml CpG ODN was added. After 24 hours, the cells were collected for flow detection and the level of IFN- 伪 in the supernatant of pDC culture was detected. The morphological changes of pDC in culture holes were observed. Results after 2 h of CBMC culture, round flat cells were found to be covered with visual field for 24 h. After 24 hours, the cells began to adhere to the wall, the cytoplasm was expanded and the volume increased. Small clusters forming round, transparent, and visible; On the 3rd day of culture, the cell volume continued to increase, most of the cells were round, some cells had small protuberances on the surface, and a small number of fusiform, tadpole, stellate or other irregular cells could be seen. The colony in the culture medium was obviously increased and increased compared with the former. On the 5th day of culture, the number of colony and the number of cells in the colony gradually decreased, while the number of circular or small protuberant suspension cells in the culture medium increased gradually. Flow cytometry showed that CD123 and BDCA-2 were detected by flow cytometry. All BDCA-4 positive cells, which were pDC. the proportion of pDC increased continuously during the culture process, which accounted for only 1.08% of the total number of CBMC at the beginning of culture. On the 4th day, it rose to 5.32 and reached the peak on the 8th day, 19.8. at the 8th day of cultivation. The level of IFN 伪 in the supernatant of pDC culture was 11 302.61 卤1745.31 g / ml. Conclusion Human CBMC was used as the initial culture cell. The combination of Flt3-L and IL-3 could induce pDCin successfully in vitro.
【作者单位】：湖南中医药大学第一附属医院传染科;
【分类号】：R329.2
【正文快照】： 每年约有100万人死于HBV感染所致的肝衰竭、肝硬化和肝细胞癌[1-2]。HBV在体内持续存在的机制十分复杂,其中机体细胞免疫功能低下是导致病毒不能有效被清除的重要原因。树突状细胞(dendrit-ic cell,DC)是目前已知的功能最强、唯一能激活初始T淋巴细胞的抗原递呈细胞(antigen p

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