体外诱导培养健康成人外周血单核细胞源树突状细胞及鉴定

目的：建立体外从健康成人外周血单核细胞（Mo）诱导培养成熟的树突状细胞（DE）的方法。方法：采用连续贴壁法分离正常人外周血单核细胞，在GM-CSF和IL-4的完全培养基中培养。培养5天后收集细胞，重新铺板后继续在TNF-α的完全培养基中培养48小时后，收集细胞和上清液，采用流式细胞术检测DC表型CD80、CD83、CD40的表达；用ELISA法检测上清液中细胞因子IL-12的浓度；用倒置显微镜动态观察DC形态变化。结果：采用连续贴壁法和用GM—CSF、IL-4和TNF—α联合培养可以从人外周血诱导培养出大量的树突状细胞，且高表达CD80、CD83、CD40，表达率分别为84．29％、90．73％、92．38％。DC分泌的细胞因子IL-12浓度为38．52±11．34pg／ml。结论：采用连续贴壁法和用GM．CSF、IL-4和TNF-α联合培养可以从人外周血诱导培养大量的树突状细胞，检测表型CD80、CD83、CD40的表达率和细胞因子IL-12浓度可以鉴定树突状细胞。

Culture and Identification of Dendritic Cells Obtained from Monocytes in Peripheral Blood in Vitro

Objective：To establish a method to generate mature dentritic cells （DC）from monocytes in peripheral blood from healthy adults in vitro. Methods：Monocytes were seperated from peripheral blood with a successive adherence method and were cultured with GM-CSF and IL-4 for 5 days. The cultured monocytes were cultured with TNF-α for another 48 h. The cultured ceils and supernatant were gathered for detection of CDS0, CD83, and CIM0 by flow cytometry and IL-12 in the supernatant by ELISA. The morphology of DC was observed with up-side-down microscope. Results： Large numbers of DC were cultured with a successive adherence method and culturing with GM-CSF, IL-4, and TNF-α. The cultured DC highly expressed CD80 ,CD83, and CD40 with a rate of 84.29% ,90.73%, and 92.38%, respectively. IL-12 in the supernatant was （38.52 ± 11.34） pg/ml. Conclusion： The successive adherence method and culturing with GM-CSF, IL-4, and TNF-α can be suitable for generating substantive DC. To detect the expression rate of CD80, CD83, and CD40 and the concentration of IL-12 may be useful to identify DC.