中国免疫学杂志Issue(5)：633-638,6.DOI:10.3969/j.issn.1000-484X.2014.05.014
小鼠骨髓源耐受性树突细胞的分离培养及功能鉴定
Isolated culture and functional identification of mouse bone marrow derived tolerogenic dendritic cells
摘要
Abstract
Objective:To establish the methods of isolated culture and functional identification of mice bone marrow derived tolerogenic dendritic cells (CD11b+F4/80 +TDCs) in vitro.Methods: Mice bone marrow cells were isolated and cultured to obtain iDCs with the simulation of mouse rmGM-CSF and rmIL-4.CD11b+F4/80 +TDCs were purified by fluorescence-activated cell sorting on day 6.The morphological changes of TDCs were observed with the inverted microscope dynamically .The expression of CD11b+F4/80 +TDCs were analyzed by the flow cytometry .Tolerogenic function of CD11b+F4/80 +TDCs was evaluated by the expression of MHCⅡ, CD83, IDO, TLR-2, IL-10 and TGF-β1.The expression of MHCⅡ was analyzed by the flow cytometry , and the expression of CD83, IDO and TLR-2 were analyzed by immune-histochemistry.The levels of IL-10 and TGF-β1 in the supernatant of CD11b+F4/80 +TDC were analyzed by ELISA .Meanwhile mature DCs ( mDCs) induced by LPS were used as control .Results:The fresh isolated bone marrow cells look like round and small under microscope .After two days of culture , cells became big and formed into clusters . Five or six days later, cells clusters increased, and the morphology of cells became irregular .At the same time, more dendrite ap-peared on the surface of cells .The percentage of CD11b+F4/80 +TDCs induced by rmGM-CSF and rmIL-4 was about 23%, and the purity of the purified BM CD11b+F4/80 +iDC was about 99%.Compared with mDCs, CD11b+F4/80 +TDCs expressed low levels of MHCⅡand CD83 and high levels of IDO, TLR-2, IL-10 and TGF-β1.Conclusion:CD11b+F4/80 +TDCs derived from mouse bone marrow could be induced successfully by rmGM-CSF and rmIL-4 in vitro.CD11b+F4/80 +TDCs showed tolerogenic function by the expressions of IL-10, TGF-β1, IDO and TLR-2.关键词
耐受性树突细胞/骨髓源/分离/功能鉴定Key words
Tolerogenic dendritic cells/Bone marrow-derived/Isolation/Functional identification分类
医药卫生引用本文复制引用
付静静,盛康亮,李影,李培培,汪庆童,陈镜宇,吴华勋,张玲玲,魏伟..小鼠骨髓源耐受性树突细胞的分离培养及功能鉴定[J].中国免疫学杂志,2014,(5):633-638,6.基金项目
本文为国家自然科学基金（No．81330081，31100640，81173075）和省自然科学基金（No．11040606M195）资助项目。 （）
