Dectin-1介导人急性单核细胞白血病细胞巨噬细胞样细胞吞噬白念珠菌的作用研究

Roles of Dectin-1 in phagocytosis of Candida albicans by macrophage-like cells derived from a human acute monocytic leukemia cell line THP-1

目的探讨Dectin-1受体在源于人急性单核细胞白血病细胞（THP-1）的巨噬细胞样细胞吞噬白念珠菌中的作用。方法以THP-1巨噬细胞样细胞为靶细胞，siRNA靶向下调Dectin-1受体表达，并与热灭活白念珠菌共培养，通过荧光显微镜计数、流式细胞仪检测其对白念珠菌的吞噬作用。应用SPSS19．0统计软件，采用单因素方差分析及t检验进行统计分析。结果细胞转染siRNA-Dectin-1后，Dectin-1mRNA相对表达量及蛋白表达水平明显降低（t=26．163，P〈0．001）。显微镜观察转染siRNA-Dectin-1和siRNA无义片段（NC）的THP-1巨噬细胞样细胞与白念珠菌共培养1、2、4h吞噬率分别为17．5％和22．1％、18．6％和24．3％、39．2％和59．1％，两两比较差异均有统计学意义（P〈0．05）；吞噬≥3个菌的细胞百分比分别为2．2％比4．7％、2．5％比5．4％、5．1％比8．3％，两两比较差异均有统计学意义（P〈0．05）。流式细胞仪检测显示，THP-1巨噬细胞样细胞与带有荧光的白念珠菌共培养30min、1h、2h、4h后，转染siRNA-Dectin-1组较转染siRNA-NC组平均荧光强度明显降低，分别为36．8和45．7、54.3和62．4、72．1和84．9、93．6和116．7，两两比较差异均有统计学意义（P〈0．05）。结论Dectin-1受体在巨噬细胞吞噬白念珠菌的效应中发挥重要作用。

Objective To investigate the roles of Dectin-1 in phagocytosis of Candida albicans （C. albicans） by macrophage-like ceils derived from a human acute monocytie leukemia cell line THP-1. Methods THP-1 maerophage-like cells served as the target cells, and were transfected with small interfering RNA （siRNA） targeting Dectin-1 to down-regulate the expression of Dectin-1 receptor （siRNA- Dectin-1 group）. THP-1 macrophage-like cells transfected with nonsense siRNA （siRNA-NC） served as a negative control group. After transfection, the THP-1 maerophage-like cells in the above 2 groups were co- cultured with heat-killed C. albicans separately. And then, fluorescence microscopy was performed to countTHP-1 macrophage-like cells phagocytosing C. albicans, and flow cytometry was used to determine the mean fluorescence intensity （MFI） of dihydrorhodamine （DHR）- 123 fluorescent ceils. Statistical analysis was done by one-way analysis of variance （ANOVA） and t test with the SPSS19.0 software. Results After transfection with siRNA-Dectin-1, the mRNA and protein expression of Dectin-1 significantly decreased in THP-1 macrophage-like cells （t = 26.163, P 〈 0.001）. After 1-, 2-, 4-hour co-culture of THP-1 macrophage- like cells with C. albicans, fluorescence microscopy showed that the phagocytosis rates of C. albicans by THP - 1 macrophage-like cells were significantly lower in the siRNA-Dectin- 1 group than in the negative control group （17.5% vs. 22.1%, 18.6% vs. 24.3%, 39.2% vs. 59.1%, respectively, all P 〈 0.05）, so were the percentage of THP- 1 macrophage-like cells phagocytosing more than 3 C. albicans cells （2.2% vs. 4.7%, 2.5% vs. 5.4%, 5.1% vs. 8.3%, respectively, all P 〈 0.05）. After 30-minute, 1-, 2- and 4-hour co-culture of THP-1 macrophage-like cells with DHR-123-labelled C. albicans, flow cytometry showed that the MFI of C. albicans-phagocytosing cells was significantly lower in the siRNA-Dectin- 1 group than in the negative control group （36.8 vs. 45.7, 54.3 vs. 62.4, 72.1 vs. 84.9, 93.6 vs. 116.7, respectively, all P 〈 0.05）. Conclusion Dectin-1 receptor plays an important role in the phagoeytosis of C. albicans by macrophages.