摘要
目的建立全血白细胞吞噬白念珠菌的流式细胞术检测方法。方法绘制白念珠菌生长曲线后,取生长对数中期的白念珠菌,以活体细胞示踪荧光探针二乙酰羧基荧光黄琥珀酰亚胺酯（CFDA-SE）标记,与以PC5标记的CD45单克隆抗体预标记的全血白细胞,在37℃分别反应10、30、60min（MOI≈10∶1）,以流式细胞术观察CD45阳性细胞对白念珠菌的吞噬情况。结果白念珠菌在液体酵母浸出粉胨葡萄糖培养基（YPD）中37℃、50mL/LCO2培养,第5~11h为对数生长期。10mmol/LCFDA-SE染色30min,可使白念珠菌均匀着色;CD45阳性细胞和标记的白念珠菌作用10、30、60min,CD45阳性细胞中中性粒细胞的吞噬率分别为（80.1±6.1）%、（83.8±7.7）%和（92.3±11.2）%;单核细胞的吞噬率为（11.2±3.6）%、（15.8±4.4）%和（27.7±6.8）%,淋巴细胞作为无明显吞噬作用内参,其吞噬率为（0.9±0.3）%、（0.8±0.4）%和（5.2±1.6）%。结论成功建立流式细胞术检测全血白细胞吞噬白念珠菌的方法,全血白细胞与白念珠菌共孵育30min是较为合适的作用时间。
Objective To establish a whole blood leukocyte phagocytosis assay for Candida albicans（C.albicans）based on flow cytometry（FCM）.Methods C.albicans of mid-logarithmic growth phase was labeled by fluorescence probe carboxyfluorescein diacetate succinimidyl ester（CFDA-SE）,and then added into CD45-PC5 pre-stained human whole blood cells at a 10∶1 multiplicity of infection（MOI） in 37℃.The cells were incubated for 10,30 and 60 minutes.Phagocytosis rate of C.albicans by the CD45 positive cells in the blood was determined by FCM.Results In yeast extract peptone dextrose medium（YPD） and under the conditions of 37℃ and 50mL/L CO2,the logarithmic growth phase of C.albicans SC5314 was from the 5th to 11 th hour.C.albicans were well stained by 10 mmol / L CFDA-SE after 30-minute incubation.After 10-,30-and 60-minute incubation with SC5314 C.albicans with CD45＋cells,the phagocytosis rates measured by FCM were（80.1 ±6.1） %,（83.8 ± 7.7） % and（92.3 ± 11.2） % for the neutrophils,（11.2 ± 3.6） %,（15.8 ± 4.4） % and（27.7 ± 6.8） % for the monocytes and（0.9 ± 0.3） %,（0.8 ± 0.4） % and（5.2 ± 1.6） % for the lymphocytes.Conclusion The method for measuring whole blood leukocyte phagocytosis of C.albicans based on FCM is successfully established,and 30 minutes are the proper incubation time for the phagocytosis assay.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2015年第4期470-473,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
河北省自然科学基金(C2010001882)
河北省卫生厅指导性课题项目(ZD20140031)
白求恩国际和平医院计划课题(201430)
关键词
流式细胞术
白念珠菌
吞噬
flow cytometry
Candida albicans
phagocytosis
