小鼠骨髓树突状细胞对白念珠菌吞噬、杀伤作用的研究  

The study of mouse bone marrow-derived dendritic cells phagocytosis and cytotoxicity against Candida albicans in vitro

作　　者：王琼[1] 史冬梅[2] 刘维达[1] 

机构地区：[1]中国医学科学院皮肤病研究所,南京210042 [2]济宁市第一人民医院皮肤科,济宁272011

出　　处：《中国真菌学杂志》2016年第4期193-200,共8页Chinese Journal of Mycology

基　　金：973计划项目(2013CB531605);国家自然科学基金(81401653);山东省自然科学基金(ZR2015HL127);济宁市医学科学计划(2014jnnk13)

摘　　要：目的体外分离培养小鼠骨髓树突状细胞(Dendritic Cell,DC)并与白念珠菌共同孵育,研究不同浓度的DC对白念珠菌的吞噬、杀伤作用,并研究随着共孵育时间的延长DC表型的变化及其吞噬、杀伤白念珠菌能力的变化。方法无菌条件下分离培养C57小鼠骨髓DC;倒置显微镜下观察DC的形态;Cellometer Mini细胞计数仪测定DC生长曲线及细胞直径变化;流式细胞仪检测DC及白念珠菌刺激后DC表面标志物CD11c及其表面分子MHCⅡ、CD40、CD80及CD86的表达;光学显微镜、激光共聚焦显微镜及流式细胞仪测定DC对白念珠菌的吞噬及杀伤作用。结果体外培养10d小鼠骨髓DC,倒置显微镜观察可见典型DC形态;随着培养时间的延长,细胞逐渐增大,培养第10天时,细胞直径分布最多的为10.2μm;生长曲线显示,DC呈倍数增长,第2~9天为细胞对数生长期,第9天左右增殖曲线达到顶峰。培养第10天,未经白念珠菌刺激的DC表面CD11c分子及CD80分子高水平表达,CD40、CD86及MHCⅡ低表达;经白念珠菌刺激后,DC表面CD11c分子、CD80、CD40、CD86及MHCⅡ均高表达,呈成熟DC表型。激光共聚焦(Laser Scanning Confocal Microscope,LSCM)及流式细胞仪(Flow Cytometer,FCM)检测DC对白念珠菌吞噬作用,当DC与白念珠菌的比例为1∶1时,随着共培养时间的延长(30min^1.5h),吞噬率无明显变化(P=0.063);当DC与白念珠菌比例为2∶1、1∶2及1∶4,随着DC细胞所占比例的减小,DC细胞吞噬作用增强,当比例为1∶4时,DC吞噬作用最强,且随着共培养时间的延长(30min^1.5h),吞噬率也明显上升,差异有统计学意义(P=0.003;P=0.002;P=0.002)。显微镜检测DC对白念珠菌杀伤率,可见随着时间的延长,各实验组的杀伤率下降(P=0.000);当DC∶白念珠菌=1∶1时,较2∶1时,杀伤率明显下降,但随着白念珠菌比例在一定范围内的增高(从1∶1到1∶4),杀伤率明显增高,差异有统计学意义(P=0.000)。结论小鼠骨髓细胞体外经Objective Mouse bone marrow-derived dendritic cells were isolated and cultured in vitro and co-incubated with Candida albicans,in order to study the phenotype,phagocytosis and cytotoxicity of which against Candida albicans under different concentration and time.Methods Dendritic cells from C57 mouse bone marrow were isolated and cultured under aseptic conditions.The morphological and the growth curve and cell diameter of DC were observed by optical microscope and Cellometer Mini cell counter.The expression levels of CD11 c,MHCII,CD40,CD80 and CD86of DC were measured by flow cytometer.The phagocytosis and cytotoxicity of DC against Candida albicans were detected by microscope,Laser Scanning Confocal Microscope（LSCM）and flow cytometry.Results Typical morphology of DC cells was observed under inverted microscope 10 days after culture.10.2μm was the most widely distributed diameter of DC as illustrated and the size of DC increased with the extension of incubation time.The growth curve showed that DC increased multiply.After inoculation,2-9days were logarithmic phase and stagnate phase was from 9days.The result of immunological phenotype detected by FCM showed that the CD11 cand CD80were highly expressed and MHCII,CD40,CD86 were lower expressed in control group.After stimulated by Candida albicans,the DC were appeared mature phenotype and CD11 c,CD80,CD40,CD86 and MHCⅡwere all highly expressed.As the ratio（DC∶Candida albicans）reduced（from 2∶1to 1∶4）,the phagocytosis effects of DC enhanced and the strongest effect appeared when the ratio was 1∶4.When the ratio was 1∶1,the phagocytosis effects of DC was no significant change（P=0.063）with the extension of co-incubation time（30min to 1.5h）.But when the ratio were 2∶1,1∶2and 1∶4,the phagocytosis effects of DC were obviously rised with the extension of co-incubation time（30min to1.5h）,a statistically significant difference（P=0.003,P=0.002,P=0.002）.When co-incubation time was prolonged,the cytotoxicity of DC was dramatic descend

关 键 词：骨髓 树突状细胞 白念珠菌 吞噬 杀伤 

分 类 号：R379.4[医药卫生—病原生物学]