摘要
目的探究白假丝酵母(Candida albicans)激活的Raw264.7细胞自噬在主要组织相容性复合体Ⅱ类分子(MHCⅡ)抗原提呈以及协同刺激分子表达中的作用。方法 C.albicans刺激Dectin-1单克隆抗体封闭或白皮杉醇阻断的Raw264.7细胞,Western blot法检测LC3Ⅱ表达量,RT-PCR检测CD80与CD86的表达。免疫荧光实验观察有无3-MA预处理的GFP-LC3-Raw264.7细胞与C.albicans共孵育后MHCⅡ与LC3在胞浆的分布,ELISA法检测有无封闭Dectin-1或3-MA预处理的Raw264.7细胞与C.albicans共孵育不同时间段后IL-6的分泌。结果阻断Dectin-1或Sky后C.albicans诱导的LC3Ⅱ表达降低。LC3、MHCⅡ与胞内C.albicans存在显著的共定位关系,阻断自噬后C.albicans与MHCⅡ的共定位明显减弱。C.albicans引发Raw264.7细胞表达CD80与CD86mRNA,封闭Dectin-1或阻断自噬后二者转录水平降低。C.albicans通过Dectin-1引发Raw264.7细胞分泌IL-6,阻断自噬对IL-6分泌无显著影响。结论 C.albicans通过Dectin-1/Sky通路激活巨噬细胞自噬,自噬体的构建促进MHCⅡ招募至胞内C.albicans,并促进协同刺激分子的表达。
Objective To explore the role of Candida albicans-induced autophagy of Raw264.7 cells in the presentation of major histocompatibility complex class II（MHCII）antigens and expression of co-stimulation molecules.Methods Raw264.7 cells were blocked with Dectin-1 monoclonal antibody or piceatannol,and stimulated with C.albicans.The expression of LC3 II protein in RAW264.7 cells was detected with Western blot assay,and the levels of CD80 and CD86 mRNA were detected by using RT-PCR.Then the GFP-LC3-Raw264.7 cells were incubated with or without 3-MA,and stimulated with C.albicans;the expression and positioning of LC3 II were observed by using immunofluorescence staining.Finally,the Raw264.7 cells were stimulated with C.albicans with or without pretreatment with Dectin-1 monoclonal antibody or 3-MA.ELISA was used to test the level of IL-6 secreted by Raw264.7 cells.Results The blocking of Dectin-1 or Sky significantly reduced the C.albicans-stimulated expression levels of LC3 II in Raw264.7 cells.The C.albicans in cytoplasm showed significant colocalization with LC3 and MHCII;blocking autophagy inhibited the colocalization of C.albicans and MHCII.C.albicans induced the expression of CD80 and CD86 mRNA in Raw264.7 cells,but blocking autophagy or Dectin-1 obviously reduced the levels of CD80 and CD86 mRNA.Finally,C.albicans stimulated the secretion of IL-6 by Raw264.7 cells via Dectin-1,while blocking autophagy did not affect the secretion of IL-6.Conclusion Dectin-1/Sky signaling pathway mediated the C.albicans-induced autophagy in Raw264.7 cells,and the formation of autophagosomes facilitated the recruitment of MHCII into C.albicans in cytoplasm and the expression of co-stimulatory molecules.
作者
张梅青
沈继录
胡典明
李志鹏
ZHANG Meiqing;SHEN Jilu;HU Dianming;LI Zhipeng(Department of Laboratory Medicine,Anqing Hospital of PLA Navy,Anqing,Hefei 246003,China)
出处
《中国微生态学杂志》
CAS
CSCD
2018年第8期911-915,共5页
Chinese Journal of Microecology
