白念珠菌诱导小鼠骨髓来源巨噬细胞焦亡的初步研究  

A preliminary study on Candida albicans-induced pyroptosis of murine bone marrow-derived macrophages

作　　者：杨璐 段志敏 何艳艳 王嘉宁 陈青[2,3] 陈旭[1,2] 李岷 Yang Lu;Duan Zhimin;He Yanyan;Wang Jianing;Chen Qing;Chen Xu;Li Min(Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs,Hospital of Dermatology,Chinese Academy of Medical Sciences and Peking Union Medical College,Nanjing 210042,China;Center for Global Health,School of Public Health,Nanjing Medical University,Nanjing 211166,China;Department of Blood Transfusion,Nanjing Drum Tower Hospital,The Affiliated Hospital of Nanjing University Medical School,Nanjing 210008,China)

机构地区：[1]中国医学科学院、北京协和医学院皮肤病研究所、江苏省皮肤病与性病分子生物学重点实验室,南京210042 [2]南京医科大学全球健康中心,南京211166 [3]南京大学医学院附属鼓楼医院输血科,南京210008

出　　处：《中华皮肤科杂志》2023年第4期301-308,共8页Chinese Journal of Dermatology

基　　金：国家自然科学基金(82103749、82173432);江苏省自然科学基金(BK20190144);南京市国家级临床医学中心培育计划项目(2019060001)。

摘　　要：目的探讨白念珠菌对小鼠骨髓来源巨噬细胞(BMDM)焦亡的影响。方法通过活细胞工作站实时观察白念珠菌[感染复数(MOI)=50,下同]体外诱导BMDM后是否发生焦亡;将BMDM分为对照组、白念珠菌组,分别用磷酸盐缓冲液和白念珠菌酵母诱导BMDM 6 h,实时荧光定量PCR检测NOD样受体热蛋白结构域相关蛋白3(NLRP3)、白细胞介素1β(IL-1β)、IL-18 mRNA表达水平,Western印迹法检测NLRP3、胱天蛋白酶1(Caspase-1)、Gasdermin D(GSDMD)的表达及剪切水平。用白念珠菌诱导BMDM不同时间(0、10、15、20及25 h)后,酶联免疫吸附试验检测IL-1β、IL-18分泌水平。白念珠菌体外诱导野生型(WT)BMDM及GSDMD敲除(KO)BMDM 15 min,采用流式细胞仪比较WT、GSDMD KO BMDM对白念珠菌的吞噬率;诱导6 h后,采用流式细胞仪及乳酸脱氢酶(LDH)释放法分别检测WT、GSDMD KO BMDM的死亡率。分别设置空白对照组、对照组、样品最大酶活性对照孔组、IL-1β组、白念珠菌组、IL-1β+白念珠菌组,用IL-1β和/或白念珠菌酵母诱导BMDM后用LDH释放法分析WT及GSDMD KO BMDM死亡率。采用非配对t检验、Kruskal-Wallis检验、方差分析等统计学方法进行统计分析。结果白念珠菌体外诱导BMDM后细胞可出现气球样变及出泡现象,证实焦亡发生;与对照组相比,白念珠菌组诱导BMDM 6 h后NLRP3、IL-1β的mRNA表达水平明显升高(t=13.02、17.51,P=或<0.001),而IL-18 mRNA表达水平无明显变化(P=0.486),且白念珠菌诱导可引起炎症小体NLRP3表达增多及Caspase-1、GSDMD剪切。白念珠菌诱导BMDM不同时间(0、10、15、20及25 h)后IL-1β的分泌水平随时间逐渐升高(H=12.90,P=0.012),而IL-18的分泌水平无明显变化(F=0.48,P=0.753),且GSDMD KO组BMDM的IL-1β分泌水平低于WT BMDM组(F=24.22,P=0.008)。白念珠菌体外诱导15 min后,GSDMD KO BMDM[(50.3±1.10)%]对白念珠菌的吞噬率低于WT BMDM[(58.53±1.19)%,t=5.09,P=0.007];白念珠菌体外诱导6 h后,流式细胞仪�Objective To investigate the effect of Candida albicans(C.albicans)on pyroptosis of murine bone marrow-derived macrophages(BMDMs).Methods Live-cell imaging was used to observe morphologic changes of in vitro C.albicans-infected BMDMs(multiplicity of infection[MOI]=50)so as to evaluate whether pyroptosis occurred.Cultured BMDMs were divided into a control group and a C.albicans group,which were treated with phosphate-buffered saline and C.albicans suspensions respectively for 6 hours;then,real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of NOD-like receptor pyrin domain containing 3(NLRP3),interleukin(IL)-1βand IL-18,and Western blot analysis to determine the protein expression and cleavage levels of NLRP3,caspase-1 and gasdermin D(GSDMD).BMDMs were cultured with C.albicans suspensions for different durations(0,10,15,20,and 25 hours),and enzyme-linked immunosorbent assay was conducted to detect secretion levels of IL-1βand IL-18.Cultured wild-type BMDMs and GSDMD-knockout BMDMs were treated with C.albicans suspensions for 15 minutes,and then rates of phagocytosis of C.albicans by wild-type BMDMs and GSDMD-knockout BMDMs were estimated by flow cytometry;after 6-hour treatment with C.albicans,flow cytometry and lactate dehydrogenase(LDH)release assay were performed to assess mortality rates of wild-type BMDMs and GSDMD-knockout BMDMs.In addition,some wild-type BMDMs and GSDMD-knockout BMDMs were separately divided into blank control group,control group,maximum enzyme activity-sample control group,IL-1βalone group,C.albicans alone group,and IL-1β+C.albicans group,and cell mortality rates were detected by the LDH release assay after treatment with IL-1βand/or C.albicans.Statistical analysis was carried out by using unpaired t test,Kruskal-Wallis test,analysis of variance,and other statistical methods.Results After in vitro treatment with C.albicans,swelling and ballooning with large bubbles blowing from the plasma membrane occurred in BMDMs,suggesting the occurrence of

关 键 词：白色念珠菌 巨噬细胞 焦亡 白细胞介素1Β 白细胞介素18 Gasdermin D 

分 类 号：R519.3[医药卫生—内科学]