CAJ | 학술논문

目的探讨人中性粒细胞能否通过C型凝集素1受体(dectin-1)识别白念珠菌细胞壁不溶性β葡聚糖来介导体外杀伤白念珠菌的活性.方法 100 mg/L β葡聚糖体外作用于中性粒细胞1、6、24 h后,实时荧光定量逆转录PCR分析dectin-1和Toll样受体2 mRNA表达水平.100 mg/Lβ葡聚糖体外分别刺激中性粒细胞15 min、2h、6h,或先用dectin-1抑制剂昆布多糖100 mg/L和50 mg/L预处理30 min后,再予100 mg/L β葡聚糖刺激2h,微量培养板荧光分析法检测中性粒细胞H2O2释放水平.昆布多糖预处理的中性粒细胞与白念珠菌体外共培养后,检测菌落形成单位(CFU).结果 β葡聚糖作用中性粒细胞1、6、24 h后,dectin-1 mRNA水平分别为2.8195±0.1669、5.4859±0.7244、3.6041±0.5372,均明显高于空白对照组(均P＜ 0.01).β葡聚糖刺激中性粒细胞15 min后H2O2水平为(64.55±15.67) μmol/L,2h时为(290.34±30.56)μmol/L,6h时为(208.54±26.88) μmol/L,与空白对照组(22.05±3.99) μmol/L比较,差异均有统计学意义(均P＜ 0.01)；100 mg/L和50 mg/L昆布多糖预处理组分别为(80.45±22.41) μmol/L和(130.42±44.55) μmol/L,与β葡聚糖刺激组相比,分别降低了73％和45％,差异均有统计学意义(P＜0.01).昆布多糖能明显抑制中性粒细胞体外杀伤白念珠菌的活性(均P＜ 0.01).结论 Dectin-1参与人中性粒细胞分泌H2O2以及对白念珠菌杀灭活性,为过继性粒细胞转输治疗系统性念珠菌感染提供初步依据.
목적 탐토인중성립세포능부통과C형응집소1수체(dectin-1)식별백념주균세포벽불용성β포취당래개도체외살상백념주균적활성.방법 100 mg/L β포취당체외작용우중성립세포1、6、24 h후,실시형광정량역전록PCR분석dectin-1화Toll양수체2 mRNA표체수평.100 mg/Lβ포취당체외분별자격중성립세포15 min、2h、6h,혹선용dectin-1억제제곤포다당100 mg/L화50 mg/L예처리30 min후,재여100 mg/L β포취당자격2h,미량배양판형광분석법검측중성립세포H2O2석방수평.곤포다당예처리적중성립세포여백념주균체외공배양후,검측균락형성단위(CFU).결과 β포취당작용중성립세포1、6、24 h후,dectin-1 mRNA수평분별위2.8195±0.1669、5.4859±0.7244、3.6041±0.5372,균명현고우공백대조조(균P＜ 0.01).β포취당자격중성립세포15 min후H2O2수평위(64.55±15.67) μmol/L,2h시위(290.34±30.56)μmol/L,6h시위(208.54±26.88) μmol/L,여공백대조조(22.05±3.99) μmol/L비교,차이균유통계학의의(균P＜ 0.01)；100 mg/L화50 mg/L곤포다당예처리조분별위(80.45±22.41) μmol/L화(130.42±44.55) μmol/L,여β포취당자격조상비,분별강저료73％화45％,차이균유통계학의의(P＜0.01).곤포다당능명현억제중성립세포체외살상백념주균적활성(균P＜ 0.01).결론 Dectin-1삼여인중성립세포분비H2O2이급대백념주균살멸활성,위과계성립세포전수치료계통성념주균감염제공초보의거.
Objective To investigate whether human neutrophils kill Candida albicans through recognition of insoluble β-glucan in cell walls of C.albicans (CalG) by dectin-1,a C-type lectin receptor.Methods Neutrophils were obtained from peripheral blood of healthy human subjects and cultured in vitro.Real-time PCR was carried out to quantify the mRNA expressions of dectin-1 and Toll-like receptor 2 (TLR2) in neutrophils challenged with CaIG of 100 mg/L for 1,6,and 24 hours.A Fluoro hydrogen peroxide (H2O2) detection kit was used to determine H2O2 levels in some neutrophils exposed to CaIG (100 mg/L) for 15 minutes,2 hours,6 hours,as well as in some neutrophils preincubated with laminarin (a dectin-1 inhibitor) of 100 mg/L and 50 mg/L for 30 minutes followed by challenge with CaIG of 100 mg/L for 2 hours.Colony forming units (CFUs) were counted after the incubation of C.albicans with neutrophils pretreated with laminarin of 100 mg/L and 50 mg/L for 30 minutes.Results The relative mRNA expression level of dectin-1 was 2.8195 + 0.1669,5.4859 + 0.7244 and 3.6041 + 0.5372 in neutrophils challenged with CaIG for 1,6 and 24 hours,respectively,significantly higher than that in unchallenged neutrophils at these corresponding time points (all P ＜ 0.01).The level of H2O2 was (64.55 + 15.67),(290.34 + 30.56),and (208.54 ± 26.88) μ mol/L respectively in neutrophils treated with CaIG for 15 minutes,2 hours,and 6 hours respectively,compared to (22.05 ± 3.99) μmol/L in untreated neutrophils (all P ＜ 0.01).The pretreatment with laminarin of 100 and 50 mg/L attenuated the release of H2O2 in CaIG-treated neutrophils by 73％ ((80.45 + 22.41) μ mol/L,P＜ 0.01) and 45％ ((130.42 + 44.55) μmol/L,P＜ 0.01),respectively,compared with neutrophils treated with CaIG only.The fungicidal activity of neutrophils against C.albicans was also significantly inhibited by pretreatment with laminarin of 50 and 100 mg/L (both P＜ 0.01).Conclusions Dectin-1 may be involved in the secretion of H2O2 as well as killing of C.albicans by human neutrophils,which may provide a preliminary evidence for adoptive transfer of neutrophils as an approach to the treatment of systemic C.albicans infection.