中华皮肤科杂志 ›› 2013, Vol. 46 ›› Issue (8): 543-546.

• 论著 • 上一篇    下一篇

C型凝集素1受体参与人中性粒细胞体外杀伤白念珠菌活性的实验研究

陈青1,曾荣2,沈永年3,胡素泉4,李岷3,刘维达3   

  1. 1. 江苏省血液中心
    2. 中国医学科学院皮肤病研究所
    3. 南京 中国医学科学院北京协和医学院皮肤病研究所
    4. 南京医科院皮研所
  • 收稿日期:2013-01-05 修回日期:2013-05-05 出版日期:2013-08-15 发布日期:2013-08-01
  • 通讯作者: 李岷 E-mail:drlimin@sina.cn
  • 基金资助:
    国家自然科学基金;江苏省自然科学基金

Dectin?鄄1, a C?鄄type lectin receptor, participates in the killing of Candida albicans by human neutrophils: an experimental study

  • Received:2013-01-05 Revised:2013-05-05 Online:2013-08-15 Published:2013-08-01
  • Supported by:
    National Natural Science Foundation of China;Natural Science Foundation of Jiangsu Province of China

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摘要： 【摘要】 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/L β葡聚糖体外分别刺激中性粒细胞15 min、2 h、6 h，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2 h，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，2 h时为（290.34 ± 30.56） μmol/L，6 h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。 【关键词】 念珠菌，白色； 中性白细胞； β葡聚糖类； 外源凝集素类，C型； 过氧化氢

关键词: 念珠菌，白色, 中性白细胞, β葡聚糖类, 过氧化氢, 外源凝集素类，C型

Abstract: CHEN Qing*, ZENG Rong, SHEN Yong-nian, HU Su-quan, LI Min, LIU Wei-da. *Jiangsu Province Blood Center, Nanjing 210042, China Corresponding author: LI Min, Email: minli08@gmail.com 【Abstract】 Objective To investigate whether human neutrophils kill Candida albicans through recognition of insoluble β-glucan in cell walls of C. albicans （CaIG） 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） μmol/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） μmol/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. 【Key words】 Candida albicans; Neutrophils; beta-Glucans; Lectins, c-type; Hydrogen peroxide