上海交通大学学报（医学版）

›› 2011, Vol. 31 ›› Issue (7): 869-.doi: 10.3969/j.issn.1674-8115.2011.07.001

• 论著（基础研究） •    下一篇

钩端螺旋体在不同宿主巨噬细胞内存活能力的比较

罗云蔓1, 黄莉莉2, 刘伯玉2, 张 彦2, 胡宝瑜2, 朱 平3, 郭晓奎2, 何 平2, 姜叙诚1   

  1. 上海交通大学 基础医学院 1.病理学教研室, 2.病原生物学教研室, 3.细胞生物学教研室, 上海 200025
  • 出版日期:2011-07-28 发布日期:2011-07-27
  • 作者简介:罗云蔓（1984—）, 女, 硕士生；电子信箱: xiaoman73@yahoo.cn。
  • 基金资助:

    国家高技术研究发展计划（“863”计划）（2006AA02Z176）；国家自然科学基金（30770820）；上海市自然科学基金（06ZR14056）；上海市卫生局科研课题(2008045)

Comparison of viability of Leptospira in macrophages from different hosts

LUO Yun-man1, HUANG Li-li2, LIU Bo-yu2, ZHANG Yan2, HU Bao-yu2, ZHU Ping3, GUO Xiao-kui2, HE Ping2, JIANG Xu-cheng1   

  1. 1.Department of Pathology, 2.Department of Microbiology and Parasitology, 3.Department of Cell Biology, Basic Medical College, Shanghai Jiaotong University, Shanghai 200025, China
  • Online:2011-07-28 Published:2011-07-27
  • Supported by:

    National High Technology and Research Development Program, “863” Program, 2006AA02Z176；National Natural Science Foundation of China, 30770820；Natural Science Foundation of Shanghai, 06ZR14056；Shanghai Municipal Health Bureau Foundation, 2008045

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摘要：

目的 通过比较钩端螺旋体（钩体）在人和小鼠单核-巨噬细胞内的存活情况，探讨固有免疫在钩端螺旋体病致病机制中的作用。方法 将钩体秋季血清群强毒株56606v株及其经体外多次传代的弱毒株56606a株在体外分别感染经佛波酯（PMA）诱导分化的人单核细胞系THP-1和小鼠单核-巨噬细胞系RAW264.7。感染后2、24、72 h，采用免疫荧光染色激光共聚焦显微镜观察并计算两种细胞的含菌细胞百分数；感染后2、12、24、48和72 h，采用Real-Time PCR技术定量检测可间接反映细胞内钩体存活数量的钩体16S rRNA的表达。结果 随着感染时间的延长，两种细胞的含菌细胞百分数逐渐降低；Real-Time PCR检测结果显示：随着感染时间的延长，两种细胞内钩体16S rRNA的表达逐渐下调，细胞内钩体的存活数量逐渐减少。结论 钩体在人和小鼠单核-巨噬细胞内均不能存活和繁殖，提示致病性钩体通过抗巨噬细胞的杀灭降解而在胞内的存活和繁殖可能不是钩体感染人类宿主的主要致病途径，其发病机制有待进一步阐明。

关键词: 钩端螺旋体, 巨噬细胞, 活性, 实时荧光定量PCR

Abstract:

Objective To explore the role of innate immunity in the pathogenesis of leptospirosis by comparing the viability of Leptospira in mononuclear macrophages of human and mouse origin. Methods Human monocytic cell lines (THP-1) treated with phorbol myfismte acetate (PMA) to be differentiated into macrophages and murine mononuclear macrophages cell lines (RAW264.7) were infected with Leptospira interrogans serovar Autumnalis strain autumnalis #56606 (56606v) and avirulent strain (56606a) in vitro. Laser scanning confocal microscopy was employed to determine the percentages of cells with intracellular leptospires in these two cell lines with immunofluorescence staining 2 h, 24 h and 72 h after infection, and Real-Time PCR was used to detect the expression of 16S rRNA of leptospires in these two cell lines 2 h, 12 h, 24 h, 48 h and 72 h after infection, which indirectly reflected the viability of intracellular Leptospira. Results The percentages of THP-1 and RAW264.7 cells with leptospires decreased with time of infection. RealTime PCR revealed that the expression of 16S rRNA of leptospires in these two cell lines decreased with time of infection, and the numbers of intracellular viable leptospires also decreased with time of infection. Conclusion Leptospira can not survive and replicate within macrophages of human or mouse origin, which indicates that anti-degradation of macrophages by pathogenic Leptospira may not be the major pathogenic mechanism of leptospirosis. Further studies are required to interpret the pathogenesis.

Key words: Leptospira, macrophages, viability, Real-Time PCR