炭疽芽孢在巨噬细胞RAW264.7内的萌发过程
李霆, 王恒樑,史兆兴, 冯尔玲, 刘润艳,黄留玉,军事医学科学院生物工程研究所 北京市 100071
李霆, 1977-04-18生,江苏省南京市人,汉族.军事医学科学院生物工程研究所2002级博士研究生,从事病原微生物功能基因组研究.
国家自然科学基金资助项目,No. 30470100
通讯作者:王恒樑,100071, 北京市丰台东大街20号,军事医学科学院生物工程研究所. wanghl@nic.bmi.ac.cn
电话: 010-66948836 传真:010-63833521
收稿日期:2005-05-08 接受日期:2005-05-25
, 百拇医药
Germination process of Bacillus anthracisendospores within macrophage RAW264.7
Ting Li, Heng-Liang Wang, Zhao-Xing Shi, Er-Ling Feng, Run-Yan Liu, Liu-YuHuang
Ting Li,Heng-Liang Wang, Zhao-Xing Shi, Er-Ling Feng, Run-Yan Liu, Liu-Yu Huang, StateKey Laboratory of Pathogen and Biosecurity, Beijing Institute ofBioengineering, Beijing 100071, China
Supported by National Natural Science Foundation of China,No.30470100
, 百拇医药
Correspondence to: Dr. Heng-Liang Wang, Beijing Institute ofBioengineering, State key laboratory of pathogen and biosecurity, 20Dongda Street, Beijing 100071, China. wanghl@nic.bmi.ac.cn
Received: 2005-05-08 Accepted:2005-05-25
Abstract
AIM: To confirm the germination process of Bacillus anthracis A16Rendospores within murine macrophage RAW264.7
, 百拇医药
METHODS: Macrophage RAW264.7 cells were infected by Bacillusanthrax A16R (pXO2-)spores at a multiplicity of infection (MOI) of 20∶1.Then the cells were harvested at different time points (1, 2, 2.5, 3, 3.5,4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 and 8 h after infection). The growth ofinfected cells was observed under light microscope by fuchsin basicmethylene blue staining.
RESULTS: The endospores began to germinate and develop intovegetative bodies 2 to 2.5 hours after infection. The vegetative bodiesentered the phase of binary fission 3.5 to 4 hours after infection. At 5to 5.5 hours, the bacillus proliferated into exponential phase andthemacrophages began to lyse 7 to 8 hours after infected.
, http://www.100md.com
CONCLUSION: For the fisrt time, fuchsin basic methylene bluestaining is used to study the germination process of Bacillus anthracisendospores within macrophage RAW264.7.
Key Words: Bacillus anthracis; Germination; Fuchsin basicmethylene blue staining; Macrophage
Li T, Wang HL, Shi ZX, Feng EL, Liu RY, Huang LY. Germination process of Bacillusanthracis endospores within macrophage RAW264.7. Shijie Huaren XiaohuaZazhi 2005;13(13):1540-1543
, 百拇医药
摘要目的:确定炭疽杆菌A16R芽孢在巨噬细胞系RAW264.7内萌发的过程.
方法:用炭疽杆菌A16R芽孢以MOI 20∶1感染RAW264.7，分别于吞噬后1、2、2.5、3、3.5、4、4.5、5、5.5、6、6.5、7、7.5、8 h取细胞爬片进行复红美蓝染色，显微镜观察.
结果:吞噬后2-2.5 h, 细胞内的芽孢开始萌发并形成繁殖体;吞噬后3.5-4 h,繁殖体开始二分裂;吞噬后5-5.5 h,繁殖体进入指数增长阶段;吞噬后7-8 h,大量的繁殖体充斥于细胞内、外，细胞开始崩解.
结论:首次采用复红美蓝染色确定了A16R炭疽芽孢在巨噬细胞RAW264.7内的萌发过程.
, 百拇医药
关键词:炭疽芽孢杆菌; 巨噬细胞; 萌发;复红美蓝染色
李霆, 王恒樑,史兆兴, 冯尔玲, 刘润艳,黄留玉. 炭疽芽孢在巨噬细胞RAW264.7内的萌发过程.世界华人消化杂志 2005;13(13):1540-1543
: 1 h; B:2 h; C:2.5 h; D:3 h; E:3.5 h; F:4 h; G:4.5 h; H:5 h; I:5.5 h; J:6 h; K:6.5 h; L:7 h; M:7.5 h; N:8 h.
3 讨论感染性疾病是病原微生物和宿主紧密相互作用的结果，只有深入了解病原微生物与宿主相互作用的分子细节才可能鉴定出病原微生物的所有毒力基因、全面理解病原微生物的致病机制和宿主的防御机制.巨噬细胞是炭疽芽孢萌发和毒力体现的关键宿主细胞，因此开展巨噬细胞反应机制的研究对于理解炭疽感染机制和宿主免疫机制意义重大.国外已有文献报道了多种菌株炭疽芽孢在巨噬细胞内存活、萌发的研究，而国内在这方面却鲜有报道.另外，本试验所采用的A16R(pXO2-)是我国特有的无荚膜水肿型活芽孢疫苗株，其皮肤炭疽的保护率为80-100%，但对死亡率高的肺炭疽保护效果不佳，其机制如何尚无报道，因此开展对炭疽杆菌A16R和巨噬细胞相互作用的研究不仅有利于深入了解炭疽感染的分子机制，还将为A16R疫苗的改良提供帮助.
, 百拇医药
炭疽芽孢感染巨噬细胞是一个动态的过程，准确界定炭疽芽孢在感染模型中结构、功能改变的时间点(段)是全面研究宿主反应机制的前提.已有文献采用了免疫荧光-激光共聚焦显微镜[5-6]、透射电镜[7]以及常规革兰染色[7-8]等方法检测了几株炭疽芽孢在巨噬细胞内的萌发情况，但都存在着过程繁琐或者不能同时显示芽孢和繁殖体等缺陷.因此，我们考虑能否应用芽孢染色法来检测芽孢在巨噬细胞内萌发的过程.常用的芽孢染色法包括孔雀石绿染色、Moller染色以及复红美蓝染色等，但前两种染色过程都需要加热，不适用于细胞爬片的染色.我们采用染色过程温和的复红美蓝染色法，对吞噬后的芽孢和萌发的繁殖体进行了清楚地着色(芽孢呈红色和繁殖体呈蓝色)，从而简便、有效地确定了A16R芽孢在巨噬细胞RAW264.7内的萌发过程，并为今后通过基因表达谱分析和比较蛋白质组等方法对宿主细胞在芽孢萌发不同时段的全局性反应进行研究奠定了基础.
4 参考文献1 Fukao T. Immune system paralysis by anthrax lethal toxin: the rolesof innate and adaptive immunity.
, 百拇医药
Lancet Infect Dis 2004;4:166-170
2 Moayeri M, Leppla SH. The roles of anthrax toxin in pathogenesis. CurrOpin Microbiol 2004;7:1924
3 Guidi-Rontani C. The alveolar macrophage: the Trojan horse of Bacillusanthracis. Trends Microbiol 2002;10:405-409
4 Hanna PC, Acosta D, Collier RJ. On the role of macrophages inanthrax. Proc Natl Acad Sci USA 1993;90:10198-10201
, 百拇医药
5 Guidi-Rontani C, Weber-Levy M, Labruyere E, Mock M. Germination of Bacillusanthracis spores within alveolar
macrophages. Molecular Microbiol 1999;31:917
6 Guidi-Rontani C, Levy M, Ohayon H, Mock M. Fate of germinated Bacillusanthracis spores in primary murine
macrophages. Molecular Microbiol 2001;42:931938
7 Dixon TC, Fadl AA, Koehler TM, Swanson JA, Hanna PC. Early Bacillusanthracis-macrophage intracellular survival
, 百拇医药
and escape. Cellular Microbiol 2000;2:453-463
8 Bergman NH, Passalacqua KD, Gaspard R, Shetron-Rama LM, QuackenbushJ, Hanna PC. Murinemacrophage
transcriptional responses to bacillusanthracis infection and intoxication. Infection Immunity 2005;73:1069-1080
编辑 潘伯荣 审读 张海宁, http://www.100md.com( 李 霆, 王恒樑, 史兆兴, 冯尔玲, 刘润艳, 黄留玉)
李霆, 1977-04-18生,江苏省南京市人,汉族.军事医学科学院生物工程研究所2002级博士研究生,从事病原微生物功能基因组研究.
国家自然科学基金资助项目,No. 30470100
通讯作者:王恒樑,100071, 北京市丰台东大街20号,军事医学科学院生物工程研究所. wanghl@nic.bmi.ac.cn
电话: 010-66948836 传真:010-63833521
收稿日期:2005-05-08 接受日期:2005-05-25
, 百拇医药
Germination process of Bacillus anthracisendospores within macrophage RAW264.7
Ting Li, Heng-Liang Wang, Zhao-Xing Shi, Er-Ling Feng, Run-Yan Liu, Liu-YuHuang
Ting Li,Heng-Liang Wang, Zhao-Xing Shi, Er-Ling Feng, Run-Yan Liu, Liu-Yu Huang, StateKey Laboratory of Pathogen and Biosecurity, Beijing Institute ofBioengineering, Beijing 100071, China
Supported by National Natural Science Foundation of China,No.30470100
, 百拇医药
Correspondence to: Dr. Heng-Liang Wang, Beijing Institute ofBioengineering, State key laboratory of pathogen and biosecurity, 20Dongda Street, Beijing 100071, China. wanghl@nic.bmi.ac.cn
Received: 2005-05-08 Accepted:2005-05-25
Abstract
AIM: To confirm the germination process of Bacillus anthracis A16Rendospores within murine macrophage RAW264.7
, 百拇医药
METHODS: Macrophage RAW264.7 cells were infected by Bacillusanthrax A16R (pXO2-)spores at a multiplicity of infection (MOI) of 20∶1.Then the cells were harvested at different time points (1, 2, 2.5, 3, 3.5,4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 and 8 h after infection). The growth ofinfected cells was observed under light microscope by fuchsin basicmethylene blue staining.
RESULTS: The endospores began to germinate and develop intovegetative bodies 2 to 2.5 hours after infection. The vegetative bodiesentered the phase of binary fission 3.5 to 4 hours after infection. At 5to 5.5 hours, the bacillus proliferated into exponential phase andthemacrophages began to lyse 7 to 8 hours after infected.
, http://www.100md.com
CONCLUSION: For the fisrt time, fuchsin basic methylene bluestaining is used to study the germination process of Bacillus anthracisendospores within macrophage RAW264.7.
Key Words: Bacillus anthracis; Germination; Fuchsin basicmethylene blue staining; Macrophage
Li T, Wang HL, Shi ZX, Feng EL, Liu RY, Huang LY. Germination process of Bacillusanthracis endospores within macrophage RAW264.7. Shijie Huaren XiaohuaZazhi 2005;13(13):1540-1543
, 百拇医药
摘要目的:确定炭疽杆菌A16R芽孢在巨噬细胞系RAW264.7内萌发的过程.
方法:用炭疽杆菌A16R芽孢以MOI 20∶1感染RAW264.7，分别于吞噬后1、2、2.5、3、3.5、4、4.5、5、5.5、6、6.5、7、7.5、8 h取细胞爬片进行复红美蓝染色，显微镜观察.
结果:吞噬后2-2.5 h, 细胞内的芽孢开始萌发并形成繁殖体;吞噬后3.5-4 h,繁殖体开始二分裂;吞噬后5-5.5 h,繁殖体进入指数增长阶段;吞噬后7-8 h,大量的繁殖体充斥于细胞内、外，细胞开始崩解.
结论:首次采用复红美蓝染色确定了A16R炭疽芽孢在巨噬细胞RAW264.7内的萌发过程.
, 百拇医药
关键词:炭疽芽孢杆菌; 巨噬细胞; 萌发;复红美蓝染色
李霆, 王恒樑,史兆兴, 冯尔玲, 刘润艳,黄留玉. 炭疽芽孢在巨噬细胞RAW264.7内的萌发过程.世界华人消化杂志 2005;13(13):1540-1543
: 1 h; B:2 h; C:2.5 h; D:3 h; E:3.5 h; F:4 h; G:4.5 h; H:5 h; I:5.5 h; J:6 h; K:6.5 h; L:7 h; M:7.5 h; N:8 h.
3 讨论感染性疾病是病原微生物和宿主紧密相互作用的结果，只有深入了解病原微生物与宿主相互作用的分子细节才可能鉴定出病原微生物的所有毒力基因、全面理解病原微生物的致病机制和宿主的防御机制.巨噬细胞是炭疽芽孢萌发和毒力体现的关键宿主细胞，因此开展巨噬细胞反应机制的研究对于理解炭疽感染机制和宿主免疫机制意义重大.国外已有文献报道了多种菌株炭疽芽孢在巨噬细胞内存活、萌发的研究，而国内在这方面却鲜有报道.另外，本试验所采用的A16R(pXO2-)是我国特有的无荚膜水肿型活芽孢疫苗株，其皮肤炭疽的保护率为80-100%，但对死亡率高的肺炭疽保护效果不佳，其机制如何尚无报道，因此开展对炭疽杆菌A16R和巨噬细胞相互作用的研究不仅有利于深入了解炭疽感染的分子机制，还将为A16R疫苗的改良提供帮助.
, 百拇医药
炭疽芽孢感染巨噬细胞是一个动态的过程，准确界定炭疽芽孢在感染模型中结构、功能改变的时间点(段)是全面研究宿主反应机制的前提.已有文献采用了免疫荧光-激光共聚焦显微镜[5-6]、透射电镜[7]以及常规革兰染色[7-8]等方法检测了几株炭疽芽孢在巨噬细胞内的萌发情况，但都存在着过程繁琐或者不能同时显示芽孢和繁殖体等缺陷.因此，我们考虑能否应用芽孢染色法来检测芽孢在巨噬细胞内萌发的过程.常用的芽孢染色法包括孔雀石绿染色、Moller染色以及复红美蓝染色等，但前两种染色过程都需要加热，不适用于细胞爬片的染色.我们采用染色过程温和的复红美蓝染色法，对吞噬后的芽孢和萌发的繁殖体进行了清楚地着色(芽孢呈红色和繁殖体呈蓝色)，从而简便、有效地确定了A16R芽孢在巨噬细胞RAW264.7内的萌发过程，并为今后通过基因表达谱分析和比较蛋白质组等方法对宿主细胞在芽孢萌发不同时段的全局性反应进行研究奠定了基础.
4 参考文献1 Fukao T. Immune system paralysis by anthrax lethal toxin: the rolesof innate and adaptive immunity.
, 百拇医药
Lancet Infect Dis 2004;4:166-170
2 Moayeri M, Leppla SH. The roles of anthrax toxin in pathogenesis. CurrOpin Microbiol 2004;7:1924
3 Guidi-Rontani C. The alveolar macrophage: the Trojan horse of Bacillusanthracis. Trends Microbiol 2002;10:405-409
4 Hanna PC, Acosta D, Collier RJ. On the role of macrophages inanthrax. Proc Natl Acad Sci USA 1993;90:10198-10201
, 百拇医药
5 Guidi-Rontani C, Weber-Levy M, Labruyere E, Mock M. Germination of Bacillusanthracis spores within alveolar
macrophages. Molecular Microbiol 1999;31:917
6 Guidi-Rontani C, Levy M, Ohayon H, Mock M. Fate of germinated Bacillusanthracis spores in primary murine
macrophages. Molecular Microbiol 2001;42:931938
7 Dixon TC, Fadl AA, Koehler TM, Swanson JA, Hanna PC. Early Bacillusanthracis-macrophage intracellular survival
, 百拇医药
and escape. Cellular Microbiol 2000;2:453-463
8 Bergman NH, Passalacqua KD, Gaspard R, Shetron-Rama LM, QuackenbushJ, Hanna PC. Murinemacrophage
transcriptional responses to bacillusanthracis infection and intoxication. Infection Immunity 2005;73:1069-1080
编辑 潘伯荣 审读 张海宁, http://www.100md.com( 李 霆, 王恒樑, 史兆兴, 冯尔玲, 刘润艳, 黄留玉)
