钩端螺旋体 LPS 和外膜蛋白诱导巨噬细胞凋亡及其与 Fas／FasL 相关性研究  

Lipopolysaccharide and outer membrane proteins of Leptospira interrogans induce macrophage apop-tosis via Fas/FasL pathway

作　　者：杜蓬[1] 刘小香[1] 严杰[2] 葛玉梅[2] 孙爱华[1] 

机构地区：[1]浙江医学高等专科学校基础医学部,杭州310053 [2]浙江大学医学院病原生物学系

出　　处：《中华微生物学和免疫学杂志》2014年第6期447-452,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金（81171534）;浙江省卫生高层次创新人才培养工程资助项目（2012-241）

摘　　要：目的：了解致病性问号钩体螺旋体（简称钩体）脂多糖（ L-LPS）和外膜蛋白（ L-OMP）诱导J774A．1小鼠巨噬细胞死亡及其与Fas/FasL相关性。方法分别采用酚水法和Triton X-114法从问号钩体黄疸出血群赖型赖株中提取L-LPS和L-OMP。采用流式细胞术检测紫外线灭活前后问号钩体赖株、多黏菌素B（PMB）处理前后L-LPS、蛋白酶K（PK）作用前后L-OMP诱导J774A．1细胞凋亡及坏死情况。采用siRNA沉默J774A．1细胞Fas或FasL基因并用实时荧光定量RT-PCR检测靶基因沉默效果。采用流式细胞术测定L-LPS或L-OMPs诱导Fas或FasL基因沉默J774 A．1细胞凋亡的作用。结果紫外线灭活前后问号钩体赖株可引起相似的J774A．1细胞早期凋亡率（55．6％和47．1％）和晚期凋亡/坏死率（7．9％和7．6％）。100 ng L-LPS或100μg L-OMP作用1×10^5 J774A．1细胞4 h后，早期凋亡率和晚期凋亡/坏死率分别为40．4％和34．0％、7．5％和6．9％，但等量PMB预处理L-LPS或PK预处理L-OMP诱导细胞凋亡或坏死的作用消失。 Fas或FasL基因沉默后，L-LPS诱导的J774A．1细胞早期凋亡率均显著下降（P＜0．05），L-OMP仅使Fas基因沉默J774A．1细胞早期凋亡率有所下降（P＜0．05）。结论 L-LPS和L-OMP可诱导Fas/FasL相关巨噬细胞凋亡，从而有利于问号钩体在宿主体内建立有效感染。Objective To investigate the role of pathogenic Leptospira interrogans lipopolysaccha-ride （L-LPS） and outer membrane proteins （L-OMP） in the apoptosis of mouse macrophages （J774A.1） and their association with Fas/FasL pathway .Methods Phenol-water extraction and Triton X-114 phase separation were used to extract L-LPS and L-OMP from L.interrogans serogroup icterohaemorrhagiae serovar Lai strain Lai, respectively.Polymyxin B （ PMB） and protease K （ PK） were used to treat L-LPS and L-OMP, respectively.J774A.1 cells were stimulated by L.interrogans strain Lai with or without ultraviolet inactivation.In parallel, the cells were stimulated by extracted L-LPS and L-OMP with or without PMB and PK treatments .The apoptosis and necrosis of J 774 A.1 cells before and after treatment were detected by flow cytometry.The siRNAs were used to silence the expression of Fas or FasL gene in J 774A.1 cells and their inhibitory effects were further validated by using real-time fluorescent quantitative RT-PCR.Flow cytometry was used to detect the effects of L-LPS or L-OMP on the apoptosis of J774A.1 cells with Fas or FasL gene-knockdown .Results L.interrogans strain Lai with or without ultraviolet inactivation could cause similar early apoptosis rates （47.1%and 55.6%） and late apoptosis/necrosis rates （7.6%and 7.9%）.The ear-ly apoptosis rates of 1×10^5 J774A.1 cells were 40.4%and 34.0%after the treatment with 100 ng of L-LPS and 100 μg of L-OMP for 4 h.The late apoptosis/necrosis rates of the cells were 7.5%and 6.9%upon the treatments with L-LPS and L-OMP, respectively.However, the apoptosis or necrosis of the cells was not ob-served when using L-LPS and L-OMP pre-treated by PMB and PK, respectively.Silenced expression of Fas or FasL gene reduced the L-LPS-induced J774A.1 cells apoptosis （P〈0.05）, while decreased early apopto-sis rate of J774A.1 cells mediated by L-OMP was only observed in Fas gene-knockdown cells （P〈0.05）. Conclusion Both L-LPS and L-OMP can cause the Fas/FasL-associated ap

关 键 词：问号钩端螺旋体 脂多糖 外膜蛋白 巨噬细胞 凋亡 

分 类 号：R377[医药卫生—病原生物学]