CAJ | 학술논문

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1×105 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。
목적：료해치병성문호구체라선체（간칭구체）지다당（ L-LPS）화외막단백（ L-OMP）유도J774A．1소서거서세포사망급기여Fas/FasL상관성。방법분별채용분수법화Triton X-114법종문호구체황달출혈군뢰형뢰주중제취L-LPS화L-OMP。채용류식세포술검측자외선멸활전후문호구체뢰주、다점균소B（PMB）처리전후L-LPS、단백매K（PK）작용전후L-OMP유도J774A．1세포조망급배사정황。채용siRNA침묵J774A．1세포Fas혹FasL기인병용실시형광정량RT-PCR검측파기인침묵효과。채용류식세포술측정L-LPS혹L-OMPs유도Fas혹FasL기인침묵J774 A．1세포조망적작용。결과자외선멸활전후문호구체뢰주가인기상사적J774A．1세포조기조망솔（55．6％화47．1％）화만기조망/배사솔（7．9％화7．6％）。100 ng L-LPS혹100μg L-OMP작용1×105 J774A．1세포4 h후，조기조망솔화만기조망/배사솔분별위40．4％화34．0％、7．5％화6．9％，단등량PMB예처리L-LPS혹PK예처리L-OMP유도세포조망혹배사적작용소실。 Fas혹FasL기인침묵후，L-LPS유도적J774A．1세포조기조망솔균현저하강（P＜0．05），L-OMP부사Fas기인침묵J774A．1세포조기조망솔유소하강（P＜0．05）。결론 L-LPS화L-OMP가유도Fas/FasL상관거서세포조망，종이유리우문호구체재숙주체내건립유효감염。
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 1×105 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 apoptosis of macrophages , which is beneficial for L.interrogans to establish the productive infection in hosts .