牙龈卟啉菌脂多糖诱导HL-60细胞分泌IL-1β、TNF-α、IL-6能力及其相关Toll样受体和信号通路的差异  

Diversity of Porphyromonad gingivalis lipopolysaccharide induced IL-1β, TNF-αand IL-6 levels in HL-60 cells and the associated Toll-like receptors and signaling pathways

作　　者：张迪亚[1] 严杰[2] 李盛来[3] 陈莉丽[1] 

机构地区：[1]浙江大学医学院附属第二医院口腔科,杭州310009 [2]浙江大学医学院病原生物学系 [3]浙江大学医学附属口腔医院

出　　处：《中华微生物学和免疫学杂志》2007年第10期954-959,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金面上项目(30471888)

摘　　要：目的探讨牙龈卟啉菌(Porphyronumas gingivalis,Pg)脂多糖诱导人粒细胞HL-60分泌IL-1β、TNF-α、IL-6能力差异及其相关Toll样受体和信号通路。方法采用酚水法提取牙龈卟啉菌ATCC33277株脂多糖(Pg-LPS)。采用ELISA试剂盒定量检测Pg-LPS作用的HL-60细胞分泌IL-1β、TNF-α和IL-6水平。采用TLR2或TLR4单克隆抗体阻断试验联合ELISA检测,了解Pg-LPS结合靶细胞上Toll样受体的类型。采用JNK、P38MAPK和NF-κB通路特异性阻断剂的阻断试验联合ELISA检测,了解Pg-LPS诱导HL-60细胞分泌IL-1β、TNF-α和IL-6的相关胞内信号传导通路。实验中采用大肠杆菌O111:B4脂多糖(E-LPS)作为对照。结果1μg/ml Pg-LPS分别作用24、48和48 h或1μg/ml E- LPS分别作用48、48和72 h,HL-60细胞分泌的IL-1β、TNF-α和IL-6水平明显升高(P<0.01);Pg-LPS诱生的TNF-α最高浓度与E-LPS相近(P>0.05),但诱生的IL-1β和IL-6最高浓度明显高于E-LPS(P <0.05)。TLR2单抗可抑制Pg-LPS诱导HL-60细胞分泌IL-1β、TNF-α或IL-6的活性(P<0.05),但E- LPS诱导HL-60细胞分泌上述3种细胞因子的活性仅可被TLR4单抗所抑制(P<0.05)。Pg-LPS诱导HL-60细胞分泌IL-1β、TNF-α和IL-6的信号通路分别为JNK和NF-κB、JNK和P38MAPK及NF-κB、JNK和P38MAPK(P<0.05),E-LPS则分别为JNK和P38MAPK及NF-κB、P38MAPK和NF-κB、P38MAPK和NF-κB(P<0.05)。结论Pg-LPS诱导HL-60细胞分泌IL-1β、TNF-α和IL-6活性高于E-LPS。TLR2和TLR4分别可能是Pg-LPS和E-LPS的受体。Pg-LPS诱导HL-60细胞合成上述细胞因子的信号传导通路与E-LPS明显不同,不同细胞因子合成的胞内信号传导通路也不尽相同。Objective To determine the diversity of Porphyromonad gingivalis （Pg） lipopolysaccharide induced IL-1β, TNF-α and IL-6 levels in HL-60 cells and the associated Toll-like receptors and signaling pathways. Melhods Pg strain ATCC33277 lipopolysaccharide （Pg-LPS） was prepared using phenol-water method. The levels of IL-1β, TNF-α and IL-6 secreted by HL-60 cells under inducement of Pg-LPS were quantitatively detected using commercial ELISA kits. The blocking test using anti-TLR2 or anti-TLR4 monoelonal antibody （McAb） plus the ELISA were used to determine the types of Pg-LPS binding Toll-like receptors （TLR） on the surface of target cells. The blocking test respectively using the specific inhibitors of JNK, P38MAPK and NF-kB signaling pathways were applied to confirm the associated signaling pathways of Pg-LPS inducing HL-60 cells to secrete IL-1β, TNF-α and IL-6 . A commercial E . coli strain O 1 1 1 ： B4 LPS was used as a control in this study . Results When 1μg/ml Pg-LPS acted for 24, 48 and 48 h or 1μg/ml E-LPS acted for 48, 48 and 72 h, the levels of IL-1β, TNF-α and IL-6 secreted by HL-60 cells were remarkably increased （ P 〈 0.01）. The maximal TNF-α concentration induced by Pg-LPS was similar to that induced by E-LPS （ P 〉 0.05）, while the maximal concentratious of IL-1β and IL-6 were obviously higher that induced by E-LPS （ P 〈 0.05）. The activity of Pg-LPS inducing HL- 60 cells to secrete the three cytokines could be blocked with TLR2 McAb alone （ P 〈 0.05）. TLR4 McAb showed the effects blocking the three cytokine secretion in HL-60 cells under inducement of E-LPS （ P 〈 0.05）. In HL-60 cells, the signaling pathways responsible for Pg-LPS inducing IL-1β, TNF-a and IL-6 were JNK and NF-s：B, all the three pathways, JNK and P38MAPK, respectively （P 〈 0.05）, while for E-LPS were all the three pathways, P38MAPK and NF-s：B, P38MAPK and NF-s：B （ P 〈 0.05）. Conclusion Pg-LPS shows higher activity inducing HL-60 cells to secrete IL-1t3, TNF-

关 键 词：牙龈卟啉菌 脂多糖 HL-60细胞 细胞因子 LPS受体 信号传导通路 

分 类 号：R378[医药卫生—病原生物学]