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目的 比较具有不同毒力特性的牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)临床菌株的多糖成分诱导人单核细胞系THP-1细胞分泌白细胞介素(interleukin,IL)Ip、IL-8和肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)的能力,分析Pg多糖成分的免疫原性,探讨Pg的毒力相关因素.方法 酚-水提取法提取Pg高毒力标准菌株W83、高毒力临床菌株SJD2和SJD12(高毒株组)、低毒力标准菌株ATCC33277及低毒力临床菌株SJD4、SJD5和SJD11(低毒株组)的多糖成分,并作用于人单核细胞系THP-1细胞,利用酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)法检测在10％和1％胎牛血清环境中,不同刺激物浓度、不同刺激时间下细胞上清液中IL-1β、IL-8和TNF-α的浓度.结果 酚-水提取法获得各菌株多糖成分,经鉴定主要含有脂多糖及荚膜多糖成分.在含10％胎牛血清培养基中,各菌株多糖提取物诱导THP-1细胞分泌IL-1β、IL-8和TNF-α均呈时间、剂量依赖性;在含1％胎牛血清的培养基中,高毒株组IL-1β[4 h:(1 639 ±497) μg/L]、IL-8[4 h:(1 648 ±513) μg/L]及TNF-α的质量浓度[4 h:(140 ±48)μg/L]均显著高于低毒株组[4h时IL-1β、IL-8及TNF-α质量浓度分别为(773 ±382)、(892 ±400)及(67±33) μg/L].结论 Pg多糖提取物具有免疫原性,可以诱导THP-1细胞分泌IL-1β、IL-8和TNF-α;各菌株诱导THP-1细胞表达细胞因子的能力可能与其毒力密切相关.

Objective To compare the ability of the polysaccharide from various Porphyromonas gingivalis(Pg) type and clinical strains in inducing THP-1 cells to produce cytokines interleukin (IL)-1 β,IL-8,and tumor necrosis factor (TNF)-or,in order to analyze the immunogenicity of Pg polysaccharide components and the virulence-associated factors of this periodontal pathogen.Methods The bacterial polysaccharide was extracted from high virulent Pg strains,W83,SJD2,SJD12 and low virulent Pg,ATCC33277,SJD4,SJD5,and SJD11 by phenol-water extraction.The extracted polysaccharide was used to stimulate the THP-1 cells with different simulation periods and doses.The level of the cytokines,including IL-1 β,IL-8 and TNF-α in the cell culture suspension was measured by enzyme-linked immunosorbent assay (ELISA).Results The polysaccharide extraction of Pg strains was composed of lipopolysaccharide (LPS) and capsular polysaccharide.The secretion of IL-1 β,IL-8 and TNF-α,produced by the THP-1 cells showed in a time-and dose-dependent manner in the medium containing 10％ fetal bovine serum.The level of these cytokines of the high virulent strains was higher than that of the low virulent strains in medium containing 1％ fetal bovine serum.Four hours after stimulation with polysaccharide extracted from high virulent strains,the levels of IL-1β,IL-8,and TNF-α in the cell suspension were (1 639 ± 497),(1 648 ± 513) and (140 ±48) μg/L,respectively,whereas for low virulent strains,the levels of IL-1β,IL-8,and TNF-o were (773 ±382),(892 ± 400) and (67 ± 33) μg/L,respectively.Conclusions Polysaccharide extracted from Pg could induced the THP-1 cells to secrete the cytokines of IL-1β,IL-8 and TNF-α.The level of the cytokines produced by the THP-1 cells associates with the bacterial virulent properties.