牙龈卟啉单胞菌不同菌株多糖诱导THP-1细胞分泌细胞因子的比较分析

The comparison of the ability of polysaccharide from different Porphyromonas gingivalis in inducing THP-1 cells to produce cytokines

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）-113, IL-8, and tumor necrosis factor （TNF）-ct, 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-113, IL-8 and TNF-a 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-1β, 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-1 β, IL-8, and TNF-α in the cell suspension were （1 639 ± 497）, （1 648 ± 513）and （140 ± 48 ） ug/L, respectively ,whereas for low virulent strains, the levels of IL-1 β, IL-8, and TNF-α were （773 ± 382）, （ 892 ±400 ） and （ 67 ± 33 ） ug/L, respectively. Conclusions Polysaccharidc 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.