内毒素耐受对人牙龈上皮细胞分泌IL-1β、IL-6和IL-8的影响

目的:观察脂多糖(lipopolysaccharides,LPS)反复刺激细胞,诱导产生的内毒素耐受对人牙龈上皮细胞(human gingival epithelial cells,HGECs)分泌细胞因子IL-1β、IL-6和IL-8的影响。方法:采用1mg/L牙龈卟啉单胞菌(Porphyromonas gingivalis,P.gingivalis)LPS或1mg/L大肠杆菌(Escherichia coli,E.coli)LPS刺激HGECs 24h,洗涤细胞后,分别采用相同的LPS再次刺激24h,构建内毒素耐受模型。采用ELISA技术检测细胞条件培养液中IL-1β、IL-6和IL-8分泌水平的变化。结果:P.gingivalis LPS或E.coli LPS刺激HGECs 24h后,3种细胞因子的分泌水平均较刺激前明显增高(P<0.05)。2种LPS重复刺激,诱导细胞耐受后,IL-6和IL-8的分泌水平较第1次刺激后明显降低(P<0.05),但P.gingivalis LPS重复刺激后,IL-1β的分泌水平与第1次刺激后无明显差别。结论:内毒素耐受能抑制HGECs分泌细胞因子IL-6和IL-8,进而可能影响牙周组织的炎症和免疫反应。     

五倍子水提取物对Pg LPS诱导单核细胞分泌IL-6的抑制作用

目的：研究百倍子水提取物对牙龈卟啉菌(Pg)内毒素(LPS)介导的人单核细胞分泌IL-6水平的影响。方法：采用健康人外周血分离培养的单核细胞以25μg/ml牙龈卟啉菌内毒素作为刺激因子，用放射免疫分析法(RIA)测定细胞培养上清中IL-6的水平，观察5种浓度瓦倍子水提取物对单核细胞分泌炎性细胞因子的影响，结果：五倍子水提取物可显著抑制牙龈卟啉菌内毒素诱导人单核细胞分泌IL-6的水平，其作用在一定范围内呈浓度依赖性。结论：五倍子水提取物能够显著抑制牙龈卟啉菌内毒素诱导人单核细胞分泌IL-6的水平，提示五倍子具有一定的抗炎作用，有助于对牙周病的防治。     

可溶性CD14对LPS诱导人牙龈成纤维细胞活化的影响

目的:观察重组表达的sCD14对LPS的炎症介导作用的影响。方法:将异硫氰酸荧光素(fluoresceinisothiocyanate,FITC)标记的LPS分别和sCD14以及阳性对照内毒素中和蛋白(endotoxin neutralizing protein,ENP)孵育后,加入人牙龈成纤维细胞培养系统中,通过流式细胞仪测定LPS与细胞结合后的荧光强度;采用ELISA检测细胞培养上清中的TNF-α和IL-6的含量。结果:sCD14和ENP可导致细胞膜平均通道荧光强度减低,LPS诱导的细胞因子TNF-α和IL-6分泌量显著下降。结论:sCD14和ENP使人牙龈成纤维细胞的LPS结合量显著下降,表明sCD14和LPS中和蛋白可竞争性结合LPS,引起细胞培养系统中游离LPS浓度下降,对细胞的炎症介导作用减小。     

内毒素对牙龈成纤维细胞mCD14表达的影响

目的:研究mCD14在牙龈成纤维细胞的膜表面分布及内毒素对mCD14在牙龈成纤维细胞膜表面表达的影响。方法:组织块法培养人牙龈成纤维细胞,利用免疫组化和Western blot方法研究mCD14在牙龈成纤维细胞的膜表面分布,同时观察内毒素对mCD14在牙龈成纤维细胞膜表面表达的影响。结果:免疫组织化学染色实验和蛋白印迹实验结果均表明mCD14在牙龈成纤维细胞的膜表面表达阳性,同时LPS可增强膜表面的mCD14的表达。结论:本实验表明正常人牙龈成纤维细胞可表达mCD14,LPS可以上调膜表面mCD14的表达,从而导致牙周损伤。     

环孢素A引发牙龈过度生长过程中IL-6的表达

目的:对体外培养的牙龈上皮细胞和成纤维细胞施加环孢素A(cyclosporin,CsA)刺激,应用免疫组化方法探讨CsA引发药物性牙龈过度生长(gingival overgrowth,GO)的病理机制。方法:对体外培养的牙龈上皮细胞和成纤维细胞分别施加浓度为600、800和1000ng/mL,作用时间为48、72h的CsA刺激。在观察细胞生长曲线及其变化的基础上,通过对细胞铺片的免疫酶染色(ABC法)定量分析和对细胞培养液的酶联免疫吸附检测(ELISA法),分别对牙龈组织细胞IL-6的表达和分泌进行测定,应用SAS 6.0软件包对数据进行统计学分析。结果:牙龈上皮细胞接受CsA刺激后,细胞数量明显增加,与对照组相比具有显著差异(P<0.05)。在接受相同条件CsA刺激下,牙龈上皮细胞和成纤维细胞胞内IL-6表达无显著差异,细胞胞内IL-6表达量与CsA作用时间、浓度间相关。接受CsA刺激的最初24h内,牙龈成纤维细胞分泌IL-6的总量在各CsA浓度实验组及对照组间均无显著差异(P>0.05);牙龈成纤维细胞接受刺激超过24h后,CsA浓度为1000ng/mL的实验组与对照组间在细胞分泌IL-6总量上有显著增加...     

牙龈卟啉菌和大肠杆菌内毒素对人牙周膜细胞分泌IL-6、TNF-α的影响

目的：研究龈卟啉菌、大肠杆菌内毒素对人牙周膜细胞（PDLC）分泌IL-6、TNF-α的影响。方法：采用细胞培养技术和ELISA方法，检测培养上清中IL-6、TNF-α水平。结果：在孵育6h后，即可在培养上清中检测到IL-6和TNF-α。IL-6在12h、TNF-α在24h内呈时间依赖性方式升高。结论：PDLC在内毒素作用下局部分泌IL-6、TNF-α，参与了牙周炎的发生、发展过程。     

不同fimA基因型牙龈卟啉单胞菌对牙龈成纤维细胞表达白细胞介素-8的影响

目的:研究不同fimA基因型牙龈卟啉单胞菌(Porphyromonas gingivalis, Pg)对人牙龈成纤维细胞表达IL- 8的影响,探讨fimA基因型与Pg致病力差异之间的关系.方法: Pg ATCC 33277(Ⅰ型),WCSP115(Ⅱ型),WCSP1.5(Ⅲ型),W83(Ⅳ型)分别与牙龈成纤维细胞在标准条件下共同孵育,于孵育后1、3、6、12 h收集细胞和上清,应用实时荧光定量RT- PCR和ELISA法分别检测牙龈成纤维细胞IL- 8 mRNA和蛋白的动态表达.结果:与对照组比较,各fimA型Pg对牙龈成纤维细胞IL- 8 mRNA和蛋白的表达均有明显的促进作用(P<0.01);其中ⅡfimA型组IL- 8 mRNA和蛋白的表达量明显高于其它fimA型组,不同时间点IL- 8 mRNA相对表达量分别为20.42±2.21～103.01±12.50,蛋白分泌水平为(137.46±4.97～323.24±13.74) pg/ml;而Ⅲ fimA型Pg组的表达水平弱于其它fimA型组,IL- 8 mRNA相对表达量为3.61±0.39～12.88±1.56,蛋白分泌水平为(44.83±3.68～189.19±87.34) pg/ml, 差异有统计学意义(P<0.05).结论:Pg可促进牙龈成纤维细胞IL- 8 mRNA和蛋白的表达,且各fimA型Pg的促进作用有所差异.提示: fimA基因型的不同可能是Pg的致病力差异的基因基础.     

IL-1α刺激人牙龈成纤维细胞产生IL-11和IL-6的比较研究

目的比较IL—1α刺激牙龈成纤维细胞（human gingival fibroblast，HGF）IL-11和IL-6的表达和调控。方法收集细胞上清液，ELISA法测定IL-11／IL-6的水平；提取细胞mRNA，实时定量RT—PCR法分析IL-11mRNA的表达。结果IL-1α显著增加HGFIL-11／IL-6的表达（P〈0．05），其中IL-6水平增加最为显著；HGF受到IL—1α刺激IL-11mRNA表达明显增强。吲哚关欣在蛋白质（P〈0．05）和mRNA水平都显著抑制了IL—1α刺激的HGFIL-11的产生，但对IL-6的产生在蛋白质水平则有不同影响，五株HGF细胞系中有四株IL-6的产生受到显著抑制（P〈0．05），另一株则表现为轻微促进IL-6的产生。IL-1α刺激IL-11的产生也都受到staurosporine的抑制（P〈0．05），而IL-6的产生在细胞株HGF-2受到显著抑制（P〈0．05），在HGF-3株则不受影响。结论IL-1α刺激HGF显著增加IL-11／IL-6的产生，IL-11与IL-6在该刺激产生过程中受内源性前列腺素及蛋白激酶C信号途径调控的机理不完全相同。     

Nod/Rip2信号通路参与牙龈卟啉单胞菌诱导牙髓细胞白细胞介素-6表达

目的探讨牙龈卟啉单胞菌刺激牙髓细胞产生细胞因子的信号转导通路。方法厌氧培养牙龈卟啉单胞菌（P.gingivalis）,胞内感染原代培养牙髓细胞,RNA抽提,实时荧光定量聚合酶链反应（qPCR）检测Nods、Rip2,ELISA检测白细胞介素-6（IL-6）的表达水平。结果牙髓细胞基础表达Nods、Rip2 mRNA及IL-6。P.gingivalis感染后2 h, Nods和Rip2 mRNA增高,达到高峰,6 h出现下降趋势。而P.gingivalis活菌刺激牙髓细胞能增强IL-6表达水平。结论P.gingivalis能激活牙髓细胞固有免疫反应,通过Nod/Rip2途径进行信号转导上调细胞因子IL-6表达。     

TLR4对牙周膜细胞分泌IL-8影响的实验研究

目的:观察TLR4在牙周膜细胞分泌IL-8过程中的作用。方法:采用RT-PCR和ELISA的方法检测IL-8mRNA和蛋白在牙周膜细胞中的表达,并观察不同剂量LPS对其表达量的影响以及抗TLR4抗体作用下IL-8mRNA和蛋白表达量的变化。结果:牙周膜细胞中存在IL-8mRNA表达,分泌少量IL-8蛋白;经LPS刺激后IL-8表达量上调,抗TLR4抗体能够拮抗这一作用。结论:牙周膜细胞表达IL-8mRNA及蛋白,在受到LPS刺激后,IL-8mRNA和蛋白的表达量增加,抗TLR4受体能够拮抗这一作用,提示TLR4参与了牙周膜细胞受到LPS刺激后分泌IL-8的过程。     

Suppressive effect of the antimicrobial peptide LL-37 on expression of IL-6, IL-8 and CXCL10 induced by Porphyromonas gingivalis cells and extracts in human gingival fibroblasts

Porphyromonas gingivalis is a major periodontogenic bacterium and possesses immunostimulatory components, such as lipopolysaccharides (LPS) and fimbriae. The host antimicrobial peptide, LL-37, suppresses proinflammatory responses of immune cells but its effect on human gingival fibroblasts (HGFs) is not known. In this study, we assessed the effect of LL-37 on the proinflammatory responses of HGFs stimulated with P. gingivalis cells and their components. Live P. gingivalis cells did not induce proinflammatory responses of HGFs, and LL-37 did not alter these responses. However, LL-37 was able to suppress the killed P. gingivalis cell-induced secretion of interleukin (IL)-6 and IL-8. LL-37 also suppressed the expression of IL6, IL8, and CXCL10 genes that was induced by P. gingivalis components, including phenol-water extracts, lipid A, and fimbriae, and the induction of phosphorylation of p38 and extracellular signal-regulated kinase (ERK) by P. gingivalis lipopolysaccharide (LPS). CAMP was found to be expressed in oral epithelial cells but not in HGFs, despite stimulation with P. gingivalis components. Therefore, LL-37 can exert a suppressive effect on P. gingivalis-induced proinflammatory responses of HGFs in a paracrine manner, suggesting that excess inflammatory responses to P. gingivalis in the gingival tissue are suppressed by LL-37 in vivo.     

Direct and indirect effects of Porphyromonas gingivalis lipopolysaccharide on interleukin-6 production by human gingival fibroblasts

We examined the production of interleukin-6 (IL-6) by human gingival fibroblasts (ATCC CRL 1292) stimulated with lipopolysaccharide (LPS) from Porphyromonas gingivalis and Escherichia coli, or supernatant of human peripheral blood adherent cell culture medium incubated in the presence of IL-1 and the same two LPS. Confluent monolayers of gingival fibroblasts were incubated with stimulants for 6 h at 37 degrees C in 5% CO2 and air. After removal of stimulants, the cell cultures were incubated for an additional 2 or 24 h in the same environment. At the end of the culture period, supernatants were collected and assayed for IL-6 activity by stimulatory IgG production with the human B-lymphoblastoid cell line CESS. The direct effect of LPS on IL-6 production by gingival fibroblasts was much weaker than the indirect one via IL-1 production by adherent cells. The stimulating effect of culture supernatants of adherent cells stimulated with LPS on IL-6 production by gingival fibroblasts was as effective as that of recombinant IL-1, when this latter was added at a concentration equivalent to that contained in the culture supernatant of adherent cells. These results suggest that, although gingival fibroblasts may be involved in the pathogenesis of chronic periodontal disease by the production of cytokines, such a role may not result from a direct stimulation by periodontopathic bacteria. The phenomenon is more likely to be mediated indirectly by IL-1 produced by infiltrating inflammatory cells.     

Interleukin 4 (IL-4) and IL-6-producing memory T-cells in peripheral blood and gingival tissues in periodontitis patients with high serum antibody titers to Porphyromonas gingivalis

We assessed cytokine production and proliferation of memory T-cells that were isolated from peripheral blood of adult periodontitis patients with high anti- Porphyromonas gingivalis titer. Memory T-cells were stimulated with P. gingivalis lipopolysaccharide, sonicates and formalin-killed whole cells. Interleukin 4 (IL-4)- and IL-6-producing cells were stained by immunocytochemically on peripheral blood smears and compared with cryostat sections of autologous gingival biopsies. Memory T-cells in the peripheral blood of patients rated significantly higher than in healthy subjects (32.3±7.1 vs 25,3±3.0%). Stimulation of patient-derived memory T-cells with P. gingivalis whole cells induced higher IL-4 production than in healthy subjects (4.4±4.1% vs 0.7±0.6%). Induction of IL-4-producing memory T-cells by P. gingivalis lipopolysaccharide and whole cells was respectively 1.37 and 1.56 times that induced by medium alone. IL-6 production did not differ between the groups. Proliferation of memory T-cells in healthy subjects tended to be more inhibited by P. gingivalis antigens than that in patients. In some patients, induction of IL-4- and IL-6-producing memory T-cells in peripheral blood and in autologous gingival biopsies tended to coincide. Memory T-cells with functional characteristics of Th2 could be a crucial cell population capable of reflecting individual susceptibility to periodontitis.     

Regulation of cytokine production in human gingival fibroblasts following treatment with nicotine and lipopolysaccharide

BACKGROUND: Patients who smoke are at increased risk for chronic periodontitis (CP). Most studies suggest that the microbial flora in these patients is similar to that found in non-smoking CP patients. Thus, the increased risk for development of CP is not dependent on an altered microbial profile, but rather to some change in the host response to these periopathogens. There is evidence that human gingival fibroblasts (HGF) derived from diseased sites produce greater amounts of interleukin (IL)-6 and IL-8 in vitro than cells derived from healthy sites. This suggests that HGF subpopulations may be selected based upon the inflammatory milieu in which they reside. The hypothesis to be tested was that the combination of nicotine and lipopolysaccharide (LPS) could regulate HGF inflammatory mediator production. METHODS: HGF cell cultures were established from explants derived from 10 patients with CP. HGF cell cultures were stimulated with 1 mM, 1 microM, or 1 nM nicotine +/- Escherichia coli or Porphyromonas gingivalis LPS. At 12, 24, or 48-hour time points, the cells were counted and the supernatant was collected for subsequent IL-6 and IL-8 determination in an enzyme-linked immunosorbent assay. RESULTS: At the 24-hour time point, 1 nM nicotine stimulated IL-6 production compared to control (P=0.02). E. coli LPS alone caused a 3- to 4-fold increase in IL-6 and IL-8 production, whereas P gingivalis LPS did not augment IL-6 or IL-8. A synergistic effect upregulating IL-6 was observed with combined treatment of 1 mM nicotine and E. coli LPS or P gingivalis LPS at the 24-hour time point (P<0.0005 and P=0.002, respectively). Similar effects were seen when IL-8 production was evaluated following HGF stimulation with high doses of nicotine and E. coli LPS or P gingivalis LPS. CONCLUSIONS: These results demonstrate that nicotine by itself can stimulate HGF IL-6 and IL-8 production. Moreover, the combination of high doses of nicotine and either E. coli or P gingivalis LPS can synergistically upregulate cytokine production. These findings support the hypothesis that a proinflammatory fibroblast phenotype may be elicited in an environment enriched with bacterial LPS and nicotine.     

IFN-γ对牙龈卟啉单胞菌内毒素诱导的细胞耐受的影响

目的：观察IFN-γ对牙龈卟啉单胞菌（porphyromonasgin givalis,P．gingivalis）脂多糖（1ipopolysaccharides,LPS）诱导人单核细胞株THP-1细胞耐受后,炎症因子IL-1β和趋化因子IL-8分泌水平的影响。方法：将THP-1细胞用1mg／LIFN-γ预先处理24h,同时采用1mg／L P．gingivalisLPS刺激该细胞24h,洗脱后,采用P．gingivalis LPS再刺激24h,构建内毒素耐受模型。采用大肠杆菌（escherichiacoli,E．coli）LPS作为阳性对照。运用ELISA技术检测细胞条件培养液中IL-1β和IL-8分泌水平的变化。结果：P．gingivalis LPS重复刺激THP-1细胞后,IL-1β分泌水平较第-次刺激后明显降低（P〈0．05）,IL-8分泌水平与第-次刺激后差别不大。经过IFN-γ预先处理的细胞,P．gingivalis LPS重复刺激诱导细胞耐受后,IL-1β分泌水平较无预处理的细胞明显增高（P〈0．05）,IL-8分泌水平无明显变化。结论：IFN-丫能够促进P．gingivalisLPS诱导的耐受细胞分泌IL-1β,进而可能影响牙周组织的炎症和免疫反应。     

牙龈卟啉单胞菌膜泡诱导牙龈上皮细胞炎性反应的体外研究

目的建立牙龈卟啉单胞菌膜泡诱导牙龈上皮细胞炎性反应的体外模型，探讨牙龈卟啉单胞菌在牙周炎中的致病作用。方法用酶联免疫吸附法检测牙龈卟啉单胞菌膜泡对牙龈上皮细胞前列腺素E2（prostaglandin E2，PGE2）分泌的影响，以实时反转录聚合酶链反应法检测牙龈卟啉单胞菌膜泡对牙龈上皮细胞环氧化物酶（cyclooxygenase，COX）-2和白细胞介素（interleukin，IL）-6、IL-8基因表达的作用。结果牙龈卟啉单胞菌膜泡浓度依赖性地促进了牙龈上皮细胞PGE2的分泌，并使COX-2、IL-6、IL-8的mRNA表达水平显著上调。结论牙龈卟啉单胞菌膜泡诱导牙龈上皮细胞发生的细胞炎性反应，可能是牙周炎发生、发展的重要因素。     

Treponema medium glycoconjugate inhibits activation of human gingival fibroblasts stimulated with phenol-water extracts of periodontopathic bacteria

Oral treponemes are well-known as causative agents of periodontal diseases; however, the details have not been fully clarified. Here, we examined the effects of Treponema medium glycoconjugate on the activation of human gingival fibroblasts using phenol-water extracts from Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum subsp. nucleatum, and Actinobacillus actinomycetemcomitans. The phenol-water extracts activated human gingival fibroblasts to mediate IL-8 production, as well as IL-8 mRNA expression, phosphorylation of p38 mitogen-activated protein kinase, and expression of intercellular adhesion molecule-1. T. medium glycoconjugate exhibited no activation of human gingival fibroblasts, while phenol-water extract-induced activation of human gingival fibroblasts was clearly inhibited by T. medium glycoconjugate. Furthermore, binding of biotinylated phenol-water extracts to CD14 in the presence of LPS-binding protein was blocked with T. medium glycoconjugate. These results suggest that T. medium glycoconjugate has an inhibitory effect on host cell activation by periodontopathic bacteria caused by binding to CD14- and LPS-binding protein.