LPS刺激对体外培养人牙龈成纤维细胞环氧合酶表达的影响

目的检测脂多糖（LPS）刺激后体外培养人牙龈成纤维细胞环氧合酶-1和环氧合酶-2（COX-1,COX-2）表达的改变,探讨牙龈成纤维细胞与牙周炎症发生发展的关系。方法组织块法原代培养正常人牙龈成纤维细胞并进行来源鉴定,MTT法检测不同浓度LPS刺激后活细胞数量的改变;以10ug/ml刺激第4代细胞,24h后用免疫细胞化学方法检测COX-1和COX-2在细胞中的表达,多功能彩色细胞分析管理系统进行图像分析和统计学分析。结果 LPS刺激可使牙龈成纤维细胞的增殖速度降低,生长曲线大致呈＂S＂形;10ug/ml LPS刺激24h后细胞数量未见明显变化。免疫细胞化学染色结果显示COX-1在LPS刺激前后均有表达但无显著差异（P〉0.05）,COX-2在LPS刺激前无表达,LPS刺激后则表达增强并有显著差异（P〈0.01）。结论 LPS对牙龈成纤维细胞生长增殖有抑制作用,不影响COX-1的表达强度,但可使COX-2表达显著增强,说明牙龈成纤维细胞对LPS刺激的反应可能影响牙周炎症进展。     

IL-1β对体外培养人牙髓成纤维细胞中基质金属蛋白酶-2的影响

目的:探讨白细胞介素-1β(interleukin-β,IL-1β)对体外培养的人牙髓成纤维细胞中基质金属蛋白酶-2(matrix metallproteinase-I,MMP-2)的影响。方法:利用免疫组化和明胶酶谱法,检测正常和经IL-1β刺激后的牙髓成纤维细胞中MMP-2的表达、分泌和活性。结果:MMP-2在正常和经IL-1β刺激后的牙髓成纤维细胞中均有表达,后者表达明显强于前者。明胶酶谱分析显示,与对照组相比,经IL-1β刺激的牙髓成纤维细胞中MMP-2的水平在48 h后持续显著升高。结论:IL-1β可能参与调节牙髓成纤维细胞合成和分泌MMP-2,从而促进牙髓炎症的发生。     

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。     

口腔癌相关成纤维细胞的体外生物学行为研究

目的探讨口腔鳞癌中癌相关成纤维细胞(carcinoma associated fibroblasts,CAFs)的体外生物学行为。方法组织块贴壁法培养CAFs及牙龈正常成纤维细胞(normal fibrolasts,NFs);通过形态观察,免疫组化染色,细胞增殖活性、贴壁率、迁移能力等测定,比较两种细胞的生物学行为差异。结果与NFs相比,CAFs特征性表达成纤维激活蛋白(fibroblast activation protein,FAP)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)及基质金属蛋白酶-2(matrix metalloprotease-2,MMP-2),具有更高的增殖活性、贴壁率及迁移能力(P<0.05)。结论口腔癌CAFs特征性表达相关蛋白,体外增殖、粘附、迁移能力均高于NFs。     

环孢素A与牙龈卟啉菌内毒素对人牙龈成纤维细胞增殖及TGF-β1分泌影响的研究

目的 探讨环孢素A(CsA)与牙龈卟啉菌内毒素(Pg LPS)对人牙龈成纤维细胞增殖及分泌转化生长因子-β1(TGF-β1)的影响。方法 用组织块法原代培养健康人牙龈成纤维细胞,分别用800 ng/ml CsA、100 ng/ml LPS、800 ng/ml CsA+100 ng/ml LPS作用于生长良好的第4~8代细胞,用MTT法测定作用后第2、3、5天的吸光值。ELISA法检测第3天时细胞上清中的TGF-β1分泌的变化。结果 细胞经CsA或LPS作用后形态无明显变化,CsA能促进细胞增殖和分泌TGF-β1,LPS作用与其相反,二者联合作用能明显促进细胞的生长及TGF-β1分泌,比单纯使用CsA更明显。结论 CsA与LPS在人牙龈成纤维细胞增殖和TGF-β1分泌方面可能有协同作用。     

牙龈卟啉单胞菌膜泡对牙龈上皮细胞MMPs基因表达的影响

目的:探讨牙龈卟啉单胞菌膜泡对牙龈上皮细胞基质金属蛋白酶(MMPs)基因表达的影响,揭示牙龈卟啉单胞菌在牙周炎中的致病作用.方法:以Real-time RT-PCR法检测牙龈卟啉单胞菌膜泡刺激下牙龈上皮细胞MMP-1和MMP-3的mRNA表达水平.结果:牙龈卟啉单胞菌膜泡显著地上调MMP-1和MMP-3 mRNA表达水平.结论:牙龈卟啉单胞菌诱导牙龈上皮细胞发生细胞炎症反应,可能是牙周炎发生、发展的重要因素.     

绿茶多酚对牙龈上皮细胞内炎症因子的抑制作用

目的:探讨绿茶多酚表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCg)对牙龈上皮细胞炎症反应的作用,从而为牙周病的预防和治疗开发安全有效的牙周炎症抑制剂提供依据。方法:通过建立牙龈卟啉单胞菌膜泡刺激牙龈上皮细胞引起细胞炎症反应的体外模型,以酶联免疫吸附法(ELISA)检测EGCg对牙龈上皮细胞分泌前列腺素E2(PGE2)的影响,以Real-time RT-PCR法检测EGCg对牙龈上皮细胞表达环氧化物酶-2(COX-2)和基质金属蛋白酶-3(MMP-3)mRNA的作用。结果:EGCg浓度依赖性抑制牙龈上皮细胞内PGE2分泌和COX-2、MMP-3 mRNA的表达水平。结论:EGCg对牙龈上皮细胞的炎症反应具有抑制作用,具有成为牙周炎症抑制剂的潜能。     

基质金属蛋白酶-2和基质金属蛋白酶-3在大鼠牙周炎模型中的表达

目的 研究不同时期、不同严重程度的大鼠牙周炎模型中基质金属蛋白酶-2（MMP-2）和基质金属蛋白酶-3（MMP-3）的表达和分布特征,探讨二者在牙周炎发生发展中的作用。方法 对大鼠牙周组织标本的石蜡切片进行MMP-2和MMP-3的免疫组化染色,并进行图像定量分析。结果 MMP-2和MMP-3在牙龈炎和牙周炎的牙龈上皮中呈强阳性表达,在牙周炎的牙周组织中,主要在牙周膜成纤维细胞、破骨细胞等细胞中呈强阳性表达。MMP-2和MMP-3的变化趋势为在健康牙周组织中呈弱阳性表达,急性龈炎和急性牙周炎中呈强阳性表达,到慢性牙周炎时期,表达较急性期有所降低。结论 MMP-2和MMP-3在牙周炎症组织中,随着炎症进程呈现不同特征的表达,可能直接参与牙周组织的炎性破坏。     

五倍子水提取物抑制内毒素诱导人牙龈成纤维细胞分泌IL-6

目的：研究不同浓度五倍子水提取物对内毒素(LPS)介导的人牙龈成纤维细胞(HGF)分泌IL-6水平的影响。方法：采用MTT比色法和放射免疫分析法(RIA)，测定五倍子水提取物对HGF增殖的影响；及对以25μg／mL内毒素作为刺激因子，HGF培养上清中IL-6水平的影响。结果：五倍子水提取物可显著抑制内毒素诱导HGF分泌IL-6水平，其作用在一定范围内呈浓度依赖性，同时当其浓度＞50μg／mL时，可抑制HGF增殖。结论：五倍子水提取物能够抑制内毒素诱导人牙龈成纤维细胞分泌IL-6的水平，浓度过高时对HGF增殖有影响，提示一定浓度(＜50μg／mL)的五倍子水提取物具有抗炎作用，有助于对牙周病的防治。     

糖基化终产物对人牙龈成纤维细胞增殖和基质金属蛋白酶-1合成的影响

目的探讨糖基化终产物（AGEs）对人牙龈成纤维细胞（HGF）增殖和基质金属蛋白酶-1（MMP-1）合成的影响,以及AGEs加重糖尿病性牙周炎的作用机制。方法采用酶消化-组织块培养法获得HGF,在培养基中加入体外合成的不同浓度AGEs,噻唑蓝（MTT）比色法检测在不同时间段下HGF增殖水平的变化;ELISA法测定HGF合成MMP-1的水平。结果各浓度组AGEs对HGF有抑制作用,呈浓度依赖关系（P〈0.05）;共培养72h后,HGF合成MMP-1量明显增加（P〈0.05）,并且随着浓度的增加而增加。结论AGEs能够抑制HGF的增殖;AGEs可以促进MMP-1合成,导致胶原降解,加速了糖尿病时牙周病进程。     

Celecoxib对细菌脂多糖促牙龈成纤维细胞生长抑制作用的研究

目的观察celecoxib对细菌脂多糖(Lipopolysaccharides,LPS)促人牙龈成纤维细胞(humangingivalfibroblasts,HGFs)生长作用的影响。方法采用细胞活性和增殖水平测定(WST)实验检测LPS及celecoxib对体外培养的HGFs生长的影响。结果LPS可促进HGFs的生长,其作用效果随LPS剂量的增加而增强,1.0,12.5、100.0LPS处理后HGFs的细胞生长率与阴性对照组相比分别为126.1%,164.5%和215.5%;celecoxib对LPS促HGFs生长的现象有明显的抑制作用,其抑制作用呈剂量依赖性关系,12.5μmol/L到100μmol/L的celecoxib处理可使LPS刺激的HGFs生长率降低至对照组的76.3%~30.3%。结论LPS可促进HGFs的生长;celecoxib能抑制LPS对HGFs的生长刺激作用。     

Lymphatic growth factors are expressed in human gingiva and upregulated in gingival fibroblasts after stimulation

1 Background

The lymphatic growth factors vascular endothelial growth factor (VEGF)‐C and ‐D are important for maintenance and growth of lymphatic vessels (lymphangiogenesis), but their localization in human gingiva is unknown. This study investigated the expression of VEGF‐C and ‐D in human gingiva and isolated human gingival fibroblasts (HGFs). In addition, the localization of their main receptor VEGFR‐3 was explored.

2 Methods

Non‐inflamed gingiva from six donors was used for immunohistochemistry or isolation of HGFs. HGFs were stimulated with either E.coli lipopolysaccharide (LPS) or IL‐6/soluble IL‐6 receptor (sIL‐6R) complex for 1, 6, and 24 hours. Quantitative real‐time polymerase chain reaction (qRT‐PCR) was used to quantify the relative changes in gene expression of VEGF‐A, ‐C, and ‐D and enzyme‐linked immunosorbent assay (ELISA) for quantification of protein levels.

3 Results

VEGF‐C, ‐D and VEGFR‐3 were seen in keratinocytes, blood vessels and in scattered single cells in gingiva. VEGFR‐3 was also found in lymphatic vessels and VEGF‐C in cells with fibroblastic appearance. Gene analysis showed no expression of VEGF‐D in the HGFs, but showed constitutive expression of VEGF‐C and ‐A. Stimulation of HGFs with LPS or IL‐6/sIL‐6R complex was followed by gene upregulation of VEGF‐C and ‐A and increased protein levels in cell culture supernatant (P ≤0.05).

4 Conclusions

The localization of VEGF‐C, ‐D, and VEGFR‐3 expression imply that signaling via VEGFR‐3 is linked to vascular homeostasis and keratinocyte function under normal conditions in gingiva. Inflammatory stimulation of HGFs upregulates VEGF‐C and ‐A expression and may contribute to angiogenesis and lymphangiogenesis.     

Down-regulation of Inflammatory Mediator Synthesis and Infiltration of Inflammatory Cells by MMP-3 in Experimentally Induced Rat Pulpitis

Introduction

Matrix metalloproteinase (MMP)-3 is a member of the MMP family that degrades the extracellular matrix. Application of MMP-3 to injured pulp tissue induces angiogenesis and wound healing, but its anti-inflammatory effects are still unclear. Here, we evaluated the anti-inflammatory functions of MMP-3 in vitro and in vivo.

Methods

Nitric oxide and inflammatory mediator synthesis in macrophages activated by lipopolysaccharide (LPS) was measured in the presence or absence of MMP-3. The mouse Mmp3 (mMmp3) expression vector containing full length cDNA sequence of mMmp3 or cDNA sequence of mMmp3 missing the signal peptide and pro-peptide regions was transfected to RAW264, a mouse macrophage cell line, and NO synthesis and inflammatory mediator expression were evaluated. Pulpal inflammation was histologically and immunohistochemically evaluated in a rat model of incisor pulpitis induced by the application of LPS for 9 hours in the presence or absence of MMP-3.

Results

NO and pro-inflammatory mediator synthesis promoted by LPS was significantly down-regulated by MMP-3 in vitro. The full length of mMmp3 down-regulated the LPS-induced NO synthesis and chemical mediator mRNA expression, however the mMmp3 missing the signal peptide failed to block the NO synthesis induced by LPS. The numbers of major histocompatibility complex class II+ and CD68+ cells, which infiltrated into the rat incisor pulp tissues in response to the topical application of LPS, were significantly decreased by the application of MMP-3 in vivo.

Conclusions

These results indicate that MMP-3 possesses anti-inflammatory functions, suggesting its potential utility as an anti-inflammatory agent for pulpal inflammation.     

Effect of phenytoin on collagen accumulation by human gingival fibroblasts exposed to TNF-alpha in vitro

OBJECTIVE: Tumor necrosis factor (TNF)-alpha is associated with chronic gingival inflammation and reported to induce gingival overgrowth (GO), while phenytoin (PHT) is also known to be a causative agent of GO. We examined the synergistic effect of PHT and TNF-alpha on collagen metabolism in human gingival fibroblasts (HGFs). MATERIALS AND METHODS: HGFs were cultured with TNF-alpha and PHT. Quantitative real-time RT-PCR was employed to determine the mRNA levels for collagen, matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and integrin subunits. Cellular collagen endocytosis was determined using a flow-cytometry. RESULTS: The proliferation of HGFs was not affected by TNF-alpha or PHT individually, whereas both synergistically increased collagen accumulation in HGFs. Further, collagen mRNA expression was not increased by TNF-alpha or PHT, although together they markedly prevented cellular collagen endocytosis, associated with the suppression of alpha2beta1-integrin mRNA expression. The mRNA expression of MMP-1 and-2 was suppressed by PHT, while TIMP-1 mRNA expression was enhanced by both TNF-alpha and PHT. CONCLUSION: Our results suggest that TNF-alpha and PHT together cause impaired collagen metabolism by suppression of enzymatic degradation with MMPs/TIMP-1 and integrin-mediated endocytosis. These synergistic effects may also be involved in TNF-alpha- and PHT-induced collagen accumulation, leading to GO.     

Nicotine and lipopolysaccharide affect cytokine expression from gingival fibroblasts

BACKGROUND: This in vitro study investigated the influence of nicotine, lipopolysaccharide (LPS), and a combination of both agents on cytokine expression from human gingival fibroblasts (HGFs). METHODS: HGFs were exposed for 48 hours to 250 microg/ml nicotine, 1 microg/ml Porphyromonas gingivalis LPS, or both. The expression of multiple cytokines was detected in the HGFs conditioned media using cytokine protein arrays. RESULTS: The untreated HGFs expressed several cytokines, which included relatively high levels of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1). They also expressed low levels of growth-regulated oncogene (GRO), IL-3, and IL-10. Nicotine had the greatest effect on the expression of GRO-alpha, IL-7, IL-10, and IL-15 compared to the untreated control. P. gingivalis LPS had the greatest effect on the expression of GRO-alpha; IL-7; IL-10; and RANTES (regulated on activation, normal T-cell expressed, and presumably secreted) compared to the untreated control. The combination of both agents had the biggest impact on the expression of GRO-alpha, IL-7, IL-10, IL-15, RANTES, and interferon-gamma (IFN-gamma) compared to the untreated control. CONCLUSION: HGFs exposed to nicotine, P. gingivalis LPS, or a combination of both agents increased the expression of multiple cytokines.