内毒素刺激对体外培养人牙龈成纤维细胞表达OPG和RANKL的影响

目的:检测内毒素(LPS)刺激后,体外培养人牙龈成纤维细胞(HGFs)表达骨保护素(OPG)和核因子-kB受体活化因子配体(RANKL)的变化,探讨其对牙槽骨吸收的影响.方法:组织块法培养正常HGFs并鉴定来源,以5 μg/ml LPS刺激第4 代细胞,在0、6、12、24、48 h各时间点用免疫细胞化学方法检测OPG...     

Thymosin beta4 is cytoprotective in human gingival fibroblasts

Thymosin beta4 (Tbeta(4)) is a naturally occurring, ubiquitous, non-toxic protein with documented wound-healing, anti-inflammatory, anti-apoptotic, and tissue-repair properties in skin, the ocular surface, and the heart. The ability of Tbeta(4) to demonstrate similar protective properties in cells of the oral cavity was analyzed using an in vitro model of cultured human gingival fibroblasts. Thymosin beta 4 significantly suppressed the secretion of interleukin-8 (IL-8) following stimulation with tumor necrosis factoralpha (TNF-alpha), suggesting that it may suppress the inflammatory response initiated by pro-inflammatory cytokines. By contrast, Tbeta(4) was not effective in protecting fibroblasts from challenge with lipopolysaccharide purified from Porphyromonas gingivalis or Escherichia coli. Thymosin beta 4 was able to protect gingival fibroblasts against the known cytotoxic effects of chlorhexidine digluconate, a mouthrinse containing chlorhexidine digluconate, and carbamide peroxide. Additionally, Tbeta(4) was able to protect gingival fibroblasts from the apoptosis that is induced by stimulation with TNF-alpha or by exposure to chlorhexidine. Because of its multifunctional roles in protecting cells against damage, Tbeta(4) may have significant potential for use as an oral heathcare aid with combined antimicrobial, anti-inflammatory, anti-apoptotic, and cytoprotective properties.     

Effects of nicotine on apoptosis in human gingival fibroblasts

Aim

Cigarette smoke is a complex mixture of more than 4700 chemical compounds including free radicals and oxidants and it is a world widely known problem to health. Nicotine is the major compound of tobacco and known as the cause of gingivitis and periodontitis. It induces intracellular oxidative stress recognized as the important agent in the damage of biological molecules. The aim of this study is to clarify the cytotoxic pathway of nicotine in human gingival fibroblasts (HGFs).

Methods

Human gingival fibroblasts stimulated by nicotine were used as an in vitro model. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect cell viability and reactive oxygen species (ROS) generation was assessed with 2,7-dichlorofluoroscein diacetate (DCF-DA). Morphological change was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) assay, stained with 4,6-diamidino-2-phenylindole (DAPI). To delineate the roles of extracellular signal-regulated kinase (ERK), P38 and c-Jun N-terminal kinase (JNK), Western blot and caspase-3 (CASP3) activity assay were performed.

Results

Exposure of the human gingival fibroblasts to nicotine reduced cell viability by time and dose dependent and increased the generation of ROS. It also showed morphological evidence of increased apoptosis, resulted in transient activation of JNK and ERK concomitant with activation of P38, and stimulated apoptosis as evidenced by CASP3 activation and Poly ADP ribose polymerase (PARP) cleavage.

Conclusion

These results suggest that nicotine induces apoptosis through the ROS generation and CASP3 dependent pathways in HGFs.     

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。     

排龈时间与药物对牙龈成纤维细胞的毒性比较研究

目的:研究不同排龈药物与不同排龈时间对人牙龈成纤维细胞(HGF)毒性作用的影响,指导临床选择最佳排龈药物和排龈时间。方法:MTT比色测定细胞活性,透射电镜观察直接损伤后细胞超微结构的变化,排龈药物pH值测定。结果:0.1%盐酸肾上腺素的细胞毒性作用最小,排龈时间5 min细胞毒性小于10 min,硫酸铁的细胞毒性最大。结论:健康患者可采用0.1%盐酸肾上腺素进行排龈;伴有心血管系统疾病的患者,可采用20%硫酸铝替代0.1%盐酸肾上腺素;临床上使用硫酸铁排龈时应适当降低其浓度。     

NH4C1 and protein metabolism in human gingival fibroblasts

Abstract – The biologic effect of ammonia was studied in cultures of fibroblasts isolated from human gingiva. NH_CI in the range 2–20 mu was found to exhibit a concentration dependent growth inhibitory effect, with a delayed action wllich was most pronounced at low concentrations. Concomitant with the growth inhibitor)'effect a significant cellular accumulation of protein was evident. No effect on protein synthesis in general, as measured by G-protine incorporation, was found, whereas some inhibitory effect on collagen biosynthesis was indicated. Secretion of C-collagen and other labeled proteins was not affected. The only pronounced effect of ammonia on metabolism of the l4C-labeled proteins was an inhibition of the intracellular degradation of newly synthesized collagen, the lysosomes being suggested as the site for this degradation.     

Triclosan reduces prostaglandin biosynthesis in human gingival fibroblasts challenged with interleukin-1 in vitro

Abstract The effect of the toothpaste ingredient triclosan (2,4,4′-trichloro-2′-hydroxyldiphenyl ether) on the prostaglandins biosynthesis in human gingival fibroblasts challenged with interleukin-1β (IL-1β) or tumor necrosis factor α (TNFα) was studied in vitro. When gingival fibroblasts were treated simultaneously with triciosan and IL-1β, the stimulatory effect of IL-1β on prostaglandin E₂ (PGE₂) and PGI₂ formation was reduced in a dose-dependent manner by triclosan. Triclosan also reduced the PGE: formation induced by TNFα. Furthermore, the capacity of IL-1β to induce release of [³H] arachidonic acid from prelabelled gingival fibroblasts was reduced in the presence of triclosan. Addition of exogenous unlabelled arachidonic acid (AA) to the cells resulted in enhanced PGE₂ formation which was reduced by triclosan. The upregulation of the metabolism of AA to PGE₂ induced by IL-lβ, was markedly reduced in the presence of triclosan. The study indicates that the stimulatory effect of IL-1β on prostanoid formation (PGE₂, PGI₂) in human gingival fibroblasts was diminished in the presence of triciosan partly at the level of phospholipase A₂ and partly at the level of cyclooxygenase. The present data that triclosan. in vitro, inhibits the production of inflammatory mediators such as prostaglandins suggests that this can be an aspect of its clinical effect on gingivitis, in addition to its antibacterial effect.     

Triclosan reduces microsomal prostaglandin E synthase-1 expression in human gingival fibroblasts

OBJECTIVE: The effect of triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) on the expression of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) and on the translocation of the nuclear factor-kappaB (NF-kappaB) in relation to prostaglandin E2 (PGE2) production was investigated in human gingival fibroblasts challenged with tumor necrosis factor alpha (TNFalpha). METHODS: Fibroblasts were established from gingival biopsies obtained from six children. COX-2 mRNA and protein expression was quantified using mRNA quantitation and enzyme immunometric assay kits. mPGES-1 mRNA was analysed by RT-PCR, mPGES-1 protein and NF-kappaB translocation by immunoblotting. PGE2 was determined by radioimmunoassay. RESULTS: The cytokine TNFalpha enhanced the expression of mRNA as well as the protein levels of both COX-2 and mPGES-1 and subsequently the production of PGE2 in gingival fibroblasts. Treatment of gingival fibroblasts with triclosan (1 microg/ml) significantly reduced the stimulatory effect of TNFalpha (10 ng/ml) on the expression of mPGES-1 at both the mRNA and the protein level by an average of 21% and 43%, respectively, and subsequently the production of PGE2 (p<0.01). Triclosan did not, however, affect the translocation of NF-kappaB or the expression of COX-2 in TNFalpha-stimulated cells. CONCLUSION: The results show that triclosan reduces the augmented biosynthesis of PGE2 by inhibiting the mRNA and the protein expression of mPGES-1 in gingival fibroblasts. This finding may partly explain the anti-inflammatory effect of the agent previously reported in clinical studies.     

Stimulatory effect of Aggregatibacter actinomycetemcomitans DNA on proinflammatory cytokine expression by human gingival fibroblasts

ObjectiveWhile different virulence factors have been reported of Aggregatibacter actinomycetemcomitans (Aa), there is little information about the stimulatory effect of its DNA. The main purpose of this study was to assess the inflammatory response of human gingival fibroblasts (HGFs) stimulated with A. actinomycetemcomitans DNA.DesignCytokine levels of IL-6, IL-1α and TNF-α were measured on the supernatant of HGFs activated with 10, 25, 50 and 100 μg/ml DNA of Aa during 24 h. Primary cultures of HGFs were infected with Aa and its DNA at different times and concentrations to compare its cytotoxic effect. Cell damage and adhesion of Aa to HGFs were evaluated under light microscopy and Scanning electron microscopy respectively.ResultsThere was a statistical difference (p < 0.05) in cytokine expression in HGFs activated by bacterial DNA with a dose dependent on IL-6 expression and a significantly elevated expression of IL-1α and TNF-α compared to Human DNA negative control. Substantial morphological alterations were observed after infection of A. actinomycetemcomitans in HGFs but not with bDNA exposure. Aggregatibacter actinomycetemcomitans showed a high rate of adhesion and cell damage to HGFs after 30 min.ConclusionsGenomic DNA of A. actinomycetemcomitans could be a factor in the pathogenesis of periodontitis that might play a major role in the inflammatory response.     

Synthesis of sulfated glycosaminoglycans by human gingival fibroblasts from phenytoin-induced gingival overgrowth in vitro

Abstract – The in vitro synthesis of sulfated glycosaminoglycans (GAGs) was studied in gingival fibroblasts from two patients exhibiting phenytoin(PHT)-induced gingival overgrowth, i.e. pseudopockets, which required surgical excision, from one patient on PHT medication not exhibiting pseudopockets and from two normal controls. The results showed that the newly synthesized GAGs were distributed to the culture medium, to a pericellular pool and to the cell fraction. Gingival fibroblasts from the PHT-induced gingival overgrowth showed a significantly increased incorporation of ³⁵SO₄^2- into GAGs compared to the other strains, and this, increase was mainly confined to the dermatan sulfate fraction. These results are in accordance with our previous biochemical studies where increased amounts of GAGs were found in gingival biopsies from the PHT-induced lesion.     

Cytotoxic effects of gingival retraction cords on human gingival fibroblasts in vitro

The objective of this study was to determine the cytocompatibility of three different extracts of gingival retraction cords and to compare the cytotoxic effect of these materials on human gingival fibroblasts. Gingival retraction cords impregnated with aluminium sulphate (Gingi-Aid), dl-adrenaline HCl (Gingi-Pak) and non-drug-impregnated cord (Gingi-Plain) were eluted with culture medium for 10 min and 24 h. Cytotoxicity was judged using a tetrazolium bromide reduction assay. Our data demonstrated that gingival retraction cords applied alone almost completely inhibited cell viability (P < 0.05). In addition, the results also showed that the eluates from aluminium sulphate-impregnated cord, dl-adrenaline HCl-impregnated cord and non-drug-impregnated cord were cytotoxic to primary human gingival fibroblast cultures (P < 0.05). The cell viability of incubation of gingival fibroblasts containing 10-min eluates of aluminium sulphate, dl-adrenaline HCl and non-drug-impregnated cord was 61, 21 and 70%, respectively. The cell viability of incubation of gingival fibroblasts containing 24 h eluates of aluminium sulphate, dl-adrenaline HCl and non-drug-impregnated cord was 68, 58 and 72%, respectively. It was found that dl-adrenaline HCl-impregnated gingival retraction cord was the most toxic gingival retraction cord among the materials tested in all cultures (P < 0.05). The cytotoxicity decreased in an order of dl-adrenaline HCl-impregnated cord > aluminium sulphate-impregnated cord > non-drug-impregnated cord. The extent or degree of the cytotoxicity depended on the materials tested. Gingival retraction cords have significant potential for gingival toxicity. Careful management of gingiva retraction cords would lower the risk of potential gingival tissue damage during clinical application procedure and thus increase the success of prosthodontic procedures.     

钛表面微沟槽形貌对人牙龈纤维细胞黏着斑蛋白表达的影响

目的 比较不同尺寸微沟槽表面对人牙龈成纤维细胞（HGF）黏着斑蛋白vinculin表达的影响,作为选择种植体穿龈部分微沟槽尺寸的依据之一。方法针对HGF大小,设计不同沟槽尺寸,利用光刻技术制作微沟槽形貌,沟槽宽度和间隔分别为15 μm、30 μm和60 μm,沟槽深度为5 μm和10 μm,分组为T15/5、T15/10、T30/5、T30/10、T60/5和T60/10,光滑钛(T0)表面为对照组。通过免疫荧光、实时荧光定量PCR和蛋白免疫印迹比较vinculin基因和蛋白水平的表达差异。采用SPSS17.0软件包对数据进行统计学分析。结果免疫荧光显示,随着沟槽宽度增加,vinculin 的绿色荧光点增多,mRNA和蛋白水平表达T60最高,T15最低。相同沟槽宽度下,深度改变对该蛋白的表达无显著影响。结论微沟槽形貌有利于vinculin蛋白的表达,表达量随着沟槽的宽度增加而增加。就vinculin蛋白的表达而言,T60组沟槽更适合作为穿龈部分的沟槽尺寸。     

Inhibitory effect of NH4Cl on secretion of collagen in human gingival fibroblasts

Abstract – The general protein synthesis in human gingival fibroblasts as measured by ¹⁴C-proline incorporation was only moderately inhibited by 10 mM NH₄Cl during incubation for 36 h. The proportion secreted as noncollagen protein and recovered from the cellular fraction as collagen was not significantly affected, whereas a pronounced inhibitory effect on the secretion of collagen was evident after 24 h. This effect was dose dependent, with a significant inhibition of collagen secretion even at 2 mM ammonia. The applied concentrations of NH₄Cl had no significant effect on the hydroxylation of prolyl residues in collagen. Ammonia had no inhibitory effect on the secretion of fibronectin, another major secretory protein from fibroblasts. When comparing different lysosomotropic agents; NH₄Cl, chloroquine and methylamine, the most prominent effect was consistently found to be an inhibition of the secretion of collagen.     

二十碳五烯酸对人牙龈成纤维细胞生物学活性及炎症因子表达的影响

目的:探索二十碳五烯酸(eicosapentaenoic acid,EPA)对人牙龈成纤维细胞(human gingival fibroblasts,HGFs)生物学活性及炎症因子表达的作用.方法:分别通过活-死细胞染色、免疫荧光染色、流式细胞术观察EPA对HGFs细胞活性、形态、细胞周期的影响,采用牙龈卟啉单胞菌(P...     

Infection of primary human gingival fibroblasts by Porphyromonas gingivalis and Prevotella intermedia

Adhesion and penetration of clinical isolates of Porphyromonas gingivalis and Prevotella intermedia in human gingival fibroblast monolayers were studied by transmission electron microscopy (TEM). Fibroblasts were cultured from biopsies of human healthy gingiva. Porphyromonas gingivalis and Prevotella intermedia were isolated from patients with periodontitis. Fibroblasts were incubated with microorganisms in an antibiotic-free medium for 24 h. Then cultures were washed to remove nonadherent bacteria. Consecutively, infected cultures were grown for another 24 h. Thereafter, the treated monolayers were prepared for TEM investigations. Internalized Porphyromonas gingivalis and Prevotella intermedia were visible after 24 h of incubation. Prevotella intermedia showed only division in cytoplasm of fibroblasts after 24 h and 48 h incubations. Infected fibroblasts revealed various morphological alterations such as extensive vacuolization and breakdown of mitochondria. These findings demonstrate that Porphyromonas gingivalis and Prevotella intermedia may invade human gingival fibroblasts and thus may damage these cells directly or due to the release of microbial cytotoxic components. Received: 15 October 1999 / Accepted: 19 November 1999     

Efficient gene transfer to periodontal ligament cells and human gingival fibroblasts by adeno-associated virus vectors

Objectives

We explored for the first time the possibility to deliver a reporter gene (Green Fluorescence Protein) to human primary periodontal ligament (PDL) cells and human gingival fibroblasts (HGF) using shuttle vectors derived from adeno-associated virus (AAV). Since AAV transduction rates on other human primary fibroblasts have been previously shown to depend on the particular cell lineage and on the employed viral serotype, we determined the most effective AAV variant for periodontal cells comparing different vector types.

Methods

AAV serotypes 1–5 encoding GFP in single stranded (ss) and self-complementary (sc) vector genome conformations were used to infect primary HGF and PDL cells. Two days post-infection, the percentage of GFP expressing cells was determined by flow cytometry.

Results

Highest transduction rates for both cell types were achieved with self-complementary vectors derived from AAV-2, resulting in GFP expression in up to 86% of PDL cells and 50% of HGF. Transgene expression could be observed by optical microscopy for 2 months after infection. Lower but detectable rates were obtained with serotypes 1, 3 and 5.

Conclusions

The efficacy demonstrated here and the safety and versatility of AAV technology indicated in previous studies clearly suggest the potential of AAV vectors as tools for gene transfer to periodontal tissues.     

Nicotine increases the collagen-degrading ability of human gingival fibroblasts

BACKGROUND AND OBJECTIVE: The objective of this study was to determine the effects that nicotine and the combination of nicotine and Porphyromonas gingivalis supernatant have on human gingival fibroblast-mediated collagen degradation. MATERIAL AND METHODS: Human gingival fibroblasts were cultured with 25-500 microg/ml of nicotine in collagen-coated six-well plates. On days 1-5, the conditioned media was collected for zymography and western blot analyses of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). The cells were then removed and the collagen cleavage visualized by Coomassie blue staining. To examine the combined effect, 250 microg/ml of nicotine and 10% v/v culture supernatant of P. gingivalis ATCC 33277 were added to the human gingival fibroblasts. The mRNA levels of multiple MMPs and TIMPs were monitored. RESULTS: Nicotine increased the human gingival fibroblast-mediated collagen cleavage. The MMP-14 and MMP-2 produced by the nicotine-treated human gingival fibroblasts more readily underwent zymogen activation. Nicotine treatment resulted in TIMP-2 redistribution to the cell surface. The mRNAs of multiple MMPs and TIMPs were unaltered by nicotine. An additive collagen cleavage effect was observed when the human gingival fibroblasts were treated with both nicotine and P. gingivalis. CONCLUSION: Nicotine increased human gingival fibroblast-mediated collagen degradation, in part through the activation of membrane-associated MMPs. Nicotine and P. gingivalis had an additive effect on human gingival fibroblast-mediated collagen degradation.     

Effect of phenytoin and nifedipine on collagen gene expression in human gingival fibroblasts

Phenytoin (PHT), a widely used anticonvulsant, and nifedipine (NF), an anti-anginal drug, cause clinically similar gingival overgrowths in some patients. The aim of this work was to investigate their effects on collagen and protein synthesis and cellular proliferation in normal human gingival fibroblasts in vitro. Gingival fibroblasts were cultured from biopsies taken from three healthy individuals during operations on maxillary canines and incubated with various concentrations of NF (100 and 200 ng/ml) and PHT (5 and 10 micrograms/ml) for up to 7 days. The results showed that NF and PHT have a specific effect in reducing total protein and collagen synthesis but do not influence cell proliferation in healthy gingival fibroblasts in vitro. In addition the level of mRNA for type I collagen was decreased after incubation of the cells with the drugs for 1 or 2 days. The decrease in the level of type I collagen mRNA seemed to be specific since the level of type IV collagenase mRNA used as a reference RNA did not decrease.     

Effects of interleukin-4 on proteoglycan accumulation in human gingival fibroblasts

In inflammatory gingival diseases, cytokines have been demonstrated to play critical roles by coordinating the stimulation of immunological and connective tissue cells. The activities of these cells, degrading and remodeling extracellular matrices, constitute the major pathological and repair processes. Thus, elucidating cellular and molecular events occurring in inflamed connective tissues is crucial for the understanding and treatment of inflammation. In order to test a hypothesis that proinflammatory cytokines affect metabolism of major extracellular matrix molecules, we studied metabolism of proteoglycans (PGs) by human gingival fibroblasts (HGF) under the influence of interleukin-4 (IL-4) as a model of gingivitis. HGF in cell culture were metabolically radiolabeled using [3H]glucosamine and [35S]sulfate in the presence or absence of IL-4, and the labeled PGs were analyzed by chromatographic techniques. The incorporation of 35S into PGs increased with IL-4 both in media and cell layer. At 100 ng/ml of IL-4, the increment of 35S incorporation over control culture was 16-39% (p<0.001) in media and 12-35% (p=0.01) in cell layer. The 35S-labeled macromolecules were PGs containing heparan sulfate (HS) and chondroitin sulfate (CS) chains. From the molecular weight and glycosaminoglycan composition analyses, versican and perlecan-type and biglycan and decorin-type were very likely to be the major PG constituents both in media and cell layer. IL-4 stimulated synthesis of versican and perlecan-type more potently than biglycan and decorin-type. With IL-4 treatment, the ratio of CSPG/HSPG decreased in media and increased in cell layer. This ratio suggested that syndecan family HSPGs were also present in HGF. In conclusion, IL-4 stimulated accumulation of CS/HSPGs in human gingival fibroblasts.