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.     

Effects of Porphyromonas gingivalis on human gingival fibroblasts from healthy and inflamed tissues

Background and Objective:  This study compared the ability of human gingival fibroblasts (HGFs) isolated from healthy and inflamed gingival tissues to degrade collagen in the presence and absence of Porphyromonas gingivalis supernatant.
Material and Methods:  Human gingival fibroblasts were cultured from explants of 21 healthy and 21 inflamed periodontal tissues. The HGFs that grew out of the explants were seeded in the center of six-well plates coated with collagen in the presence and absence of 10% P. gingivalis supernatant. An inflamed and a healthy cell line were also evaluated with Arg-gingipain. After 6 days, Coomassie Blue was used to visualize the collagen cleavage.
Results:  The collagen was totally cleaved in 12 (aggressive) of the 21 cell lines isolated from the inflamed tissues in the presence of P. gingivalis . The remaining nine cell lines (non-aggressive) cleaved only the collagen underneath the cell colonies in the presence of P. gingivalis . Of the healthy tissues, five (aggressive) of the 21 cell lines cleaved all the collagen and 16 cell lines (non-aggressive) only cleaved the collagen underneath the cell colonies in the presence of P. gingivalis . All the collagen was cleaved by an aggressive cell line and only the collagen underneath the cell colonies was cleaved by a non-aggressive cell line in the presence of Arg-gingipain.
Conclusion:  The collagen in the wells was more readily cleaved by the inflamed than by the healthy cell lines, and the difference was statistically significant ( p  = 0.0278). Arg-gingipain gave identical results to the P. gingivalis supernatant.     

静磁场对人牙龈成纤维细胞碱性磷酸酶活性的影响

目的:研究不同磁路设计的磁性附着体所产生静磁场对人牙龈成纤维细胞酶学的相关作用.方法:使用自主设计的磁场加载系统,产生不同强度的静磁场,对体外培养的人牙龈成纤维细胞进行不同时间的磁场加载.通过与对照组的比较,探讨磁场对该类细胞碱性磷酸酶活性的影响.结果:改变加载强度(120 mT、10 mT、0mT)或加载时间(1、3、5个加载周期),静磁场对人牙龈成纤维细胞碱性磷酸酶活性均无显著性影响(P＞0.05).结论:静磁场对牙龈成纤维细胞碱性磷酸酶活性没有影响,初步提示开放及闭合磁路系统磁性附着体所产生静磁场,对牙龈成纤维细胞的相关酶不存在生物学效应.     

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目的    探讨烟草提取液（ST）和尼古丁液对人牙龈成纤维细胞（human gingival fibroblast,HGF）的影响。方法    2009年6—12月于昆明医学院口腔实验室,用含不同浓度的ST和尼古丁液的培养液体外培养HGF 5 d,应用噻唑蓝（MTT）快速比色法,检测细胞生长增殖情况。结果    与对照组相比,ST质量浓度从5 g／L开始时,抑制HGF的增殖,差异有统计学意义（P < 0.05）,随着浓度的加大,抑制作用更明显,呈浓度依赖性。同样,高质量浓度的尼古丁液从0.01 g／L开始对细胞增殖有抑制作用,并呈浓度依赖性,差异有统计学意义（P < 0.05）。结论    高浓度的ST和尼古丁均能抑制HGF的增殖,低浓度时则对细胞的增殖无影响。烟草对口腔黏膜上皮细胞的增殖呈现抑制作用,并随着浓度的加大,抑制作用愈发明显。     

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目的 探讨中药骨碎补对体外培养的人牙龈成纤维(HGF)细胞超微结构、碱性磷酸酶(ALP)活性的影响。方法 2005年9月至11月于河北医科大学第四医院实验中心将体外培养的HGF细胞分别与质量浓度为10、50、100、500、1000 μg/mL的骨碎补共同孵育，四甲基偶氮唑盐(MTT)比色法测定ALP活性，选出ALP活性最强的骨碎补质量浓度作为最佳浓度，并作用于培养细胞。电镜下观察细胞的超微结构改变。结果 在骨碎补各质量浓度范围内，实验组ALP活性均高于对照组(〖WTBX〗P〖WTBZ〗均<005)，骨碎补质量浓度为100 μg/mL时ALP活性最强。与对照组比较，实验组细胞器数量增加，细胞体较大，有丰富的胞浆，胞浆内粗面内质网数量明显增多，线粒体较为发达，细胞核及核仁体积较大。结论 骨碎补在一定质量浓度范围内可使ALP活性增强，细胞器数量增加，功能增强。     

hBMP2基因转染对人牙龈成纤维细胞生物学特性的影响

目的分析hBMP2基因转染人牙龈成纤维细胞的生物学特性.方法用脂质体转染法将hBMP2基因转入人牙龈成纤维细胞内,经G418筛选后,获得阳性克隆.以原位杂交和免疫组化对转染细胞进行鉴定;进一步观察细胞形态、生长特性、碱性磷酸酶活性、骨钙素合成以及体外形成矿化结节的能力.结果hBMP2基因转染后,人牙龈成纤维细胞有hBMP2 mRNA的转录和蛋白表达;部分细胞由原来的长梭形转化为多角形,碱性磷酸酶活性和骨钙素合成均明显高于对照组(两组均P＜0.01);在矿化液作用下,能形成体外矿化结节.但细胞的增殖特性无明显变化.结论外源性hBMP2基因能够在人牙龈成纤维细胞表达,并促进其向成骨细胞方向转化,而对细胞的生长特性无明显影响.     

Immunochemical detection of CD14 on human gingival fibroblasts in vitro

The activation of monocytes and macrophages induced by lipopolysaccharide has been shown to contribute to the binding of lipopolysaccharide and lipopolysaccharide-binding protein complex to the cell surface CD14 molecule. To clarify the mechanism of the lipopolysaccharide-induced modulation of the function of gingival fibroblasts, we investigated the effect of anti-CD14 on interleukin 6 (IL-6) production on human gingival fibroblasts in vitro. Immunochemical staining revealed weak positivity for CD14 on fibroblasts from healthy gingiva, while strong positivity for CD14 was found on fibroblasts from inflamed gingiva. Western blot profiles of the fibroblasts and monocytes showed a CD14-positive reaction at 55 kDa. Fluorescein isothiocyanate-conjugated Escherichia coli lipopolysaccharide bound to fibroblasts more strongly in the presence of 10% fetal bovine serum than without serum. This binding, as well as IL-6 production, was blocked by anti-CD14 monoclonal antibody. The results showed that CD14 was present on human gingival fibroblasts, which suggests that lipopolysaccharide modulation of gingival fibroblast function depends on CD14.     

The inhibition of DNA synthesis by prostaglandin E2 in human gingival fibroblasts is independent of the cyclic AMP-protein kinase A signal transduction pathway

In this study we attempted to clarify the mechanism of the inhibitory effects of PGE₂ on DNA synthesis in Gin-1 (fibroblasts derived from healthy human gingiva) from the aspect of the cyclic AMP-dependent protein kinase signal transduction pathway. PGE₂ upregulated intracellular cyclic AMP accumulation and inhibited DNA synthesis in Gin-1 in a dose-dependent manner. When the PGE₂-induced intracellular cyclic AMP accumulation was further enhanced by treatment with the cyclic AMP-phosphodiesterase inhibitor, IBMX, the inhibitory effect of PGE₂ on DNA synthesis was also enhanced. Furthermore, when we examined the effects of forskolin, an activator of cyclic AMP production, on intracellular cyclic AMP accumulation and DNA synthesis, similar results were obtained. However, inhibitors of cyclic AMP-dependent protein kinase (protein kinase A) such as HA1004 did not diminish the inhibitory effect of PGE₂ on DNA synthesis in Gin-1. These results suggest that in Gin-I, PGE₂-induced cyclic AMP accumulation may not lead to the activation of protein kinase A or protein kinase A activity may not relate directly to the growth inhibitory effect of PGE₂, and that PGE₂ does not inhibit DNA synthesis through the cyclic AMP-protein kinase A signal transduction pathway in Gin-1.     

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目的    探讨精氨酸-甘氨酸-天冬氨酸（RGD）肽修饰纯钛表面对人牙龈成纤维细胞（human gingival fibroblasts,HGF）和上皮细胞（human gingival epithelial cells,HGE）初期黏附行为的影响。方法    应用羰基二咪唑（1,1′-carbonyldiimidazole,CDI）活化法将含RGD的短肽共价连接到纯钛表面,免疫荧光法检测钛表面RGD肽。评价RGD接枝与未接枝纯钛表面对HGF和HGE初期黏附的影响。结果    RGD肽可以通过CDI活化方法接枝到纯钛表面,HGF和HGE在RGD接枝钛表面黏附和增殖的细胞数量显著高于未接枝钛表面,差异有统计学意义（P < 0.01）。结论    生物活性肽RGD接枝纯钛表面可有效促进HGF和HGE在其表面的黏附。     

糖基化终产物对人牙龈成纤维细胞2种基质金属蛋白酶表达的影响

目的研究糖基化终产物(advanced glycation end products,AGEs)对人牙龈成纤维细胞(human gingivalfibroblasts,HGF)表达基质金属蛋白酶-1(matrix matelloproteinase-1,MMP-1)和基质金属蛋白酶-8(matrix matallo-proteinase-8,MMP-8)的影响,探讨糖尿病加速牙周炎发展的可能机制。方法将培养的HGF随机分成6组:空白对照组只加入培养液;阴性对照组仅加入含50μg/mL人血清白蛋白(human serum albumin,HSA)的培养液;4个含AGE-HSA的实验组分别加入含0.5、5、50、100μg/mL AGE-HSA的培养液。培养24、48、72 h后,分别应用酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)和实时定量聚合酶链反应(real-time quantitativepolymerase chain reaction,QPCR)检测细胞MMP-1、MMP-8的蛋白和mRNA表达。结果 100μg/mL AGE-HSA组培养24、48和72 h后,MMP-1水平明显高于阴性对照组、空白对照组及0.5μg/mL AGE-HSA组,差异具有统计学意义(P<0.05)。各浓度AGE-HSA组MMP-1 mRNA水平均显著高于阴性对照组,差异有统计学意义(P<0.05)。各浓度AGE-HSA组MMP-8水平与阴性对照组相比差异均无统计学意义(P>0.05),MMP-8 mRNA表达均为阴性。结论 AGEs可能通过促进HGF合成MMP-1,介导胶原降解,从而加重糖尿病患者的牙周组织破坏,尚不能说AGEs影响MMP-8的表达。     

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...     

Zoledronic acid and geranylgeraniol regulate cellular behaviour and angiogenic gene expression in human gingival fibroblasts

The mevalonate pathway (MVP) and the anti‐angiogenic effect of bisphosphonates have been shown to play a role in the pathogenesis of bisphosphonate‐related osteonecrosis of the jaw (BRONJ). This study determined the effect of the bisphosphonate, zoledronic acid and the replenishment of the MVP by geranylgeraniol on human gingival fibroblasts. Cell viability, apoptosis, morphological analysis using transmission electron microscopy, and gene expression for vascular endothelial growth factor A, bone morphogenic protein 2, ras homologue gene family member B, epiregulin and interferon‐alpha were conducted. Results showed cellular viability was decreased in the presence of zoledronic acid and the co‐addition of zoledronic acid with geranylgeraniol restored cell viability to control levels. Caspase 3/7 was detected in zoledronic‐acid‐treated cells indicating apoptosis. Transmission electron microscopy revealed dilation of the rough endoplasmic reticulum with zoledronic acid and the appearance of multiple lipid‐like vesicles following the addition of geranylgeraniol. Zoledronic acid significantly (P < 0.05, FR > ±2) up‐regulated vascular endothelial growth factor A, bone morphogenic protein 2, ras homologue gene family member B and epiregulin at one or more time points but not interferon‐alpha. Addition of geranylgeraniol resulted in a reduction in the expression of all five genes compared with zoledronic‐acid‐treated human gingival fibroblasts. The study concluded geranylgeraniol partially reversed the effects of zoledronic acid in human gingival fibroblasts both at the cellular and genetic levels, suggesting the regulation of these genes is mediated via the mevalonate pathway.     

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.     

水蛭素对牙龈成纤维细胞碱性成纤维细胞生长因子和转化生长因子-β1表达的影响

目的 观察水蛭素对人牙龈成纤维细胞（HGFs）碱性成纤维细胞生长因子（bFGF）及转化生长因子-β1（TGF-β1）表达变化的规律,探讨水蛭素影响牙龈改建的可能作用机制。方法 体外培养并鉴定HGFs,利用不同浓度的水蛭素分别作用于正常（对照组）和受长期机械外力作用后增生的HGFs（实验组）,通过实时定量聚合酶链反应法和免疫细胞化学法检测TGF-β1及bFGF的表达。结果 未加入水蛭素时,受长期机械外力作用后,实验组促进HGFs增殖胶原合成的TGF-β1表达升高,而抑制胶原合成的bFGF表达降低（P<0.05）。加入水蛭素干预增生的HGFs后,可正向调节bFGF表达,而负向调节TGF-β1的表达（P<0.05）。结论 外力作用干扰了HGFs胶原合成与降解之间的平衡,水蛭素可能通过调节这一平衡而促进牙龈改建过程。     

DHA对人牙龈成纤维细胞生物学活性及炎症因子表达的作用

目的 探索二十二碳六烯酸(docosahexaenoic acid,DHA)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 分别采用活死细胞染色法、荧光染色法、流式细胞术观察DHA对细胞活性、细胞形态、细胞周期的影响;DHA预处理HGFs后,利用...