硝苯地平对牙龈成纤维细胞增殖和DNA合成的影响

目的：研究钙离子通道拮抗剂硝苯地平对人牙龈成纤维细胞的影响,方法：以硝苯地平1200．0、360．0、108．0和32．4μg/L作体外培养牙龈成纤维细胞细胞计数观察,并以^3H-胸腺嘧啶脱氧核苷（^3H-TDR)细胞内掺入法评价DNA合成量。结果：细胞计数在实验的第6、7d组间差异有极显著性。主要表现为硝苯地平1200．0μg/Lt 360.0μg/L组,细胞计数明显高于低浓度组,各组间DNA合成量差异有极显著性。进一步分析差异存在于高浓组织与其他各之间。结论：高浓度硝苯地平有刺激牙龈成纤维细胞增殖的作用。     

硝苯地平导致牙龈增生机制的研究

目的:在牙龈成纤维细胞(gingival fibroblasts(GFs))与牙龈角质形成细胞(gingival kerati-nocytes,GKs)共培养下,研究硝苯地平导致牙龈增生的可能机制。方法:用不同浓度(0、0.2、20μg/mL)硝苯地平处理GFs和GKs的共培养体系,3 d后用逆转录PCR、酶联免疫吸附实验以及流式细胞计数等方法观察角质细胞生长因子(keratinocyte growth factor,KGF)及其受体(keratinocyte growth factor receptor,KGFR)的表达。结果:在共培养条件下,硝苯地平导致GFs表达KGF显著增高,并呈浓度依赖性。同时硝苯地平能促进GKs中KGFR基因表达上调,膜KGFR蛋白表达水平在硝苯地平浓度为0.2μg/mL时上调。结论:间充质-上皮作用途径之一的角质细胞生长因子-受体间相互影响,在硝苯地平导致牙龈增生中起着一定的作用;体外GFs-GKs共培养模型能够在更接近体内环境的状态下进一步研究牙龈增生的机制。     

环孢素A和肿瘤坏死因子α对人牙龈成纤维细胞增殖的影响

目的 研究环孢素A(cyclosporinA)和肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)对体外培养人牙龈成纤维细胞增殖的影响,探讨牙龈炎症与药物性牙龈增生的关系及环孢素A所致牙龈增生的相关机制.方法用原代培养的方法获取5名健康人的牙龈成纤维细胞,体外培养、传代后取其中1个生长良好的组织块4～8代细胞用于实验.按以下条件进行实验分组:A组:空白对照组;B1组:10 μg/L环孢素A,B2组:50 μg/L环孢素A,B3组:250 μg/L环孢素A,B4组:1250 μg/L环孢素A;C组:5μg/L TNF-α; D1组:10 μg/L环孢素A+5μg/L TNF-α,D2组:50 μg/L 环孢素 A+5μg/L TNF-α,D3组:250 μg/L环孢素A+5μg/L TNF-α,D4组:1250 μg/L环孢素A+ 5 μg/L TNF-α.将条件培养液分别作用于牙龈成纤维细胞,培养3、5、7d后用甲基噻唑基四唑法测定细胞的增殖情况.结果不同质量浓度的环孢素A作用于成纤维细胞后,细胞的增殖受到抑制,A值下降,其中B1、B2、B3组与A组相比差异无统计学意义,B4组与A组相比差异具有统计学意义(P =0.001);5μg/L的TNF-α作用于成纤维细胞可以刺激细胞的增殖,A值(0.542)与A组(0.441)相比显著升高(P＜0.01).环孢素A和TNF-α共同作用于成纤维细胞后,D1、D2、D3组A值均较A组升高,但均较C组显著降低(P＜0.05).D4组细胞增殖显著增加,与C组相比差异具有统计学意义(P＜0.01).结论环孢素A对成纤维细胞的增殖无促进作用,高浓度时可抑制细胞的增殖;TNF-α可以促进成纤维细胞的增殖;高浓度环孢素A与TNF-α共同作用于成纤维细胞可促进成纤维细胞增殖,提示在一定浓度下环孢素A可能放大了TNF-α刺激成纤维细胞增殖的效应.     

黄芩苷对牙龈成纤维细胞和牙周膜细胞上ICAM-1表达调节的研究

目的 :观察黄芩苷对IL - 1β诱导的牙龈成纤维细胞 (humangingivalfibrobl -asts ,HGF)和牙周膜细胞 (peri odontalligamentcells ,PDLcells)上细胞间粘附分子 - 1表达的影响。方法 :应用体外细胞培养和细胞免疫组化及图像分析方法。结果 :正常牙龈成纤维细胞和牙周膜细胞上细胞间粘附分子 - 1表达阴性或弱阳性 ;1μg/mLIL - 1β能诱导细胞间粘附分子 - 1在牙龈成纤维细胞和牙周膜细胞上强阳性表达 ;0 .1μg/mL黄芩苷对IL - 1β诱导的牙龈成纤维细胞和牙周膜细胞上细胞间粘附分子 - 1的表达有显著抑制作用 (P <0 .0 1)。结论 :黄芩苷可抑制IL -1β诱导的细胞间粘附分子 - 1在牙龈成纤维细胞和牙周膜细胞上表达 ,提示黄芩苷具有抗细胞粘附的作用     

维甲酸诱导牙周韧带细胞分化的研究

目的研究并比较维甲酸对牙周韧带细胞和牙龈成纤维细胞向成骨样细胞分化潜能的影响。方法体外培养牙周韧带细胞与牙龈成纤维细胞,通过生化法、原位杂交及RT- PCR分别检测维甲酸作用前后碱性磷酸酶（ALP）活性的变化及其mRNA的表达情况。结果正常牙周韧带细胞与牙龈成纤维细胞的ALP活性有明显差别,前者高于后者;原位杂交及RT- PCR均检测到牙周韧带细胞有mRNA表达而牙龈成纤维细胞无表达。维甲酸作用后牙周韧带细胞ALP活性明显升高,mRNA表达信号相应增强,且在5×10- 6 mol/L组效果最明显;牙龈成纤维细胞ALP活性亦有升高,在mRNA水平仅RT- PCR检测到5×10- 6 mol/L组有较弱的表达,其他组及原位杂交均未见表达。结论牙周韧带细胞和牙龈成纤维细胞在向成骨方向的分化潜能上存在差异。     

尼古丁和烟草浸提液对人牙龈成纤维细胞生长和贴附能力的影响

目的：体外观察尼古丁和烟草浸提液(ST)对人牙龈成纤维细胞的生长和贴附影响。方法：用不同浓度的尼古丁和ST作用于人牙龈成纤维细胞，用MTT法检测细胞的生长和贴附情况。结果：随尼古丁和ST浓度的增加，细胞的生长率逐渐下降，尼古丁和ST对细胞生长半抑制浓度(IC50)分别为0．095g／L和11．88g／L；与对照组相比，当尼古丁及ST浓度分别大于0．01g/L和6．2g／L时差异有显著意义。而对细胞贴附的影响表明，随着尼古丁和ST浓度的增加，细胞的贴附率逐渐下降，尼古丁和ST对细胞贴附的IC50分别是0．6g／L和10．33g/L；与对照组相比，当尼古丁及ST浓度分别大于0．1g／L和12．4g／L时差异有显著意义。结论：尼古丁和浸提取液均可抑制人牙龈成纤维细胞的生长和贴附，提示尼古丁和烟草在牙周病发病中起作用。     

维拉帕米对正常牙龈成纤维细胞增殖的影响

目的体外评价维拉帕米对正常牙龈成纤维细胞增殖的影响。方法分离、培养正常牙龈成纤维细胞。用0、0．1、1、10、100μmol／L5个浓度的维拉帕米分别作用于第5代正常牙龈成纤维细胞，采用甲基噻唑基四唑法检测细胞的增殖，流式细胞仪评价细胞生长周期。结果100μmol／L维拉帕米作用66h后，检测A值为0．046，与无药物作用组（A值为0．088）相比差异有统计学意义（P〈0．01）。100μmol／L维拉帕米作用18h后，69％的细胞处于G0-G1期，27％处于S期，而无药物作用组分别为41％和49％。两者间差异有统计学意义（P〈0．001）。结论100μmol／L维拉帕米在体外通过阻滞细胞生长周期抑制正常牙龈成纤维细胞的增殖。     

人牙龈成纤维细胞诱导分化为神经样细胞的实验研究

目的 探讨作为结缔组织的人牙龈成纤维细胞,分化为神经样细胞的可能性.方法 留取阻生齿拔除后,切取的正常牙龈组织,体外原代培养人牙龈成纤维细胞,采用抗vimentin、GFAP和MAP-2进行细胞鉴定.采用低糖DMEM,含1mmol/L β-ME,20μmol/L RA,2％DMSO诱导培养人牙龈成纤维细胞向神经样细胞分...     

釉基质蛋白对牙周细胞覆盖体外创面的影响

目的：评价体外创面模型环境中,釉基质蛋白对牙周膜细胞、牙龈成纤维细胞覆盖创面能力的影响。方法：利用在盖玻片上形成的人牙周膜和牙龈成纤维细胞的融合培养物,以机械方法去除7mm宽的细胞层条带,形成体外创面模型。受损培养物在含有10％胎牛血清的培养液中继续培养2、6、9d,同时以100μg／ml的釉基质蛋白分别刺激牙周膜、牙龈成纤维细胞,并与不加釉基质蛋白者作对照。玻片经同定后作甲紫染色。以计算机辅助图像分析系统对细胞覆盖体外创面的面积进行百分比定量,采用SAS6、12软件包进行样本均数的t检验。结果：对照组组内牙周膜、牙龈成纤维细胞覆盖创面的面积存在差异,牙龈成纤维细胞覆盖创面面积在创面形成后第9天显著高于牙周膜细胞,差异有显著性（P〈0.05）。在加用100μg／ml釉基质蛋白的条件下,牙周膜细胞覆盖体外创面的面积较对照组明显增加．创面形成后第6、9天,牙周膜、牙龈成纤维细胞覆盖体外创面面积均无显著差异（P〉0．05）。结论：牙龈成纤维细胞覆盖创面的能力高于牙周膜细胞。釉基质蛋白有增进创面愈合,特别是牙周膜细胞创面覆盖的作用。     

IL—1ra对IL—1β诱导人牙龈成纤维细胞分泌IL—6的拮抗作用

目的：研究白细胞介素－1受体拮抗剂对IL－1β所介导生物学功能的影响，为进一步临床应用提供理论依据。方法：采用细胞培养技术和ELISA夹心法。结果：经IL－1β单独作用，牙龈成纤维细胞培养上清液中IL－6含量明显增高，当一定浓度的IL－1ra与1μg/L IL-1β共同作用人牙龈成纤维细胞后，培养上清液中IL－6的含量比单独IL－1β作用组低。结论：IL－1ra能够抑制IL－1诱导的人牙龈成纤维细胞分泌IL－6。     

姜黄素对环孢素A诱导激活的大鼠牙龈成纤维细胞TGF-β1/Smad3通路的抑制作用

目的：体外实验研究姜黄素（curcumin,Cur）对环孢素A（cyclosporine A,CsA）作用大鼠牙龈成纤维细胞TGF-β1/Smad3通路的影响,为进一步探讨Cur抑制CsA所致药物性牙龈增生的发生机制提供理论依据。方法：使用CCK-8法观察0、5、10、20、30 μmol/L Cur对大鼠牙龈成纤维细胞增殖的影响, 20 μmol/L Cur+200 ng/mL CsA共同作用对大鼠牙龈成纤维细胞增殖的影响。实时荧光定量PCR检测20 μmol/L Cur+200 ng/mL CsA共同作用下,牙龈成纤维细胞中转化生长因子TGF-β1、Smad3、平滑肌肌动蛋白α-SMA和I型胶原蛋白COL-I的mRNA表达变化;蛋白免疫印迹实验检测TGF-β1、Smad3、p-Smad3、α-SMA和COL-I的蛋白表达变化。细胞划痕实验观察20 μmol/L Cur+200 ng/mL CsA 对牙龈成纤维细胞迁移能力的影响。采用SPSS 23.0 软件包对数据进行统计学分析。结果：大鼠牙龈成纤维细胞在20 μmol/L Cur+200 ng/mL CsA共同作用下,细胞增殖和迁移能力明显降低;20 μmol/L Cur显著下调了牙龈成纤维细胞中TGF-β1、α-SMA 和COL-I的mRNA 表达,蛋白免疫印迹实验提示,TGF-β1、p-Smad3、α-SMA 和COL-I的表达同样显著下调。结论：Cur可能通过抑制CsA激活的牙龈成纤维细胞TGF-β1/Smad3 信号通路,从而降低牙龈成纤维细胞增殖、迁移、平滑肌肌动蛋白和胶原分泌,改善牙龈增生。     

Gingival fibroblasts grown from cyclosporin-treated patients show a reduced production of matrix metalloproteinase-1 (MMP-1) compared with normal gingival fibroblasts, and cyclosporin down-regulates the production of MMP-1 stimulated by pro-inflammatory cytokines

BACKGROUND AND OBJECTIVE: Cyclosporin-induced gingival overgrowth arises from an alteration in collagen homeostasis and is enhanced by inflammatory changes in the gingival tissues. The aim of this study was to investigate the interaction among interleukin-1, oncostatin M, cyclosporin and nifedipine in promoting the up-regulation of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase by gingival fibroblasts. MATERIAL AND METHODS: Fibroblast cultures (n = 5) were obtained from healthy controls and from patients with cyclosporin-induced gingival overgrowth, and cells were harvested between the fourth and ninth passages. Cells were stimulated with interleukin-1 and oncostatin M, alone or in combination, and with different concentrations of cyclosporin (0-2000 ng/mL) and nifedipine (0-200 ng/mL). MMP-1 and tissue inhibitor of metalloproteinase-1 production was determined using an enzyme-linked immunosorbent assay technique. A CyQuant cell proliferation assay was used to determine the DNA concentration in the sample. RESULTS: Fibroblasts obtained from patients with cyclosporin-induced gingival overgrowth produced significantly lower levels of MMP-1 than control fibroblasts (p < 0.001); tissue inhibitor of metalloproteinase-1 levels were significantly lower (p < 0.05), and the ratio of MMP-1 to tissue inhibitor of metalloproteinase-1 was reduced, in the conditioned medium of patients with cyclosporin-induced gingival overgrowth compared with controls. Interleukin-1 and oncostatin M produced a significant increase in the up-regulation of MMP-1, which was reversed when cyclosporin and nifedipine were added to the cell cultures (p < 0.05). CONCLUSION: Pro-inflammatory cytokines significantly up-regulate MMP-1 in cultured gingival fibroblasts. Up-regulation is attenuated by both cyclosporin and nifedipine. The interaction may account for the synergism between inflammation and cyclosporin-induced gingival overgrowth.     

The effect of IL-1alpha and nifedipine on cell proliferation and DNA synthesis in cultured human gingival fibroblasts

The effect of nifedipine and interleukin-alpha (IL-1alpha) on the cell proliferation and DNA synthesis was studied in human gingival fibroblasts derived from 5 patients who developed gingival overgrowth (nifedipine responders) and 5 patients who did not develop gingival overgrowth (nifedipine non-responders) in response to nifedipine. Epidermal growth factor was used as a positive control. The fibroblasts derived from nifedipine responders tended to have a numerically greater rate of cell proliferation and DNA synthesis (3H-thymidine incorporation) than those from nifedipine nonresponders in the presence of nifedipine and IL-lalpha. Fibroblasts derived from nifedipine responders showed significantly higher cell proliferation rate in the presence of nifedipine and IL-1alpha, than nifedipine or IL-lalpha alone on both the second and the fourth day of incubation (P < 0.05). A combination of IL-1alpha and epidermal growth factor also showed significantly greater cell proliferation than IL-lalpha alone on the second day (P < 0.05). The DNA synthesis rate with a combination of nifedipine and IL-1alpha was higher than that for nifedipine alone on the second day (P < 0.01), and IL-1alpha alone on the fourth day (P < 0.05) in gingival fibroblasts originating from nifedipine responders. These results suggest that the interaction between nifedipine and gingival inflammation might play an important role in the pathogenesis of nifedipine-induced gingival overgrowth.     

Flutamide inhibits nifedipine‐ and interleukin‐1β‐induced collagen overproduction in gingival fibroblasts

Lu H‐K, Tseng C‐C, Lee Y‐H, Li C‐L, Wang L‐F. Flutamide inhibits nifedipine‐ and interleukin‐1β‐induced collagen overproduction in gingival fibroblasts. J Periodont Res 2010; 45: 451–457. © 2010 John Wiley & Sons A/S Background and Objective: To understand the role of the androgen receptor in gingival overgrowth, the effects of flutamide on interleukin‐1β‐ and nifedipine‐induced gene expression of connective tissue growth factor (CTGF/CCN2) and collagen production in gingival fibroblasts were examined. Material and Methods: Gingival fibroblasts from healthy subjects and patients with dihydropyridine‐induced gingival overgrowth (DIGO) were used. Confluent cells were treated with nifedipine, interleukin‐1β or both. The mRNA expression was examined using real‐time polymerase chain reaction, and the concentration of total soluble collagen in conditioned media was analysed by Sircol Collagen Assay. In addition, the protein expressions of androgen receptor, CTGF/CCN2 and type I collagen in gingival tissue were determined by western blot. Results: Interleukin‐1β was more potent than nifedipine in stimulating CTGF/CCN2 and procollagen α1(I) mRNA expression, and there was an additive effect of the two drugs. Healthy cells exhibited an equal or stronger response of procollagen α1(I) than those with DIGO, but DIGO cells displayed a stronger response in the secretion of soluble collagen in the same conditions. Flutamide, an androgen receptor antagonist, inhibited stimulation by nifedipine or interleukin‐1β. Additionally, the protein expressions of androgen receptor and type I collagen were higher in DIGO gingival tissue than those in healthy gingival tissue. Conclusion: The data suggest that both nifedipine and interleukin‐1β play an important role in DIGO via androgen receptor upregulation and that gingival overgrowth is mainly due to collagen accumulation. Flutamide decreases the gene expression and protein production of collagen from dihydropyridine‐induced overgrowth cells.     

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.     

Cigarette smoke condensate affects the collagen-degrading ability of human gingival fibroblasts

Background and Objective: Cigarette smoke condensate, the particulate matter of cigarette smoke, is composed of thousands of chemicals, including nicotine. Cigarette smoking is a risk factor for periodontal disease. This study investigated the influence of cigarette smoke condensate on the collagen‐degrading ability of human gingival fibroblasts and its mechanism. Material and Methods: Human gingival fibroblasts were exposed for 72 h to various concentrations of total particulate matter cigarette smoke condensate. Cell proliferation and cytotoxicity were evaluated using water‐soluble tetrazolium‐1 and lactate dehydrogenase, respectively. The collagen‐degrading ability of human gingival fibroblasts was evaluated in collagen‐coated six‐well plates. Conditioned media and membrane extracts were collected for zymography and western blot analyses of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Results: Cell proliferation decreased and cytotoxicity increased in human gingival fibroblasts with increasing concentrations of cigarette smoke condensate. Cell proliferation decreased by more than 50% (p < 0.05) when the concentrations of total particulate matter cigarette smoke condensate were above 200 μg/mL, and cytotoxicity increased to more than 30% (p < 0.05) when the concentrations of total particulate matter cigarette smoke condensate were above 400 μg/mL. Cigarette smoke condensate increased the collagen‐degrading ability of human gingival fibroblasts, especially at a concentration of 100 μg/mL (1.5‐fold increase, p < 0.05) compared with the control. Cigarette smoke condensate increased the production of proMMP‐1, proMMP‐2, MMP‐14 and TIMP‐1, and decreased the production of TIMP‐2, in conditioned media. Furthermore, compared with the control group, cigarette smoke condensate increased the production of MMP‐2, MMP‐14 and TIMP‐2 in membrane extracts, especially at concentrations of 50–100 μg/mL. Conclusion: Cigarette smoke condensate affects human gingival fibroblast proliferation and is toxic at total particulate matter cigarette smoke condensate concentrations of ≥ 400 μg/mL. Cigarette smoke condensate can increase the collagen‐degrading ability of human gingival fibroblasts by altering the production and localization of MMPs and TIMPs.     

Gingival crevicular fluid can degrade Emdogain and inhibit Emdogain‐induced proliferation of periodontal ligament fibroblasts

Laaksonen M, Salo T, Vardar‐Sengul S, Atilla G, Han Saygan B, Simmer JP, Baylas H, Sorsa T. Gingival crevicular fluid can degrade Emdogain and inhibit Emdogain‐induced proliferation of periodontal ligament fibroblasts. J Periodont Res 2010; 45: 353–360. © 2009 The Authors. Journal compilation © 2009 Blackwell Munksgaard Background and Objective: Emdogain^® (EMD), consisting mostly of amelogenin, is used in periodontal therapy to regenerate lost connective tissue. Emdogain is applied onto periodontally affected root surfaces, where it becomes exposed to proteolytic enzymes. In this study, we aimed to find out whether gingival crevicular fluid or matrix metalloproteinases (MMPs) could degrade EMD, and whether this degradation has consequences for in vitro cell proliferation. Material and Methods: We studied the effects of 156 gingival crevicular fluid samples collected from subjects with different stages of periodontal disease and from healthy control subjects and the effects of MMP‐1, ‐2, ‐8, ‐9, ‐13 and ‐14 on the degradation of EMD using EMD‐embedded zymography. The effects of gingival crevicular fluid with or without EMD and the effects of amelogenin on the proliferation of cultured periodontal ligament fibroblasts were studied by cell proliferation enzyme‐linked immunosorbent assay kit. Results: Degradation of Emdogain induced by gingival crevicular fluid was greater in samples from all stages of periodontal diseases compared with healthy control samples. Of the MMPs studied, only MMP‐2 and MMP‐8 showed limited EMD‐degrading activities. One hundred micrograms per millilitre of EMD increased proliferation of periodontal ligament fibroblasts on average by 24% (confidence interval 0.60–0.64) and at 200 μg/mL by 30% (confidence interval 0.62–0.68) compared with control fibroblasts (confidence interval 0.48–0.52). However, gingival crevicular fluid (10 μg/mL) together with 100 μg/mL EMD induced the proliferation only by 6% (confidence interval 0.51–0.55) and with 200 μg/mL EMD by 12% (confidence interval 0.54–0.58). Amelogenin at 200 μg/mL decreased the proliferation of periodontal ligament fibroblasts by 54% (confidence interval 0.22–0.25). Conclusion: We suggest that diseased gingival crevicular fluid containing various proteases leads to degradation of EMD and decreased proliferation of periodontal ligament fibroblasts.     

Wnt1、β-catenin在硝苯地平引起的药物性牙龈增生中的表达

目的:探讨Wnt/β-catenin信号通路相关蛋白Wnt1、β-catenin在硝苯地平引起的药物性增生的牙龈组织中的表达.方法:选择正常牙龈对照组、(未服药)高血压牙龈增生组、硝苯地平引起的药物性牙龈增生组各10例,用Western-Blot和RT-PCR检测牙龈组织中Wnt1、β-catenin的表达.结果:药物性牙龈增生组的牙龈组织中Wnt1、β-catenin蛋白及mRNA表达水平显著高于正常牙龈对照组(P＜0.05)和高血压牙龈增生组(P＜0.05).结论:硝苯地平引起的药物性牙龈增生的牙龈组织中Wnt1、β-catenin表达水平增高,可能通过Wnt/β-catenin信号通路促进牙龈成纤维细胞的增殖导致牙龈纤维化.