白藜芦醇对高糖环境下牙龈上皮细胞TLR4表达及炎症因子分泌的影响

目的:研究白藜芦醇对高糖环境下牙龈上皮细胞TLR4表达及炎症因子分泌的影响,探讨白藜芦醇对伴糖尿病牙周炎患者的治疗作用及相关分子机制.方法:体外培养牙龈上皮细胞,按照作用方式不同分为正常对照组、高糖组和高糖+白藜芦醇组.荧光定量PCR检测TLR4表达情况;取第3代牙龈上皮细胞,高糖环境下采用或不采用白藜芦醇处理24 h,随后用100 ng/mL LPS处理2 h,ELISA检测IL-1β 、IL-6、IL-8和TNF-α分泌;Western印迹法检测TLR4信号通路下游分子NF-κB p65、p38MAPK和STAT3磷酸化.采用SPSS 17.0软件包对数据进行统计学分析.结果:白藜芦醇可以逆转高糖环境下牙龈上皮细胞TLR4水平的升高,同时抑制高糖环境下LPS诱导的IL-1β、IL-6、IL-8和TNF-α表达.Western印迹结果显示,白藜芦醇也可抑制TLR4信号通路下游分子NF-κB p65、p38MAPK和STAT3磷酸化.结论:白藜芦醇通过负向调控TLR4信号通路减轻高糖环境下牙龈上皮细胞炎症因子的分泌.     

葡萄籽原花青素对牙龈上皮细胞炎症介质表达的影响

研究葡萄籽原花青素（GSPs）预处理对脂多糖（LPS）诱导的人牙龈上皮细胞（HGECs）炎症介质表达的影响。     

高糖状态下人牙龈上皮细胞的生物学特性

目的观察高糖状态对人牙龈上皮细胞生物学特性的影响。方法原代培养人牙龈上皮细胞(h GEC),取第三代细胞分为正常组(培养液含5.5 mmol/L D-葡萄糖)、高糖组(培养液含25 mmol/L D-葡萄糖)和甘露醇组(含5.5 mmol/L D-葡萄糖及19.5 mmol/L甘露醇的培养基),采用MTS法检测细胞的增殖情况,流式细胞术检测细胞的生长周期及凋亡情况,划痕实验检测细胞迁移能力。采用重复测量的方差分析进行统计分析。结果高糖组、正常组、甘露醇组三组h GEC相比较,细胞增殖活性(F=5.053,P=0.052)、细胞周期分布(F=1.252,P=0.351)及凋亡细胞比例(F=0.325,P=0.717)差异均无统计学意义;划痕24 h时,糖尿病组的划痕迁移率显著低于正常组(F=54.453,P=0.000)。结论高糖状态下h GEC增殖及凋亡无显著性改变,但细胞迁移能力出现显著下降。     

牙龈卟啉单胞菌脂多糖对高脂血症兔腹腔巨噬细胞炎症相关因子基因表达的影响

目的 比较牙龈卟啉单胞菌脂多糖(Porphyromonas gingivalis on lipopolysaccharide,Pg-LPS)对健康和高脂血症兔腹腔巨噬细胞炎症相关因子基因表达的影响.方法 将健康新西兰兔12只随机分为2组,分别给予普通饲养喂养(健康兔)和高脂饲料喂养,6周后建立高脂血症模型(高脂兔).采用腹腔灌洗法分别获得健康和高脂兔巨噬细胞,将健康和高脂兔巨噬细胞均随机分为对照组、Pg-LPS组(1μg/mL)及阳性对照组大肠杆菌-脂多糖(Escherichia,E.coli-LPS)组(1μg/mL),24 h后采用实时PCR法分别检测各组巨噬细胞中C-反应蛋白(C-reaction protein,CRP)、白介素1β(interleukin-1β,IL-1β)、白介素6(interleukin-6,IL-6)、白介素8(interleukin-8,IL-8)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)mRNA的表达水平.结果 高脂兔对照组巨噬细胞的CRP、IL-1β、IL-6、IL-8、TNF-α水平高于健康兔对照组巨噬细胞;相较于对照组,高脂兔和健康兔Pg-LPS组巨噬细胞的CRP、IL-1β、IL-6、IL-8、TNF-α表达均升高,且高脂兔Pg-LPS组巨噬细胞以上因子的表达水平高于健康兔Pg-LPS巨噬细胞(P<0.05).结论 Pg-LPS可增强高脂血症兔巨噬细胞炎症相关因子mRNA的表达.     

细菌脂多糖诱导人牙龈上皮细胞β防御素-4表达的实验研究

目的观察不同浓度细菌脂多糖(BLPS)作用下原代培养的人牙龈上皮细胞(HGECs)β防御素-4(BD-4)的表达。方法体外原代培养HGECs,经免疫细胞化学鉴定,加入10、20、40、80、160μg/m L的BLPS,采用免疫组织化学和Western blotting检测BD-4蛋白在HGECs中的定位和表达变化。结果 HGECs组织块法原代细胞生长状态好,第3代细胞进行鉴定,Ck(AE1/AE3)(+)/Vimentin(-)。HGECs免疫细胞化学染色结果显示,HGECs胞浆可见棕黄色颗粒状着色,阳性染色弥漫分布于胞浆,随着BLPS浓度的增加表达更为强烈。Western Blotting结果显示,BD-4表达水平随BLPS浓度增加而增高。BD-4蛋白在BLPS浓度为160μg/m L表达的值最高,组间比较差异有统计学意义(P<0.01)。结论随着BLPS对牙龈上皮炎症刺激加重,BD-4表达增强,提示了BD-4在牙周炎症进程中的作用。     

牙龈卟啉单胞菌脂多糖对人脐静脉内皮细胞表达趋化因子RANTES和分形素的影响

目的 观察牙龈卟啉单胞菌脂多糖（Pg-LPS）对人脐静脉内皮细胞（HUVEC）表达调节活化正常T细胞表达和分泌的趋化因子（RANTES）和分形素的影响。方法 应用不同质量浓度（200、500、1 000 ng·mL^-1）的Pg-LPS分别处理HUVEC 1、6、12、24 h,利用实时荧光定量聚合酶链反应（RT-PCR）及酶联免疫吸附（ELISA）法检测RANTES及分形素mRNA及蛋白质的表达变化。结果 在Pg-LPS与HUVEC共同培养1、6和12 h时,除培养时间为12 h、Pg-LPS质量浓度为200 ng·mL^-1组的RANTES mRNA和1 h、200 ng·mL^-1组RANTES蛋白表达与对照组无明显差异外,其余各实验组RANTES蛋白和mRNA表达量及分形素mRNA表达量均高于对照组,差异有统计学意义（P<0.05）;6 h时,二者mRNA表达量达到峰值,分别为对照组的4.88倍和6.20倍;刺激6 h后,RANTES蛋白和mRNA表达量及分形素的mRNA表达量均降低,24 h时,仅Pg-LPS质量浓度为500 ng·mL-1组与对照组相比有统计学差异（P<0.05）;分形素蛋白的表达量则仅在Pg-LPS浓度为1 000 ng·mL-1刺激6、12 h时与对照组相比有统计学差异（P<0.05）。结论 Pg-LPS感染具有上调HUVEC表达趋化因子RANTES和分形素的作用,可能在牙周炎促进动脉粥样硬化发生、发展的过程中起一定作用。     

黄芩苷对氧化应激诱导牙龈上皮细胞凋亡的保护作用研究

目的：研究黄芩苷对氧化应激诱导牙龈上皮细胞凋亡的保护作用及其机制。方法采用过氧化氢刺激人牙龈上皮细胞系构建细胞凋亡模型,并应用黄芩苷进行干预,分别检测细胞增殖率,细胞凋亡水平及蛋白激酶B（protein kinase B,Akt）?糖原合酶激酶?3β（glycogen synthase kinase3β,GSK3β）蛋白表达水平。结果黄芩苷能有效地缓解氧化应激介导的牙龈上皮细胞增殖抑制,缓解凋亡发生,促进Akt?GSK3β信号通路磷酸化水平的提高。结论黄芩苷对氧化应激诱导的牙龈上皮细胞凋亡具有显著保护作用,该作用可能与调控Akt?GSK3β信号通路有关。     

牙龈卟啉单胞菌脂多糖引起牙周炎症的机制和特点

革兰阴性厌氧菌牙龈卟啉单胞菌和牙周炎密切相关，脂多糖是革兰阴性菌外膜中的主要结构成分，具有广泛的生物学活性，被认为是革兰阴性菌的主要毒力因子之一。本文就牙龈卟啉单胞菌脂多糖的化学组成和结构，所启动炎症信号通路及其起始受体，引起牙周炎症的机制和特点作一综述。     

Green tea catechins inhibit Porphyromonas gulae LPS-induced inflammatory responses in human gingival epithelial cells

ObjectivesTo determine the anti-inflammatory effects of green tea catechins in immortalized human gingival epithelial cells (Ca9-22) stimulated with Porphyromonas gulae lipopolysaccharide (LPS).MethodsCa9-22 cells were incubated with P. gulae LPS (10 μg/ml) with or without green tea catechins, epigallocatechin-3-gallate (EGCg), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and epicatechin (EC) (each at 50 μM), for 6 or 24 h. Real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay were used to determine the induction of cyclooxygenase 2 (COX2), tumor necrosis factor alpha (TNF-ɑ), interleukin 6 (IL-6), and IL-8. Furthermore, the expression of toll-like receptors (TLRs) 2 and 4 was examined using real-time PCR and western blotting analysis, and phosphorylation of the p38 and ERK1/2 was examined using western blotting analysis.ResultsAt the mRNA and protein levels, EGCg, EGC, ECG, and EC were found to significantly inhibit COX2, TNF-ɑ, IL-6, and IL-8. Furthermore, the levels of ERK1/2 and p38 phosphorylation induced by P. gulae LPS were decreased following the addition of each of the catechins, as well as TLR2 and 4 mRNA and protein.ConclusionsThese findings indicate that green tea catechins are potent inhibitors of in?ammatory responses induced by P. gulae LPS, and may also be useful for prevention and/or attenuation of periodontitis.     

釉基质蛋白对人牙龈上皮细胞增殖的影响

目的:研究釉基质蛋白(enamelmatrixproteins,EMPs)对体外培养的人牙龈上皮细胞增殖活性的影响。方法:乙酸提取法获取EMPs,取龈切术中切除的牙龈组织,采用酶消化法培养牙龈上皮细胞。将第1代牙龈上皮细胞按照22500个/孔接种,分为4组:对照组(不加EMPs)及EMPs分别为50、100、200μg/ml的实验组。采用MTT法检测各组细胞数量,对每个时间点的各组实验数据进行方差分析。结果:人牙龈上皮细胞能在EMPs覆盖的平皿上生长。统计学分析表明,不同浓度的EMPs在早期对牙龈上皮细胞的增殖无显著影响;从第3天开始,当培养液中EMPs浓度为200μg/ml时,牙龈上皮细胞增殖显著受抑。结论:EMPs影响牙龈上皮细胞的增殖,并存在剂量和时间依赖性。     

Th2 cytokines efficiently stimulate periostin production in gingival fibroblasts but periostin does not induce an inflammatory response in gingival epithelial cells

Objectives

This study aims to clarify whether gingival fibroblasts produce periostin in response to Th2 cytokines which are elevated in periodontitis lesion and, if so, whether periostin affects the inflammatory response and matrix-protein metabolism.

Design

Human gingival fibroblasts, periodontal ligament cells and the gingival epithelial cell line epi4 were stimulated with interleukin-4 (IL-4), IL-13, tumour necrosis factor-α (TNF-α) and Porphyromonas gingivalis lipopolysaccharide (LPS). Periostin expression was analysed by real-time polymerase chain-reaction (PCR) and Western blotting. The expression of the IL-4 receptor α-chain was evaluated by immunocytochemistry. The effect of periostin on the production of inflammatory cytokines and the expression of matrix protein-related genes was analysed by real-time PCR and enzyme-linked immunosorbent assay (ELISA).

Results

While IL-4 and IL-13 significantly induced periostin production in gingival fibroblasts and periodontal ligament cells, no effect was observed in epi4 cells. No stimulatory effect of TNF-α or P. gingivalis LPS on the production of periostin was observed. The effect of periostin on the production of inflammatory cytokines was weak in gingival fibroblasts; however, little or no effect was observed on periodontal ligament cells or epi4 cells. No significant effect of periostin on the expression of matrix protein-related genes was found.

Conclusion

The results suggest that gingival fibroblasts may be a source of periostin in periodontitis lesions but periostin has only a limited role either in the inflammatory response or in matrix-protein metabolism. Thus, the role of periostin in the cellular interaction between epithelial and mesenchymal cells in gingiva may be distinct from that of skin.     

重组猪釉原蛋白对人牙龈上皮细胞生物学活性的影响

目的:探讨重组25kDa猪釉原蛋白(recombinant porcine amelogenin,rPAm)对人牙龈上皮细胞(human gingival epithelial cells,HGEC)黏附、增殖和迁移的影响。方法:采用不同浓度的rPAm(0、5、10、20μg/mL)刺激第2代HGEC,在相应的时间点,应用细胞计数法测定黏附和增殖的效果,以创面愈合模型测定细胞迁移效果,采用GraphPad Prism软件对数据进行统计学分析。结果:在黏附实验中,rPAm对HGEC的黏附有抑制作用,且效果随着时间的递增和rPAm浓度的增高而加强;在增殖实验中,随着时间的递增和rPAm浓度的增高,HGEC增殖能力下降;在迁移实验中,rPAm能抑制HGEC迁移,20μg/mL效果最好,24h后呈现剂量和时间依赖性。结论:rPAm能显著抑制人牙龈上皮细胞的黏附、增殖和迁移,存在剂量和时间依赖关系。     

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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。     

Radioimmunoassay quantification of adrenomedullin in human gingival crevicular fluid

OBJECTIVE: To investigate whether adrenomedullin (ADM), a multifunctional peptide with key roles in host antimicrobial defence and inflammation, was present and quantifiable in human gingival crevicular fluid (GCF) and to study its relationship with periodontal health and disease. DESIGN: GCF samples (30s) were collected using perio-paper strips from one diseased site in 21 subjects with periodontal disease and one healthy site from 19 control subjects with no evidence of periodontal disease. Samples were analysed by radioimmunoassay using a specific anti-human ADM antibody. RESULTS: Measurable adrenomedullin-like immunoreactivity (ADM-LI) was present in all the GCF samples collected. ADM-LI was significantly higher in periodontitis sites (mean 493.6 pg) than in control healthy sites (mean 248.5 pg), p = 0.0016. CONCLUSION: It is concluded that ADM is present in GCF at levels at which it could have an antibacterial role in the gingival crevice and modulate the pathophysiology of periodontal inflammation.     

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。     

Cytotoxic effects of short-chain carboxylic acids on human gingival epithelial cells

Previous studies showed that foods that are retained on the dentition can accumulate high levels of short-chain carboxylic acids (acetic, formic, lactic and propionic). Since gingival epithelium is the first periodontal tissue to be challenged by oral factors, a study was undertaken to determine whether short-chain carboxylic acids can affect epithelial cells in vitro. Immortalized human oral epithelial cells were grown in supplemented keratinocyte growth medium at 37°C, and the effects of short-chain carboxylic acids were determined with tetrazolium-based and trypan blue exclusion assays. Low concentrations of short-chain carboxylic acids inhibited the growth of human oral epithelial cells, while higher concentrations led to cell death. The effects of short-chain carboxylic acids on the cells were dose-dependent and varied among the individual acids (propionate >formate >lactate >acetate). Growth inhibition was partly reversible and growth resumed after removal of the acids. However, the time needed for recovery of the cells increased with short-chain carboxylic acids concentration, consistent with progressively greater damage to the cells at higher short-chain carboxylic acids concentrations. The observed effects of short-chain carboxylic acids on gingival cells in vitro supported our hypothesis that short-chain carboxylic acids can damage the integrity of gingival epithelium in situ.