淫羊藿苷对体外培养的人牙龈成纤维细胞的作用研究

目的 研究淫羊藿苷对体外培养的人牙龈成纤维细胞(human gingival fibroblasts,HGF)的作用.方法 体外培养HGF,用MTT法检测不同浓度的淫羊藿苷(0.001、0.01、0.1 μg/ml)、不同时间(24、48、72、96 h)作用下HGF的增殖水平.结果 淫羊藿苷(0.001～0.1 μg/ml)对HGF增殖具有促进作用(P<0.01),0.01 μg/ml 浓度作用最明显.结论 淫羊藿苷有促进HGF增殖的作用.     

人牙龈上皮细胞上清液对牙髓细胞增殖及分化影响的研究

目的:研究体外培养的人牙龈上皮细胞(human gingival epithelium cells,HGECs)上清液对人牙髓细胞(human dental pulp cells,HDPCs)增殖及分化的影响,探讨在体外建立HGECs和HDPCs共培养模型的可行性。方法:采用组织块法分别进行HGECs和HDPCs原代培养,收集第2、4、6d的HGECs上清液,均以10%、30%、50%浓度作用于HDPCs,MTT法测定HDPCs增殖情况,通过测定细胞裂解液中ALP水平研究对HDPCs分化的影响,以加入相应浓度的上皮细胞培养基K-SFM为对照。结果:加入不同浓度的第2、4、6d的HGECs上清液后HDPCs的增殖均上升,并有促进HDPCs分化的作用,而对照组则抑制了HDPCs增殖和分化。结论:体外培养HGECs的上清液能够促进HDPCs增殖和分化。     

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单核细胞THP-1表达CC类趋化因子受体2的影响

目的 研究牙龈卟啉单胞菌脂多糖(Porphyromonas gingivalis lipopolysacchearide,Pg-LPS)对单核细胞THP-1表达CC类趋化因子受体2(CC chemokine receptor 2,CCR2)的影响,从分子水平探讨牙周炎与心血管疾病之间的关系.方法 采用不同质量浓度[0 μg/L(空白对照组)、10 μg/L(T1组)、100 μg/L(T2组)、1000μg/L(T3组)]Pg-LPS处理THP-1细胞1、4和24 h,流式细胞术和实时荧光定量PCR法检测THP-1表达CCR2蛋白和mRNA的变化.结果 除1h的T1组CCR2受体蛋白表达量(55.74 ±0.96)外,所有实验组不同时间点CCR2的蛋白和mRNA表达水平均显著高于空白对照组(P ＜0.05);24 h时T1 ～ T3组CCR2的蛋白表达量(52.56±0.61、40.98±0.86、26.50±0.67)和mRNA表达量(0.095±0.006、0.070±0.004、0.046±0.004)均显著低于空白对照组CCR2蛋白和mRNA表达(56.99±0.44、0.104 ±0.003),差异均有统计学意义(P＜0.05);所有实验组CCR2蛋白和mRNA表达水平均在4h时增加,24 h时降低,与空白对照组相比差异均有统计学意义(P＜0.05).结论 Pg-LPS作用于单核细胞的早期阶段,可呈浓度依赖性刺激单核细胞表面CCR2受体的表达,促进单核细胞的趋化作用;牙周致病菌导致的单核细胞趋化作用增强可能是牙周炎促进动脉粥样硬化斑块形成的原因之一.     

硝苯地平对人牙龈成纤维细胞内Ca2+含量影响的激光共聚焦显微观察

目的:探讨硝苯地平对牙龈成纤维细胞内Ca2 含量的影响。方法:体外培养人牙龈成纤维细胞,钙离子指示剂F luo-3染色,激光扫描共聚焦显微镜下观察加入不同浓度硝苯地平(NIF终浓度分别为1200、32.4 ng/mL)后荧光亮度的变化。结果:硝苯地平能抑制DHP类钙通道激动剂Bay K8644引起的牙龈成纤维细胞内钙离子含量增加(P<0.05),不同浓度硝苯地平的作用之间没有剂量-效应关系。结论:硝苯地平引起牙龈增生可能与抑制牙龈成纤维细胞内Ca2 含量有关。     

淫羊藿苷抑制牙龈卟啉单胞菌超声提取物对人牙周膜细胞增殖及骨保护素表达的影响

目的 探讨不同浓度淫羊藿苷抑制牙龈卟啉单胞菌超声提取物对人牙周膜细胞增殖及骨保护素（OPG）表达的影响.方法 体外培养人牙周膜细胞,用四唑盐（MTT）法检测不同浓度淫羊藿苷（0、0.001、0.01、0.1、1 μg/ml）及50μg/ml牙龈卟啉单胞菌超声提取物,不同时间（24、48、72h）作用下人牙周膜细胞的增殖水平.用RT-PCR及Western blot检测48h人牙周膜细胞骨保护素mRNA和蛋白的表达.结果 淫羊藿苷从0.01～1μg/ml对人牙周膜细胞增殖及骨保护素mRNA和蛋白的表达有促进作用（P＜0.01）,浓度为0.1μg/ml作用最显著.结论 淫羊藿苷可抑制牙龈卟啉单胞菌超声提取物对人牙周膜细胞增殖及骨保护素mRNA和蛋白表达的影响,促进人牙周膜细胞增殖及骨保护素mRNA和蛋白表达.     

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.     

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。     

牙骨质基质胍提取物促牙龈成纤维细胞、成骨细胞粘附研究

目的 :鉴定牙骨质基质胍提取物内牙骨质蛋白的生物活性。方法 :制取牙骨质基质胍提取物 ,检测人牙龈成纤维细胞、MC3T3 E1成骨细胞两种细胞在含牙骨质基质提取物浓度分别为 2 .5、5、10、2 0μg/ml的DMEM培养液中孵化 1h的贴壁率并以加牛血清蛋白 1mg/ml为空白对照组。检测两种细胞在含牙骨质基质提取物浓度 10 μg/ml的DMEM培养液中孵化 30、60、90、12 0min贴壁率。 结果 :不同浓度的牙骨质基质提取物均可促进牙龈成纤维细胞和成骨细胞的粘附 ,并呈浓度依赖性 ,尤以 10 μg/ml的牙骨质基质提取物的浓度为较佳浓度。细胞的贴壁率随牙骨质基质提取物作用时间的延长逐步升高 ,并以 90min为较理想的作用时间。结论 :人牙骨质基质胍提取物含有可促进牙龈成纤维细胞和成骨细胞粘附的牙骨质活性蛋白     

依那西普对牙龈卟啉菌内毒素作用下原代培养牙周膜细胞影响的实验研究

目的　观察依那西普（Etanercept）对牙龈卟啉菌内毒素（lipopolysaccharide,LPS)作用下体外培养的人牙周膜细胞(HPDLCs)的增殖能力的影响。方法　体外原代培养人牙周膜细胞,经免疫细胞化学鉴定,加入LPS和1 μg/ml Etanercept+LPS,LPS浓度分别为10、50、100、200、300 μg/ml检测（吸光度）A值。采用MTT法测定细胞增殖能力。结果　 人牙周膜细胞原代细胞生长状态良好。第3代细胞进行鉴定,Vimentin染色呈阳性,Ck(pan)染色呈阴性。MTT法检测加入梯度浓度的LPS 24 h后,HPDLCs增殖率随着LPS浓度的增加,对HPDLCs的增殖抑制逐渐增强,200 μg/ml LPS能最大程度抑制HPDLCS的增殖,抑制率为46.21%(P<0.01)。Etanercept + LPS组对HPDLCs细胞作用相对增殖率与LPS组比较（P＜0.01）,能明显抑制LPS对HPDLCs的抑制作用。结论　TNF-α抑制剂Etanercept能明显抑制牙周膜细胞在内毒素刺激下的死亡。     

In Vitro Study on Regeneration of Periodontal Tissue Microvasculature Using Human Dedifferentiated Fat Cells

Background: Human dedifferentiated fat cells (HDFATs) may be a new cell type suitable for regenerative therapies. The aim of this study is to assess the potential of HDFATs for vascular regeneration of periodontal tissue. To do this, HDFATs and human gingival endothelial cells (HGECs) were cocultivated, and vascular regeneration was examined in vitro. Methods: HDFATs were isolated from subcutaneous adipose tissue, and HGECs were isolated from gingival cells using anti‐cluster of differentiation 31 antibody‐coated magnetic beads. HDFATs were cocultured with HGECs in microvascular endothelial cell growth medium‐2 (EGM‐2MV) for 7 days. Expression of endothelial cell (EC) markers, the formation of capillary‐like tubes, and the expression of pericyte markers were determined. Results: HDFATs, cultured in EGM‐2MV or cocultured with HGECs, expressed EC markers. HDFATs in both conditions initiated tube formation within 5 hours of seeding and formed extensive capillary‐like structures within 12 hours. These structures disintegrated within 24 hours when cells were cultured in EGM‐2MV alone, whereas cocultured HDFATs maintained tubes for >24 hours. Cocultured HDFATs significantly increased expression of pericyte markers, a cell type associated with microvasculature. Conclusion: HDFATs possess the ability to express EC markers, and coculture with HGECs promotes differentiation into pericytes involved in the maturation and stabilization of the microvasculature.     

内毒素耐受对人牙龈上皮细胞分泌IL-1β、IL-6和IL-8的影响

目的:观察脂多糖(lipopolysaccharides,LPS)反复刺激细胞,诱导产生的内毒素耐受对人牙龈上皮细胞(human gingival epithelial cells,HGECs)分泌细胞因子IL-1β、IL-6和IL-8的影响。方法:采用1mg/L牙龈卟啉单胞菌(Porphyromonas gingivalis,P.gingivalis)LPS或1mg/L大肠杆菌(Escherichia coli,E.coli)LPS刺激HGECs 24h,洗涤细胞后,分别采用相同的LPS再次刺激24h,构建内毒素耐受模型。采用ELISA技术检测细胞条件培养液中IL-1β、IL-6和IL-8分泌水平的变化。结果:P.gingivalis LPS或E.coli LPS刺激HGECs 24h后,3种细胞因子的分泌水平均较刺激前明显增高(P<0.05)。2种LPS重复刺激,诱导细胞耐受后,IL-6和IL-8的分泌水平较第1次刺激后明显降低(P<0.05),但P.gingivalis LPS重复刺激后,IL-1β的分泌水平与第1次刺激后无明显差别。结论:内毒素耐受能抑制HGECs分泌细胞因子IL-6和IL-8,进而可能影响牙周组织的炎症和免疫反应。     

Irsogladine maleate regulates epithelial barrier function in tumor necrosis factor-α-stimulated human gingival epithelial cells

Fujita T, Yumoto H, Shiba H, Ouhara K, Miyagawa T, Nagahara T, Matsuda S, Kawaguchi H, Matsuo T, Murakami S, Kurihara H. Irsogladine maleate regulates epithelial barrier function in tumor necrosis factor‐α‐stimulated human gingival epithelial cells. J Periodont Res 2012; 47: 55–61. © 2011 John Wiley & Sons A/S Background and Objective: As epithelial cells function as a mechanical barrier, the permeability of the gingival epithelial cell layer indicates a defensive capability against invasion by periodontal pathogens. We have reported the expression of claudin‐1 and E‐cadherin, key regulators of permeability, in the gingival junctional epithelium. Irsogladine maleate (IM) is a medication for gastric ulcers and also regulates Aggregatibacter actinomycetemcomitans‐stimuated chemokine secretion and E‐cadherin expression in gingival epithelium. In this study, we have further investigated the effects of IM on the barrier functions of gingival epithelial cells under inflammatory conditions. Material and methods: We examined the permeability, and the expression of claudin‐1 and E‐cadherin, in human gingival epithelial cells (HGECs) stimulated with tumor necrosis factor (TNF)‐α, with or without IM. Results: TNF‐α increased the permeability of HGECs, and IM abolished the increase. TNF‐α reduced the expression of E‐cadherin in HGECs, and IM reversed the reduction. In addition, immunofluorescence staining showed that TNF‐α disrupted claudin‐1 expression in HGECs, and IM reversed this effect. Conclusion: The results suggest that IM reverses the TNF‐α‐induced disruption of the gingival epithelial barrier by regulating E‐cadherin and claudin‐1.     

Effect of interleukin-17 on the expression of chemokines in gingival epithelial cells

The role of interleukin (IL)-17 in cellular communication in inflammation has been well described, and a positive correlation between the severity of periodontitis and the level of IL-17 was reported. Although epithelial cells are a major target of IL-17, little is known about the effect of IL-17 on the production of chemokines by human gingival epithelial cells (HGECs). We evaluated the effects of IL-17 on the expression of CXCL8 and CCL2 by HGECs using quantitative real-time PCR and ELISA. In addition, the role of the nuclear factor (NF)-κB signalling pathway in the IL-17-mediated expression of chemokines was assessed using a specific inhibitor. Stimulation with IL-17 up-regulated the expression of CXCL8 mRNA but not of CCL2 mRNA in HGECs, whereas tumour necrosis factor-α (TNF-α) elevated the expression of mRNA for both chemokines. Stimulation with IL-17 up-regulated the secretion of CXCL8 protein, but not the secretion of CCL2 protein. The effect of IL-17 on CXCL8 production was suppressed using an anti-IL-17R Ig, suggesting a role for a specific receptor-ligand interaction. Inhibition of the NF-κB signalling pathway demonstrated that NF-κB activation is required for the CXCL8 expression in HGECs. In conclusion, IL-17 is involved in the regulation of the innate immune response in HGECs by inducing CXCL8 production.     

低温等离子体促进人牙龈上皮细胞在钛表面的黏附

目的 研究低温等离子体(NTAP)处理人牙龈上皮细胞(HGECs)后的生物学行为。方法 将HGECs传代培养,待生长活性最佳的第3～5代细胞,消化重悬后经NTAP处理适当时间(0,10,20, 30,60 s)后,接种在钛盘表面,加入口腔角质细胞培养基(OKM),1%双抗、5%CO₂和37℃孵箱条件下培养不同时间(4,12,24,48 h)贴壁生长,每组中各培养时间设置5个复孔。使用细胞计数试剂盒-8 (CCK-8)评估各组黏附细胞的数量;扫描电子显微镜(SEM)观察各组细胞在钛片表面的形态;实时荧光定量聚合酶链式反应(qRT-PCR)检测各组细胞层粘连蛋白α3 (Laminin α3)、整合素蛋白β4 (Integrin β4)和网蛋白(Plectin)黏附相关基因的表达情况以及Western blot观察各组细胞黏附相关蛋白表达的变化情况。结果 在0～20 s内,随着NTAP作用时间的增加,细胞的黏附数量增加;但在20～60 s内,随着作用时间的增加,细胞的黏附数量逐渐减少：证明NTAP处理时间为20 s时最有利于HGECs在钛表面的黏附。CCK-8显示：在设...     

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。     

Hyperglucose Contributes to Periodontitis: Involvement of the NLRP3 Pathway by Engaging the Innate Immunity of Oral Gingival Epithelium

Background: The NLRP3 inflammasome is essentially a family of intracellular innate immune sensors that can respond to bacterial challenge and initiate early host immunity responses. However, the involvement and possible molecular mechanism of the NLRP3 pathway in the context of chronic periodontitis (CP) and diabetes mellitus have yet to be fully elucidated. Methods: Gingival tissues were collected from patients with CP and/or type 2 diabetes mellitus (T2DM), and the expression of NLRP3 and interleukin (IL)‐1β was analyzed by immunohistochemistry. To explore the possible molecular mechanism, human gingival epithelial cells (HGECs) were established in vitro and challenged with lipopolysaccharide (LPS) and/or high glucose. High extracellular K⁺ was applied as an inhibitor of NLRP3. The NLRP3 pathway was analyzed by immunocytochemistry and quantitative polymerase chain reaction. Results: Compared with control individuals, NLRP3 and IL‐1β were significantly upregulated in oral gingival epithelium of patients with CP and/or T2DM (P <0.05). The expression of NLRP3 was significantly upregulated in HGECs when stimulated in vitro by LPS or high glucose (P = 0.00). The simultaneous stimulation of LPS and high glucose contributed to significant upregulation of NLRP3 expression versus LPS or high glucose alone (P = 0.00). Although expression of caspase 1 and IL‐1β protein were increased in HGECs when stimulated by LPS, they were partially inhibited after the NLRP3 was successfully blocked. Conclusion: For patients with T2DM and CP, hyperglycemic status may exacerbate the inflammation state of gingival tissue by activating the NLRP3 pathway, and this abnormal host inflammatory response may contribute to further tissue breakdown.