热休克蛋白27在烟草烟雾提取物介导的牙龈成纤维细胞损伤中的表达变化

目的 观察在不同质量分数烟草烟雾提取物（CSE）干预下,热休克蛋白（HSP）27在人牙龈成纤维细胞（HGFs）损伤过程中的表达。方法 取原代培养并经过鉴定的3~8代HGFs,采用细胞划痕试验检测不同刺激质量分数（0、2.5%、5.0%、12.5%、25.0%、50.0%）的CSE对HGFs体外迁移的影响,并采用Western blot方法检测HSP27在HGFs中的表达。结果 CSE质量分数越高,细胞的迁移能力越弱;HSP27在正常HGFs中呈弱阳性表达,在CSE刺激后的HGFs中呈强阳性表达,且随CSE质量分数的增高,HSP27的相对表达量有逐渐增高的趋势,与细胞迁移能力相反。结论 HSP27在CSE介导的HGFs损伤中表达升高,在CSE介导的上皮损伤中有重要作用。     

Butyrate,a bacterial metabolite,induces apoptosis and autophagic cell death in gingival epithelial cells

Tsuda H, Ochiai K, Suzuki N, Otsuka K. Butyrate, a bacterial metabolite, induces apoptosis and autophagic cell death in gingival epithelial cells. J Periodont Res 2010; 45: 626–634.©2010 John Wiley & Sons A/S Background and Objective: Butyrate is produced by some types of anaerobic periodontal bacteria. Millimolar concentrations of butyrate are found in mature dental plaque from periodontitis patients. Although butyrate reportedly has a variety of effects in many mammalian cells, its effect on gingival epithelial cells is not well known. In this study, we investigated the effect of butyrate on gingival epithelial Ca9‐22 cell death. Material and Methods: Death of Ca9‐22 cells was assessed after treating the cells with or without butyrate. A SYTOX Green dye, which exhibits strong green fluorescence once it enters dead cells through ruptured cell membranes, was used for cell death detection. Phosphatidylserine redistribution was measured using fluorescein isothiocyanate‐labeled annexin V. The activity of caspase‐3 was measured as the amount of cleaved substrate peptide. Anti‐apoptotic bcl‐2 mRNA expression was measured using real‐time RT‐PCR. Western blotting and fluoromicroscopic analysis with anti‐microtubule‐associated protein 1 light chain 3 (LC3) antibodies were performed for detection of autophagy. Results: Stimulation with millimolar concentrations of butyrate for 48 h induced Ca9‐22 cell death. The stimulation also caused increased caspase‐3 activity, phosphatidylserine redistribution and bcl‐2 down‐regulation, suggesting butyrate‐induced apoptosis. However, the pan‐caspase inhibitor, Z‐VAD‐FMK, did not inhibit cell death completely. This implies the existence of other types of cell death. In addition, markers of autophagy, namely, the conversion of LC3‐I to LC3‐II and increased LC3 accumulation, were observed. Moreover, inhibition of autophagy by 3‐methyladenine suppressed the butyrate‐induced cell death, suggesting that butyrate could induce cell death through autophagy. Conclusion: These data suggest that butyrate induces apoptosis and autophagic cell death.     

Effects of cigarette smoke and nicotine on cell viability,migration and myofibroblastic differentiation

Silva D, Cáceres M, Arancibia R, Martínez C, Martínez J, Smith P. Effects of cigarette smoke and nicotine on cell viability, migration and myofibroblastic differentiation. J Periodont Res 2012; 47: 599–607. © 2012 John Wiley & Sons A/S Background and Objective: Several studies have analysed the role of nicotine as a prominent agent affecting wound repair in smokers. However, tobacco smoke contains several components that may alter gingival wound healing. The present study aimed to analyse the roles of cigarette smoke condensate (CSC) and nicotine on cell viability, cell migration/invasion and myofibroblastic differentiation using primary cultures of human gingival fibroblasts. Material and Methods: To compare the effects of CSC and nicotine, gingival fibroblasts were stimulated with CSC (0.4–500 μg/mL) and the corresponding nicotine concentrations (0.025–32 μg/mL) present in research cigarettes (1R3F). Cell viability was evaluated through the MTS assay. Cell migration and invasion were assessed through scratch wound assays, collagen nested matrices and transwell migration. α‐Smooth muscle actin production was evaluated by western blotting. Results: Cigarette smoke condensate at 50 μg/mL induced a moderate increase in cell viability, whereas the corresponding nicotine concentration (3.2 μg/mL) did not produce this response. Cigarette smoke condensate at 250 μg/mL, but not nicotine at 16 μg/mL (the corresponding nicotine concentration), induced cell death. Both nicotine and CSC stimulated cell migration (50 μg/mL CSC; 3.2 μg/mL nicotine). At 150 μg/mL, CSC inhibited cell migration; however, the corresponding concentration of nicotine (9.6 μg/mL), did not have this effect. Although both nicotine and CSC inhibited α‐smooth muscle actin production, only the latter induced a statistically significant effect on this response. Conclusion: Cigarette smoke condensate may stimulate cell survival and migration at low concentrations and inhibit these cell responses at higher levels of exposure. Moreover, CSC may interfere in myofibroblastic differentiation. These results show that cigarette smoke, but not nicotine, may significantly alter cell viability, cell migration and myofibroblastic differentiation in gingival mesenchymal cells.     

人血清白蛋白对在纯钛表面粘附的人牙龈上皮细胞形态的影响

目的：探讨人血清白蛋白(human serum albumin,HSA)对在纯钛表面粘附的人牙龈上皮细胞(human gingival cpithelial cclls,HGE)形态的影响。方法：HGE原代培养3—5代细胞接种于纯钛试件表面，部分纯钛试件用50g/L HSA预孵育，扫描电镜观察HGE的形态学特征。结果 ：细胞接种后第4h、12h、24h，HSA预孵育组及对照组的纯钛试件表面培养的HGE，细胞形态没有显著性差异。结论：人血清白蛋白对粘附在纯钛表面的人牙龈上皮细胞的早期形态学特征无显著影响。     

高浓度唑来膦酸对人牙周膜干细胞凋亡及成骨分化影响的实验研究

目的：研究高浓度唑来膦酸对体外分离的健康人牙周膜干细胞增殖、凋亡和成骨分化的影响。方法：体外分离培养健康人牙周膜干细胞,用不同浓度的唑来膦酸（5μmol/L、10 μmol/L）处理,不加药组为空白对照。 以 CCK8 检测细胞增殖,流式细胞技术检测细胞凋亡,ALP、茜素红染色及半定量分析检测细胞体外成骨分化,荧光定量PCR检测成?标志物I型胶原(COL1A1)和骨钙素（OCN）基因的表达,免疫荧光检测 OCN和 COL1A1 蛋白的表达。结果： 唑来膦酸可呈计量依赖性抑制人牙周膜干细胞增殖;唑来膦酸用药组细胞的凋亡率显著?于对照组,且随着药物浓度的增加,凋亡百分比逐渐上升;ALP和茜素红染?结果表示唑来膦酸用药组细胞的体外成骨分化能力较对照组显著下降;荧光定量PCR和免疫荧光结果提示,药物处理后OCN和 COL1A1的基因表达和蛋白表达均降低。结论：高浓度唑来膦酸显著抑制人牙周膜干细胞的增殖及体内外成骨分化,并诱导其凋亡。     

重组人乳铁蛋白抑制口腔癌KB细胞增殖及诱导细胞凋亡

目的 研究重组人乳铁蛋白(recombinate human lactoferrin,rhLF)对人口腔KB细胞增殖及凋亡的影响,为口腔癌患者提供新的有效治疗途径提供参考。方法 采用CCK-8法检测不同浓度(0、12.5、25、50、100、200 μg/mL)的rhLF分别处理KB细胞24、48、72 h后对细胞增殖的影响;Immunofluorescence及Western blotting方法检测rhLF处理前后DNA损伤相关蛋白γ-H2AX的表达。并分别用0、50、200 μg/mL的rhLF处理KB细胞,14 d后观察其对集落克隆形成的影响;0、200 μg/mL rhLF处理KB细胞48 h后,收集细胞并使用线粒体膜电位检测试剂盒(JC-1)检测rhLF处理前后线粒体膜电位的变化。Annexin V-PI染色检测rhLF对口腔癌KB细胞凋亡的影响;Western blotting检测0、50、200 μg/mL的rhLF处理口腔癌KB细胞48 h后Parp、Caspase-3的表达情况。结果 rhLF处理KB细胞的增殖有显著的抑制作用。Immunofluorescence结果显示rhLF处理后γ-H2AX的表达明显增加,Western blotting结果也显示rhLF处理后γ-H2AX的表达成浓度依赖性增加。rhLF抑制KB细胞集落克隆的形成;JC-1荧光检测结果表明红绿荧光的相对比例降低,这种红绿荧光的相对比例降低提示膜电位下降;0、50、200 μg/mL的rhLF处理口腔癌KB细胞48 h的凋亡率为2.02%、7.60%、48.07%;同时rhLF刺激口腔癌KB细胞能诱导Parp、Caspase-3的激活。结论 rhLF能显著抑制KB细胞的增殖并且诱导其细胞凋亡,其机制与线粒体凋亡途径的激活应激有关。     

白细胞介素-1β对遗传性牙龈纤维瘤和正常牙龈上皮细胞β-防御素的影响

目的:比较白细胞介素1β(IL-1β)刺激体外培养的正常牙龈、遗传性牙龈纤维瘤(HGF)上皮细胞β-防御素(HBD)表达的差异,探讨该差异与遗传性牙龈纤维瘤发病机制的可能相关性。方法:体外培养3例遗传性牙龈纤维瘤病人和6例正常人牙龈上皮细胞,经0,0.01,0.1,1,10,100 ng/mL的IL-1β分别刺激12、24、36、48 h,提取细胞总RNA,逆转录后,采用HBD-1、2、3特异性引物经PCR扩增,以β-肌动蛋白为内参,计算HBD-1、2、3与内参扩增产物相对值,对HBD-1、2、3进行半定量分析,采用SPSS软件单向方差统计分析法分析结果。结果:HBD-1 mRNA在正常牙龈和遗传性牙龈纤维瘤上皮细胞中呈固有表达,不受IL-1β刺激的影响。IL-1β可上调两种牙龈上皮细胞HBD-2、3的表达,在浓度为0.1～10 ng/mL时,作用时间大于12 h,受刺激组与未受刺激组差异有显著性(P<0.01)。相同浓度IL-1β作用不同时间时,正常牙龈上皮细胞中HBD-2、3的表达水平显著高于遗传性牙龈纤维瘤上皮细胞中表达水平(P<0.05,P<0.01)。且随作用时间延长差异更加显著。结论:遗传性牙龈纤维瘤病变组织和正常牙龈组织中β-防御素的表达有差异,这种差异可能与遗传性牙龈纤维瘤上皮细胞的分化及发病机制相关。     

牙龈卟啉单胞菌上调钙结合蛋白1促进牙龈上皮细胞增殖

目的 探究钙结合蛋白1在牙龈卟啉单胞菌(P.gingivalis)影响牙龈上皮细胞增殖和凋亡中的作用.方法 P.gingivalis感染CA9-22细胞,在感染24 h后,采用实时荧光定量聚合酶链反应、免疫印迹法和免疫荧光法检测钙结合蛋白1(CALB1)的表达.通过RNA干扰法抑制CALB1表达,BrdU分析检测细胞增...     

Induction of apoptosis in oral epithelial cells by Candida albicans

During infection, interactions between Candida albicans and oral epithelial cells result in oral epithelial cell death. This is clinically manifested by the development of oral mucosal ulcerations generally associated with discomfort. In vitro studies have shown that C. albicans induces early apoptotic alterations in oral epithelial cells; however, these studies have also shown that treatment of infected cells with caspase inhibitors does not prevent their death. The reasons for these contradictory results are unknown and it is still not clear if C. albicans stimulates oral epithelial signaling pathways that promote apoptotic cell death. Activation of specific death pathways in response to microbial organisms plays an essential role in modulating the pathogenesis of a variety of infectious diseases. The aim of this study was to (i) characterize C. albicans‐induced apoptotic morphological alterations in oral epithelial cells, and (ii) investigate the activation of apoptotic signaling pathways and expression of apoptotic genes during infection. Candida albicans induced early apoptotic changes in over 50% of oral epithelial cells. However, only 15% of those showed mid‐late apoptotic alterations. At the molecular level, C. albicans caused a loss of the mitochondrial transmembrane potential and translocation of mitochondrial cytochrome c. Caspase‐3/9 activities increased only during the first hours of infection. Moreover, poly[ADP ribose] polymerase 1 was cleaved into apoptotic and necrotic‐like fragments. Finally, five anti‐apoptotic genes were significantly upregulated and two pro‐apoptotic genes were downregulated during infection. Altogether, these findings indicate that epithelial apoptotic pathways are activated in response to C. albicans, but fail to progress and promote apoptotic cell death.     

Impact of resveratrol on bone repair in rats exposed to cigarette smoke inhalation: histomorphometric and bone-related gene expression analysis

This study investigated the effect of resveratrol on bone healing and its influence on the gene expression of bone-related markers in rats exposed to cigarette smoke. Two calvarial defects were created in each of 60 rats, which were assigned equally (n = 20) to three groups: (1) resveratrol (10 mg/kg) + smoke exposure (SMK + RESV); (2) placebo + smoke exposure (SMK + PLA); or (3) placebo + no smoke exposure (NS + PLA). Substances were administered daily for 30 days following surgery. Smoke inhalation was started 7 days before surgery and continued for 30 days after surgery. One defect was processed for histomorphometric analysis and the other was used for mRNA quantification of bone-related gene expression by qPCR. The remaining defect was smaller in the SMK + RESV (2.27 ± 0.61 mm, P = 0.0003) and NS + PLA (2.17 ± 0.74 mm, P = 0.0005) groups than in the SMK + PLA group (3.12 ± 0.47 mm). Higher levels of Runx2 were observed in the NS + PLA group than in the smoke exposure groups (vs. SMK + PLA, P = 0002; vs. SMK + RESV, P = 0.052); levels of Lrp-5 were also higher in the no smoke exposure group (vs. SMK + RESV, P = 0.009; vs. SMK + PLA, P = 0.003). Resveratrol therapy decreased RANKL/OPG expression when compared to placebo (SMK + RESV vs. SMK + PLA, P = 0.017). Dkk1 levels were decreased in the SMK + RESV group when compared to the SMK + PLA (P = 0.006) and NS + PLA groups (P = 0.005). In conclusion, resveratrol optimizes the repair of critical-sized bone defects, up-regulating the gene expression of important bone remodelling markers in rats exposed to cigarette smoke inhalation.