Adjunctive low-dose doxycycline therapy effect on clinical parameters and gingival crevicular fluid tissue plasminogen activator levels in chronic periodontitis

Objective and design: The present study examined effectiveness of low-dose doxycycline (LDD) in combination with nonsurgical therapy on gingival crevicular fluid (GCF) tissue plasminogen activator (t-PA) levels and clinical parameters in chronic periodontitis (CP) a over 12-month period. Methods: GCF samples were collected, probing depth (PD), clinical attachment level (CAL), gingival index (GI) and plaque index were recorded at baseline, 3, 6, 9 and 12 months. CP patients (n = 65) were randomized to LDD or placebo groups. LDD group received LDD (20 mg) b.i.d for 3-months plus and root planing (SRP), while placebo group was given placebo capsules b.i.d for 3-months plus SRP. GCF t-PA levels were determined by ELISA. Friedman, Wilcoxon and Mann-Whitney test was used for statistical analysis. Results: Significant improvement was observed in all clinical parameters in both groups over 12-month period (p < 0.01). LDD group had lower PD, CAL and GI scores than placebo group at 6, 9 and 12-months (p < 0.05). GCF t-PA levels reduced in both groups over 12-month period (p < 0.01). LDD group had lower GCF t-PA levels than placebo group at 6 and 9-months (p < 0.05). Conclusions: These results provide additional information about usefulness of LDD therapy as an adjunct to nonsurgical therapy in long-term management of periodontitis. Received 8 May 2006; returned for revision 13 June 2006; accepted by J. Di Battista 12 July 2006     

IDDM牙周病患者龈沟液IL－6的检测与分析

目的：研究胰岛素依赖性糖尿病（IDDM）牙周病患者、单纯牙周病患者和牙周健康者（对照组）龈沟液（GCF）白细胞介素－6（IL－6）水平及其与糖代谢状况的关系。方法：用ELISA法检测龈沟液IL－6水平,同时检测受试者的糖化血红蛋白（HbA1c）浓度。结果：IDDM牙周病组GCF中IL－6水平明显高于单纯牙周病组及对照组。 GCF中IL－6水平与反映糖代谢控制状况的糖化血红蛋白无相关性。结论：糖尿病可使牙周病患者龈沟液IL－6水平显著增高。检测龈沟液IL－6水平的变化,对探讨牙周病发病机制、预防和指导治疗均有一定的临床价值。     

复方多西环素缓释凝胶对实验性牙周炎龈沟液IL-4和 TNF-α的影响

目的：研究复方多西环素缓释凝胶对实验性牙周炎大鼠牙龈指数（ GI）、牙周袋深度（ PD）、附着丧失程度（AL）及龈沟液中白介素-4（IL-4）和肿瘤坏死因子-α（TNF-α）水平的影响。方法选用Wister大鼠42只,随机分为3组：正常对照组（N组）,阳性对照组（C组）和实验组（T组）,每组14只。 C组和T组建立牙周炎模型后,C组给米诺环素软膏治疗,T组给复方多西环素缓释凝胶治疗,均为每周上药1次共4次,并分别在治疗前和治疗后记录GI、PD、AL及龈沟液中IL-4和TNF-α水平。结果治疗后,C组和T组GI、PD、AL均较治疗前显著下降（ P ＜08．05）。牙周炎大鼠龈沟液中IL-4水平显著低于正常组（ P ＜0．05）,治疗后显著增高（ P ＜0．05）,且T组明显高于C组（ P ＜0．05）;牙周炎大鼠龈沟液中TNF-α水平显著高于正常组（ P ＜0．05）,治疗后显著降低（ P ＜0．05）,T组和C组治疗后差异无统计学意义（ P ＞0．05）。结论复方多西环素缓释凝胶能明显改善牙周炎临床症状,显著降低龈沟液中TNF-α水平,提高龈沟液中IL-4水平。     

2型糖尿病患者龈沟液中基质金属蛋白酶-1的检测和临床意义

目的 比较T2DM患者、T2DM合并牙周病患者与正常对照者龈沟液（GCF）中基质金属蛋白酶-1（MMP-1）和GCF量,以及T2DM患者GCF中MMP-1量与糖代谢状况的关系. 方法 使用滤纸条法采集GCF.选取T2DM组、T2DM合并牙周病组各30例,正常对照组30名,ELISA测定GCF中MMP-1含量,运用高效液相色谱法检测HbA1c水平. 结果 T2DM、T2DM合并牙周病组GCF量高于正常对照组[（2.81±1.58）、（3.56±2.96）vs（1.02±0.65） μl/样本]（P＜0.01）.T2DM合并牙周病组GCF中MMP-1量高于T2DM、正常对照组[（5.02±0.16）vs（3.01土0.09）、（2.24±0.11） μl/样本]（P＜0.01）.T2DM组与正常对照组GCF中MMP-1量比较差异无统计学意义（P＞0.05）.T2DM合并牙周病组GCF中MMP-1量与HbA1c水平无相关性（r=0.153,P=0.151）. 结论 3组GCF及MMP-1水平在T2DM合并牙周病组中最高,在对照组最低,提示牙周炎症是影响结果的主要原因.MMP-1水平在T2DM组中低于T2DM合并牙周病组,提示T2DM患者不只是MMP-1水平改变而导致牙周病的易感,可能是多个因素共同作用的结果.     

GCF低表达宫颈癌HeLa细胞系的构建及辐射对其调控IER5基因表达的初步探究

目的 构建GCF低表达的宫颈癌HeLa细胞系,在⁶⁰Co γ射线照射下初步探究其细胞增殖以及调控宫颈癌放射敏感基因IER5的表达。 方法 经带有荧光标记的GCF低表达的质粒稳定转染HeLa细胞, G418筛选,蛋白印迹法、实时定量PCR法鉴定得到GCF-shRNA-HeLa单克隆细胞系。倒置显微镜观察细胞形态变化,CCK-8法探究不同剂量照射后的细胞增殖情况,在不同照射剂量下用蛋白印迹法检测GCF-shRNA-HeLa及对照细胞中IER5蛋白的表达情况。 结果 成功构建稳定表达的GCF-shRNA-HeLa单克隆细胞系,显微镜下观察GCF-shRNA-HeLa细胞比正常HeLa细胞体积小,细胞间隙较大,其中梭形细胞较多。蛋白印迹法及实时定量PCR验证了GCF-shRNA-HeLa的蛋白表达及mRNA转录水平均低于对照组(P<0.05)。辐射情况下实验结果显示在同一照射剂量、同一时间下GCF-shRNA-HeLa细胞比control对照细胞增殖缓慢(P=0.014)。不同剂量照射情况下蛋白印迹法检测HeLa细胞、control-shRNA-HeLa与GCF-shRNA-HeLa细胞IER5蛋白表达情况,结果同一辐照剂量下GCF沉默的细胞系中IER5的表达量比对照细胞高, 4Gy照射的GCF-shRNA-HeLa的IER5表达量最高(P<0.05)。 结论 本实验构建了低表达GCF的HeLa细胞系,初步探讨GCF作为IER5基因抑制性转录因子的作用。结合临床基因靶向治疗,可寻求可以降低GCF表达的方式,通过上调IER5表达这一双效途径来加速宫颈癌细胞凋亡。     

慢性牙周炎非手术治疗前后龈沟液中CRP、sICAM-1含量比较

目的:测定慢性牙周炎龈沟液中CRP、sICAM-1的含量及牙周非手术治疗对其水平的影响。方法:选择22例中重度慢性牙周炎患者,进行牙周非手术治疗,并于治疗前后收集龈沟液,用ELISA法测定CRP、sICAM-1的水平,与正常对照组比较。结果:慢性牙周炎患者龈沟液CRP、sICAM-1水平较正常者增高(p<0.05);经牙周非手术治疗后,牙周临床指标明显改善,CRP、sICAM-1水平下降,接近牙周正常者水平(p<0.05)。结论:慢性牙周炎龈沟液CRP、sICAM-1的水平升高,牙周非手术治疗可有效地降低其在龈沟液中的表达。     

Cytokine levels in crevicular fluid are less responsive to orthodontic force in adults than in juveniles

OBJECTIVES: Bone remodelling during orthodontic tooth movement is related to the expression of mediators in gingival crevicular fluid (GCF). No information is available concerning the effect of age on the levels of these mediators in GCF. The purpose of this study was to quantify three mediators (prostaglandin E2, interleukin-6 and granulocyte-Macrophage Colony-Stimulating Factor) in GCF during orthodontic tooth movement in juveniles and adults. MATERIAL AND METHODS: A total of 43 juvenile patients (mean age 11 +/- 0.7 year), and 41 adult patients (mean age 24 +/- 1.6 year) took part in the study. One of the lateral incisors of each patient was tipped labially, the other served as control. GCF samples were taken before force activation (t0) and 24 h later (t24). Mediator levels were determined by radioimmunoassay (RIA). RESULTS: PGE2 concentrations were significantly elevated at t24 in juveniles and adults, while concentrations of IL-6 and GM-CSF were significantly elevated only in juveniles. Total amounts of all three mediators in GCF significantly increased at t24 in both groups. CONCLUSIONS: In early tooth movement, mediator levels in juveniles are more responsive than levels in adults, which agrees with the finding that the initial tooth movement in juveniles is faster than in adults and starts without delay.     

Quantitative determination of lysophosphatidic acids (LPAs) in human saliva and gingival crevicular fluid (GCF) by LC-MS/MS

Lysophosphatidic acid (LPA) is a phospholipid mediator that plays multiple cellular functions by acting through G protein-coupled LPA receptors. LPAs are known to be key mediators in inflammation, and several lines of evidence suggest a role for LPAs in inflammatory periodontal diseases. A simple and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method has been developed and validated to quantify LPA species (LPA 18:0, LPA 16:0, LPA 18:1 and LPA 20:4) in human saliva and gingival crevicular fluid (GCF). LPA 17:0 was used as an internal standard and the LPA species were extracted from saliva by liquid-liquid extraction using butanol. Chromatography was performed using a Macherey-Nagel NUCLEODUR^® C8 Gravity Column (125 mm × 2.0 mm ID) with a mixture of methanol/water: 75/25 (v/v) containing 0.5% formic acid and 5 mM ammonium formate (mobile phase A) and methanol/water: 99/0.5 (v/v) containing 0.5% formic acid and 5 mM ammonium formate (mobile phase B) at a flow rate of 0.5 mL/min. LPAs were detected by a linear ion trap-triple quadrupole mass spectrometer with a total run time of 8.5 min. The limit of quantification (LOQ) in saliva was 1 ng/mL for all LPA species and the method was validated over the range of 1-200 ng/mL. The method was validated in GCF over the ranges of 10-500 ng/mL for LPA 18:0 and LPA 16:0, and 5-500 ng/mL for LPA 18:1 and LPA 20:4. This sensitive LC-MS/MS assay was successfully applied to obtain quantitative data of individual LPA levels from control subjects and patients with various periodontal diseases. All four LPA species were consistently elevated in samples obtained from periodontal diseases, which supports a role of LPAs in the pathogenesis of periodontal diseases.     

Detection of gingival crevicular fluid MMP-8 levels with different laboratory and chair-side methods

Objective:  The aim of the study was to compare four methods for gingival crevicular fluid (GCF) matrix metalloproteinase (MMP)-8 detection.
Methods:  Matrix metalloproteinase-8 levels from 20 GCF samples from two periodontally healthy subjects, 18 samples from two patients with gingivitis and 45 samples from six patients with moderate to severe periodontitis, altogether 83 samples, were analysed using (1) a time-resolved immunofluorometric assay (IFMA), (2) an MMP-8 specific chair-side dip-stick test, (3) a dentoAnalyzer device and (4) the Amersham ELISA kit. Western immunoblot using same monoclonal anti-MMP-8 as in IFMA and dentoAnalyzer was used to identify molecular forms of MMP-8 in GCFs.
Results:  Correlation between IFMA and dentoAnalyzer results calculated with Spearman's correlation coefficient was 0.95 ( P  = 0.01). The chair-side dip-stick test results were well in line with these assays. Periodontitis sites with unstable characteristics were differentiated with these methods. The Amersham ELISA results were not in line with the findings by other methods.
Conclusions:  Immunofluorometric assay and dentoAnalyzer can detect MMP-8 from GCF samples and these methods are comparable. Using Western immunoblot, it was confirmed that IFMA and dentoAnalyzer can detect activated 55 kDa MMP-8 species especially in periodontitis-affected GCF. dentoAnalyzer is among the first quantitative MMP-8 chair-side testing devices in periodontal and peri-implant diagnostics and research.