伴牙周炎2型糖尿病大鼠模型的建立及RANKL、OPG的研究

目的：建立一种模拟人类伴牙周炎的2型糖尿病动物模型,探讨2型糖尿病与牙周炎之间的关系及RAN-KL、OPG在其中所起的作用。方法：将大鼠随机分为4组,正常对照组（A组）、喂饲高脂高糖＋注射链脲佐菌素的方法建立2型糖尿病组（B组）、结扎方法建立牙周炎组（C组）及结扎＋高脂高糖＋链脲佐菌素建立2型糖尿病牙周炎组（D组）,第8周末检测血糖、牙周情况及OPG、RANKL的表达。结果：2型糖尿病伴牙周炎动物模型成功建立,其牙周炎程度为D组〉C组（P〈0．05）,血糖水平D组〉B组（P〈0．05）,龈沟液RANKL／OPG比值均较A组增大（P〈0．05）、D组与c组无统计学差异（P〉0．05）。结论：结扎＋高脂高糖＋链脲佐菌素的方法可成功建立伴牙周炎2型糖尿病大鼠动物模型,2型糖尿病与牙周炎二者协同作用互相促进。     

糖尿病大鼠牙周组织病理改变的机制研究

目的研究糖尿病大鼠牙周组织的病理改变及CD4、CD8、核因子-κB受体活化子配体（receptor activator of nuclear factor-κ Bligand,RANKL）、骨保护素（osteoprotegerin,OPG）在糖尿病大鼠牙周组织中的改变。方法 38只SD大鼠随机分成2组,糖尿病组30只,对照组8只。采用一次性静脉注射链尿佐菌素（streptozotocin,STZ）的方法建立大鼠糖尿病模型,动物于分组饲养16周后处死,制作牙周组织石蜡切片,HE染色,荧光免疫组化检测牙周组织中CD4、CD8及RANKL、OPG的表达。采用激光扫描共聚焦显微镜半定量分析CD4、CD8及RANKL、OPG,对糖尿病组与对照组的差异进行比较。结果糖尿病组与对照组CD4灰度值分别为4.87±4.08、2.71±3.35;CD8灰度值分别为5.57±5.64、1.64±1.17;RANKL灰度值分别为2.14±3.45、1.13±5.09;差异均有统计学意义。与对照组相比,糖尿病组OPG表达降低无统计学意义。结论糖尿病可能通过改变免疫功能调节以及RANKL/OPG调节途径促进牙周炎的发生发展。     

糖尿病大鼠牙周组织中免疫调节相关信号通路研究

目的观察糖尿病大鼠模型牙周组织中CD4、CD8、核因子-κB受体活化子配体(receptor activator forNF-κB ligand,RANKL)及骨保护素(osteoprotegerin,OPG)表达的变化。方法 38只健康雄性SD大鼠,随机选取30只,高热量饲料喂养1个月后,建立肥胖模型,随后每只大鼠按55 mg/kg一次性静脉注射链脲佐菌素(strepto-zotocin,STZ),建立糖尿病模型组;其余8只普通饲料喂养1个月后,一次性静脉注射相同剂量的pH值4.5、0.1 mol/L柠檬酸缓冲液,设为对照组。建模第16周处死大鼠,解剖上颌骨,制作3μm厚连续切片,荧光免疫组化检测牙周组织中CD4、CD8及RANKL、OPG的表达。采用激光扫描共聚焦显微镜(laser scanning confocal micro-scope,LSCM)采集图像,行灰度值分析,比较CD4、CD8、RANKL、OPG在糖尿病组和对照组大鼠牙周组织中的表达差异。结果糖尿病组CD4、CD8、RANKL的表达均高于对照组,差异有统计学意义(P<0.05);糖尿病组与对照组相比OPG表达降低,差异无统计学意义。结论糖尿病可能通过RANKL/OPG调节途径促进牙周炎的发生发展。     

伴牙周炎糖尿病患者血清和龈沟液中C反应蛋白的检测及意义

目的:了解龈沟液和血清中C反应蛋白(C-reactive protein,CRP)水平与牙周炎症状况、血糖控制情况的关系.方法:检测伴慢性牙周炎2型糖尿病组、慢性牙周炎组、2型糖尿病组和健康人群组血清和龈沟液中CRP浓度、糖化血红蛋(glycosylated hemoglobin,HbAlc)水平,记录牙周探诊深度(probing depth,PD)和龈沟出血指数(sulcus bleeding index,SBI).采用SPSS10.0软件包进行独立样本t检验和Spearman相关分析.结果:伴慢性牙周炎2型糖尿病组、慢性牙周炎组、2型糖尿病组血清CRP均比正常对照组显著升高(P＜0.01),且伴慢性牙周炎2型糖尿病组血清CRP水平最高,显著高于慢性牙周炎组(P＜0.01)和2型糖尿病组(P＜0.05);4组的龈沟液CRP水平均远低于血清水平,且4组间无显著差异(P＞0.05);血清CRP与龈沟液CRP无相关性(P＞0.05);血清CRP与PD、SBI和HbAlc显著相关(P＜0.01).结论:CRP可能参与了牙周炎和2型糖尿病之间的互相影响;龈沟液CRP水平不能反映牙周病炎症程度和糖尿病病情.     

胰岛素治疗对糖尿病大鼠牙槽骨RANKL/OPG mRNA影响的研究

目的通过研究胰岛素治疗对糖尿病大鼠牙周组织病理改变及牙槽骨中NF-κB受体活化因子配体(Receptor activator of nuclear factor-κB ligand,RANKL)和骨保护素(osteoprotegerin,OPG)mRNA水平比值情况的影响,探讨糖尿病影响牙周病时牙槽骨吸收的机理。方法将12只大鼠采用静脉注射链脲佐菌素的方法建立糖尿病模型,并随机分为治疗组和对照组。治疗组给予胰岛素皮下注射,对照组注射等量生理盐水。分别于实验开始时、造模成功后和8周后处死时测量大鼠体重和血糖。右下磨牙区牙周组织脱钙后HE染色观察组织病变状况;应用RT-PCR检测左下磨牙区牙槽骨RANKL和OPG mRNA表达情况,并比较两组大鼠RANKL/OPG比值差异。结果胰岛素治疗组较糖尿病组牙周组织炎症反应减轻,牙槽骨吸收减弱;血糖值(P<0.05)及RANKL/OPGmRNA比值(P<0.01)降低。结论胰岛素治疗可能增加牙周组织修复和再生能力,降低糖尿病大鼠的牙槽骨RANKL/OPGmRNA比值。提示血糖水平增高可能是影响糖尿病大鼠的牙槽骨吸收危险因素之一。     

环氧合酶-2-1195GA多态性与2型糖尿病对慢性牙周炎易感性的影响

目的:研究环氧合酶-2(COX-2)-1195GA多态性与2型糖尿病对牙周炎易感性的影响。方法:采用病例对照实验设计,选择46例单纯2型糖尿病患者、76例糖尿病伴牙周炎患者、158例单纯牙周炎患者以及112例健康对照者。应用聚合酶链反应—限制性内切酶片段长度多态性基因分析方法(PCR-RFLP),采用卡方检验比较COX-2基因-1195GA位点的基因型和等位基因频率在各组间的分布差异,并计算其与牙周炎的危险度(OR)。结果:与健康组比较,COX-2基因-1195GA位点的AA基因型以及等位基因A在牙周炎组和糖尿病伴牙周炎组中存在统计学差异(P=0.053和P=0.017);2型糖尿病对慢性牙周炎的危险度为1.667(95%CI 0.658～4.222,P=0.281),AA+GA基因型对慢性牙周炎的危险度为2.027(95%CI 1.135～3.621,P=0.017),两者叠加效应对慢性牙周炎的危险度为2.130(95%CI 1.104～4.108,P=0.024),差异具有统计学意义。结论:COX-2基因的-1195GA多态性可能提高了2型糖尿病患者对慢性牙周炎的易感性。     

Wnt3a、β-Catenin、OPG、RANKL在慢性根尖周炎合并2型糖尿病根尖周组织中的表达

目的 探讨2型糖尿病对慢性根尖周炎骨代谢可能存在的影响。方法 选取健康牙齿牙周膜组织、慢性根尖周炎患牙根尖周软组织、慢性根尖周炎合并2型糖尿病患牙根尖周软组织各20例。采用H&E染色法和免疫组织化学法检测各组根尖周组织炎症浸润情况及Wnt3a、β-连环蛋白（β-Catenin）、骨保护素（OPG）、RANKL的表达。结果 Wnt3a在慢性根尖周炎组表达水平低于健康对照组(P>0.05);在慢性根尖周炎合并2型糖尿病组表达水平高于健康对照组(P>0.05);在慢性根尖周炎组和慢性根尖周炎合并2型糖尿病组中的表达水平比较有统计学差异(P<0.05)。β-Catenin在慢性根尖周炎组、慢性根尖周炎合并2型糖尿病组表达水平高于对照组(P<0.05)。OPG、RANKL、RANKL/OPG在对照组、慢性根尖周炎组、慢性根尖周炎合并2型糖尿病组表达水平呈升高趋势,OPG与RANKL/OPG在各组间比较均有统计学差异(P<0.05),RANKL在慢性根尖周炎组、慢性根尖周炎合并2型糖尿病组的表达水平均高于对照组,且比较有统计学差异（P<0.05）。结论 ...     

伴2型糖尿病慢性牙周炎患者TNF-α基因携带频率的研究

目的:通过对伴2型糖尿病慢性牙周炎患者、不伴全身系统性疾病的慢性牙周炎患者以及健康对照组中TNF-α基因携带频率的分析,探讨病例组和对照组在该基因携带频率上的差异,并比较各组牙周临床指标和易感等位基因的关系。方法:采用牙周探针,对112例伴2型糖尿病慢性牙周炎患者(DM组)、99例单纯慢性牙周炎患者(CP组)以及健康对照组进行牙周临床指标检查和TNF-α-308基因型(TNF1/2)检测。采用SPSS13.0软件包对数据进行χ2检验和方差分析。结果:在DM组和轻中度CP组之间,轻中度DM组和重度CP组之间,重度DM组和轻中度CP组之间,重度DM组和重度CP组之间,TNF2的阳性基因型分布均有统计学差异(P<0.05)。携带等位基因TNF2的DM组和CP组的牙周探诊深度、临床附着丧失均分别显著高于只携带等位基因TNF1的DM组和CP组患者(P<0.05)。结论:携带TNF-α-308等位基因TNF2可能会增加人群牙周炎的易感性,并且在2型糖尿病和牙周炎协同作用过程中具有重要作用。     

2型糖尿病伴牙周炎患者牙周炎症程度与血糖水平分析

目的:分析2型糖尿病伴牙周炎患者牙周炎症程度对血糖水平的影响。方法:将213例2型糖尿病伴慢性牙周炎患者,根据根据牙周探诊深度(Probing depth,PD)和牙周附着丧失(Attach ment Loss,AL)的程度分为2组:2型糖尿病伴轻度慢性牙周炎组、2型糖尿病伴中重度慢性牙周炎组,分别测定血清h s-CRP、FPG水平并比较。结果:2型糖尿病伴中重度慢性牙周炎组较2型糖尿病伴轻度慢性牙周炎组血清FPG、hs-CRP水平升高,两组间FPG(t=7.144,P0.001)、h s-CRP(t=13.493,P0.001)差异具有统计学意义,而两组间年龄(t=-0.369,P=0.712)、性别(P=0.819)、饮酒(P=0.697)、吸烟(P=0.223)差异无统计学意义,偏相关分析发现PD与FPG(P0.001)、h s-CRP(P0.001)呈正相关。结论:牙周炎症可能会使2型糖尿病患者FPG、h s-CRP水平升高。     

2型糖尿病伴牙周炎糖化血红蛋白与 C-反应蛋白的关系

检测89例2型糖尿病伴慢性牙周炎患者血清超敏C-反应蛋白(hs-CRP)、糖化血红蛋白(Hb A1c)水平,并按Hb A1c水平分为≥7.0%组及<7.0%组。Hb A1c≥7.0%组的2型糖尿病伴慢性牙周炎患者hs-CRP及探诊深度(PD)、附着丧失(AL)均显著高于<7.0%组(P<0.01),2型糖尿病伴慢性牙周炎患者hs-CRP与牙周炎的严重程度呈显著正相关。     

Diabetes modulates gene expression in the gingival tissues of patients with chronic periodontitis

AIM: This study evaluated whether diabetes modulates gene expression [interleukin (IL)-1beta, IL-1ra, IL-6, IL-8, IL-10; tumor necrosis factor (TNF)-alpha; interferon (IFN)-gamma, receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG)] in sites with periodontitis. MATERIALS AND METHODS: Gingival biopsies were harvested and divided into three groups--Control group: systemically and periodontally healthy subjects (n = 10); Periodontitis group: systemically healthy subjects diagnosed with chronic periodontitis (n = 20); Diabetes group: type 1 diabetic subjects, diagnosed with chronic periodontitis (n = 20). Total RNA was obtained and analyzed by quantitative polymerase chain reaction. RESULTS: Data analysis demonstrated that, except for OPG, mRNA levels for all factors were increased by inflammation (P < 0.001). Interleukin-1beta, IL-1ra, IL-6, IL-8, IFN-gamma, and RANKL mRNA levels were higher in the diabetic group when compared with the control non-periodontitis group (P < 0.05), whereas IL-10 and OPG were lower (P < 0.05). No difference was observed for TNF-alpha between diabetic and control groups (P > 0.05). Diabetes lowered IL-1beta, IL-8, IL-10, TNF-alpha, RANKL, and OPG mRNA levels in sites with comparable type of periodontitis (P < 0.001). Moreover, increased RANKL:OPG and IL-6:IL-10 ratios were found. CONCLUSION AND CLINICAL RELEVANCE: Taken together, these data suggest that decreased levels of IL-10 and OPG may play an important role in the periodontal breakdown in diabetic patients.     

Markers of bone destruction and formation and periodontitis in type 1 diabetes mellitus

Aim: To determine plasma concentrations of bone metabolism markers in type 1 diabetes mellitus patients and non-diabetic and to evaluate the influence of periodontitis on biomarkers of bone formation in these patient groups.
Methods: Plasma concentrations of receptor activator of nuclear factor- κ B ligand (RANKL), osteoprotegerin (OPG), C-terminal telopeptide of type 1 collagen and osteocalcin were measured in type 1 diabetes mellitus patients ( n =63) and non-diabetics ( n =38) who were also subdivided on the basis of their periodontal status.
Results: Diabetics had significantly lower osteocalcin concentrations, lower RANKL to OPG ratios and higher OPG concentrations (as shown by other researchers) than non-diabetics. The ratio of RANKL to OPG was altered by the periodontal status. Osteocalcin had a negative correlation and OPG a positive correlation with the percentage of glycated haemoglobin in the blood.
Conclusion: Because, osteocalcin, a biomarker of bone formation, is lower in patients with periodontitis and in patients with type 1 diabetes mellitus with and without periodontitis than in non-diabetics without periodontitis, this might indicate that diabetics are less able to replace bone lost during active bursts of periodontitis and explain the greater severity of disease seen in studies of patients with diabetes.     

Gingival crevicular fluid levels of RANKL and OPG in periodontal diseases: implications of their relative ratio

AIM: Receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) are a system of molecules that regulate bone resorption. This study aims to compare the levels of RANKL, OPG and their relative ratio in gingival crevicular fluid (GCF) of healthy and periodontal disease subjects. MATERIAL AND METHODS: GCF was obtained from healthy (n=21), gingivitis (n=22), chronic periodontitis (n=28), generalized aggressive periodontitis (n=25) and chronic periodontitis subjects under immunosuppressant therapy (n=11). RANKL and OPG concentrations in GCF were measured by enzyme-linked immunosorbent assays. RESULTS: RANKL levels were low in health and gingivitis groups, but increased in all three forms of periodontitis. OPG levels were higher in health than all three periodontitis, or gingivitis groups. There were no differences in RANKL and OPG levels between chronic and generalized aggressive periodontitis groups, whereas these were lower in the immunosuppressed chronic periodontitis group. The RANKL/OPG ratio was significantly elevated in all three periodontitis forms, compared with health or gingivitis, and positively correlated to probing pocket depth and clinical attachment level. CONCLUSION: GCF RANKL and OPG levels were oppositely regulated in periodontitis, but not gingivitis, resulting in an enhanced RANKL/OPG ratio. This ratio was similar in all three periodontitis groups and may therefore predict disease occurrence.     

Overexpression of interleukin-1beta and interleukin-6 may play an important role in periodontal breakdown in type 2 diabetic patients

BACKGROUND AND OBJECTIVE: This study evaluated whether the biochemical changes associated with type 2 diabetes modulate the expression of interleukin-1beta, interleukin-6, interleukin-8, and interferon-gamma in sites with chronic periodontitis. MATERIAL AND METHODS: Biopsies were harvested and divided into three groups: group 1, systemically and periodontally healthy subjects (n = 10); group 2, systemically healthy subjects with moderate-to-severe chronic periodontitis (probing depth > 6 mm) (n = 20); and group 3, type 2 diabetic subjects with periodontitis (n = 20). Cytokine levels were assessed in the gingival tissues by enzyme-linked immunosorbent assay analysis. RESULTS: Data analysis demonstrated that the interleukin-1beta, interleukin-6, interleukin-8, and interferon-gamma levels were higher in the presence of periodontal inflammation than in the absence of inflammation, regardless of systemic status. The interleukin-1beta and interleukin-6 levels were higher in diabetic subjects (group 3) than in systemically healthy patients with comparable types of periodontitis (group 2). No difference was observed for the interleukin-8 and interferon-gamma levels between groups 2 and 3. CONCLUSION: Within the limits of this study, it was concluded that type 2 diabetes was associated with increased expression of interleukin-1beta and interleukin-6 in periodontally inflamed tissues of diabetic patients, relative to nondiabetic subjects, and that such overexpression may be involved in the mechanisms by which type 2 diabetes enhances periodontal destruction.     

Identification of the osteoprotegerin/receptor activator of nuclear factor-kappa B ligand system in gingival crevicular fluid and tissue of patients with chronic periodontitis

BACKGROUND AND OBJECTIVE: Recent findings have suggested that osteoclastogenesis is directly regulated by receptor activator of nuclear factor-kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG). However, no studies have described interactions of OPG/RANKL and the gp130 cytokine family in periodontal disease. This study aimed to identify and quantify OPG/RANKL in the gingival crevicular fluid (GCF) and connective tissue of patients with periodontitis, and to clarify possible correlations with disease severity and interleukin-6 (IL-6) cytokines. MATERIAL AND METHODS: Ninety-five sites in 20 patients with generalized chronic periodontitis were divided into four groups by site based on probing depth (PD) and bleeding on probing (BOP). In periodontitis patients, GCF was obtained using sterile paper strips from clinically healthy sites (PD 6 mm with BOP, n = 27). Fourteen clinically healthy sites from four periodontally healthy individuals were used as the control group. The levels of OPG, RANKL and two gp130 cytokines - IL-6 and oncostatin M (OSM) - in the GCF were determined by an enzyme-linked immunosorbent assay (ELISA) and are expressed as total amounts (pg/site). Immunohistochemical localization of OPG- and RANKL-positive cells was also performed on gingival connective tissues harvested from patients with periodontitis (inflammatory group, n = 8 biopsies) and from non-diseased individuals (healthy group, n = 8 biopsies). RESULTS: GCF RANKL, but not OPG, was elevated in diseased sites of patients with periodontitis. However, the expressions of OPG and RANKL showed no correlation with disease severity (r = 0.174 and 0.056, respectively), but the content of RANKL in the GCF was significantly positively correlated with those of IL-6 (r = 0.207) and OSM (r = 0.231) (p < 0.01). Immunohistochemical staining showed that RANKL-positive cells were significantly distributed in the inflammatory connective tissue zone of diseased gingiva, compared with those of samples from non-diseased persons (p < 0.01). However, few OPG-positive cells were found in connective tissue zones of either the diseased gingiva or healthy biopsies. CONCLUSION: These findings imply that in this cross-sectional study of GCF, RANKL, IL-6 and OSM were all prominent in periodontitis sites, whereas OPG was inconsistently found in a few samples of diseased sites but was undetectable in any of the control sites. The results also imply that the expression of RANKL was positively correlated with IL-6 and OSM in the GCF.     

人健康和炎性牙槽骨成骨细胞COX-2、PGE2、OPG,RANKL的表达

目的：初步探讨COX-2、PGE2、OPG和RANKL在牙周炎发病机制中所起的作用及其相互关系。方法：采用组织块法对人健康和牙周炎性牙槽骨进行体外培养和传代,加入含100μg/LrhOPG的不含胎牛血清的DMEM2mL。严格按照ELISA试剂盒说明进行操作,获得各指标的检测数值。结果：牙周炎组RANKL、PGE2、COX-2的表达较健康组明显增高,OPG的表达较健康组明显降低。加入rhOPG后,牙周炎组RANKL、PGE2、COX-2的表达明显降低;OPG表达明显增加。健康组RANKL、PGE2、COX-2表达明显降低;OPG表达增加。结论：RANKL、PGE2、COX-2可促进牙周炎的发生、发展,而OPG对牙周炎的发生、发展可起抑制作用,同时表明人工重组OPG可能协同其内源性OPG来共同抑制RANKL、PGE2、COX-2的活性。     

Ⅱ型糖尿病伴牙周炎病人牙龈组织中VEGF表达及MVD计数的研究

目的:探讨Ⅱ型糖尿病伴牙周炎病人牙龈组织中血管内皮生长因子(VEGF)的表达水平及其作用。方法:选取Ⅱ型糖尿病伴重度牙周炎(DP)病人、单纯重度慢性牙周炎(CP)病人、健康对照者(N)各15例,分别切取牙龈组织,用免疫组化染色方法检测牙龈组织中VEGF表达和MVD计数。结果:DP组牙龈组织中VEGF表达和MVD计数均显著高于CP组和N组(P<0.05);CP组高于N组(P<0.05)。结论:Ⅱ型糖尿病伴重度慢性牙周炎病人牙龈组织中VEGF表达水平和MVD计数明显升高。