程序性死亡受体1及其配体在大鼠牙周炎发展中的时序性表达及意义

目的 研究程序性死亡受体1（programmed death-1,PD-1）及其配体（programmed death ligand 1,PD-L1）在大鼠牙周炎发展中的时序性表达及意义。方法 SD大鼠随机分为对照组和模型组,按照测定时间不同随机分为4个亚组,分别为A组（造模1周）、B组（造模2周）、C组（造模3周）和D组（造模4周）,每个亚组各8只大鼠。对各模型组大鼠采用“丝线结扎+接种牙龈卟啉单胞菌脂多糖”的方法建立大鼠上颌实验性牙周炎模型,对各组大鼠牙周进行组织病理检查和骨吸收面积测定,采用RT-PCR法对各组大鼠牙周组织中肿瘤坏死因子α（tumor necrosis factor alpha, TNF-α）、白细胞介素1β（interleukin-1, IL-1β）、白细胞介素6（interleukin 6, IL-6）及转化生长因子β（transforming growth factor beta, TGF-β）mRNA进行测定,采用Western免疫印迹法对各组大鼠牙周组织PD-1和PD-L1蛋白表达水平进行测定。采用SPSS 22.0 软件包对数据进行统计学分析。结果 建模期间,模型组大鼠第一磨牙釉-牙骨质界到牙槽嵴顶(amelocemental junction-alveolar crest, ACJ-AC)距离和根分叉区骨吸收面积逐渐增加（P<0.05）,与相应时间点对照组相比,差异有统计学意义（P<0.01）。建模期间,模型组大鼠牙周组织中TNF-α、IL-1β和IL-6 mRNA表达水平持续升高（P<0.05）,TGF-β mRNA表达水平持续降低（P<0.05）,并且与相应时间点对照组相比,差异有统计学意义（P<0.01）。建模期间,模型组大鼠牙周组织中PD-1和PD-L1蛋白表达水平持续升高（P<0.05）,与相应时间点对照组相比,差异显著（P<0.01）。疾病发展过程中,大鼠牙周组织中PD-1和PD-L1蛋白表达水平与牙周组织炎症介质TNF-α和IL-6 mRNA表达水平持续正相关（P<0.05）。结论 PD-1作为免疫抑制分子与其受体PD-L1可促进牙周炎症进展,其作用可能通过调节TNF-α和IL-6的表达来实现。调节PD-1和PD-L1的表达,可作为治疗牙周炎症相关性疾病的新靶点。     

雌激素受体和孕激素受体在女性牙周炎患者牙龈组织中的表达

目的观察比较女性牙周炎患者及正常女性牙龈组织雌激素(ER)和孕激素(PR)的表达分布,为进一步阐明ER和PR与女性牙周炎的关系提供实验依据。方法收集正常对照组牙龈标本30例,中、重度牙周炎组34例,按年龄各自分成3组,标本行免疫组化处理(Envision法),显微镜下观察,统计分析。结果ER和PR在细胞胞浆及胞核中均有表达,无规律地分布于牙龈上皮层中,阳性样本的染色强度强弱不一。正常女性牙龈组织中ER及PR阳性率较低,女性牙周炎患者牙龈组织中ER阳性率明显升高(P<0.01),PR阳性率无明显改变(P>0.05),与年龄无明显关系。结论女性牙龈组织中ER的表达可能与牙周炎相关,雌激素对牙龈组织除了起间接作用外,还可通过ER直接影响牙周组织;而孕激素对牙周组织可能以间接影响为主。     

受体相互作用蛋白激酶1在小鼠实验性牙周炎模型中的作用

目的:研究受体相互作用蛋白激酶1(RIP1)在小鼠实验性牙周炎模型中的作用和可能机制.方法:提取牙周炎患者及牙周健康者的牙龈组织,Western blot检测RIP1蛋白的表达.在RIP1条件性基因敲除(RIP1-/-)小鼠和C57野生型小鼠上颌第二磨牙建立牙周炎模型,用Micro-CT扫描成像分析对比两组小鼠实验侧牙...     

辛伐他汀对牙周炎大鼠正畸牙移动保持阶段MMP-1表达影响的研究

目的 在牙周炎大鼠正畸牙移动后保持阶段,观察辛伐他汀对牙周组织中MMP-1表达的影响.方法 55只4周龄牙周炎大鼠分为11组,每组5只,近中移动双侧上颌第一磨牙21天后,分为空白对照组(1组,加力后直接处死),阴性对照组(5组),实验组(5组).阴性对照组和实验组内各小组分别保持1、3、7、14、21天,阴性对照组于保持前一天开始每天腹腔注射生理盐水,实验组每天注射辛伐他汀.免疫组化染色定量检测大鼠第一磨牙牙周组织中的MMP-1的表达.结果 各组近中侧牙周组织中MMP-1的表达量,早期呈逐渐增加的趋势,7天后逐渐减少.各组远中侧未保持者表达量在加力结束7天后逐渐增加,14天后开始减少.在同一时间点,实验组MMP-1表达量明显低于对照组.结论 牙周炎大鼠正畸保持阶段,全身给予辛伐他汀能降低炎症反应,抑制牙周膜纤维的降解,可能缩短正畸牙移动后的保持时间.     

细胞间粘附分子1及其受体在成人牙周炎袋上皮区的表达

目的 探讨成人牙周炎牙龈组织中白细胞通过上皮进入龈沟液的粘附机制。方法 采用碱性磷酸酶－抗碱性磷酸酶免疫组化方法,对细胞间粘附分子／白细胞功能相关抗原１在正常牙龈和成人牙周炎牙龈组织中的表达进行研究。结果 正常牙龈和成人牙周炎牙龈的结合上皮,沟内／袋上皮根部均表达ＩＣＡＭ－１,且染色强度呈方向变化;ＬＦＡ－１仅局限表达于白细胞表面,成人牙周炎袋上皮下方结缔组织及袋上皮细胞间隙中ＬＦＡ－１阳性白细胞     

大鼠实验性牙周炎治疗前后血清中IL-18与sTREM-1的浓度变化

目的:观察大鼠实验性牙周炎基础治疗前后血清中IL-18与sTREM-1水平的变化,进而探讨IL-18与sTREM-1水平变化与牙周炎治疗效果间的关系。方法:选取2个月大的健康雄性Wistar大鼠30只,同等条件下饲养,随机分为3组,每组10只。正常对照组(A组)不做其他处理;牙周炎组(B组)与牙周炎治组(C组),将消毒尼龙丝线放置在双侧上颌第二磨牙牙颈部,同时配合牙周炎食谱诱导大鼠牙周炎模型。建模成功后C组去除丝线进行基础治疗,2周后对大鼠进行腹主动脉取血并处死,采用酶联免疫吸附法(ELISA)测定大鼠血清中IL-18和sTREM-1水平。结果:通过HE染色观察各组牙周组织学改变,牙周炎组(B组)大鼠血清中IL-18及sTREM-1水平高于正常对照组(A组)和牙周炎治疗组(C组)(P<0.05),且牙周炎治疗组大鼠血清中IL-18及sTREM-1水平高于对照组(P<0.05)。结论:牙周炎时IL-18、sTREM-1水平明显增高,且经基础治疗后明显降低。表明IL-18、sTREM-1水平可以为牙周炎的诊断和预测提供指导,为临床工作者提供准确有效的信息,同时也为临床治疗提供新的治疗靶点。     

TNF-α、sICAM-1在慢性间歇低氧条件下大鼠牙周炎发病机制中的作用

目的：探讨慢性间歇低氧和常氧条件下大鼠血清和牙龈组织中肿瘤坏死因子-α（TNF-α）、可溶性细胞间黏附分子-1（sICAM-1）在牙周炎发病机制中的作用。方法：将32只雄性 SD 大鼠随机分为4组（n ＝8）：常氧对照组（A 组）、常氧牙周炎组（B 组）、低氧对照组（C 组）、低氧牙周炎组（D 组）。采用正畸丝结扎双侧上颌第二磨牙和牙周炎食谱的方法建立牙周炎模型,常氧组和低氧组分别在常氧和模拟阻塞性呼吸暂停低通气综合征条件下饲养8周,检测各组动物牙周组织各项临床指标,用 ELISA 法检测动物血清和牙龈组织中 TNF-α、sICAM-1表达。结果：低氧牙周炎组中 TNF-α、sICAM-1浓度显著高于其余各组 P ＜0．05。血清和牙龈组织中 TNF-α、sICAM-1与附着丧失（AL）成正相关（P ＜0．05）。结论：慢性间歇低氧条件下 TNF-α、sICAM-1在牙周组织中的表达水平升高,加重了牙周炎症破坏程度,同时促使炎症反应向外周血管扩散。     

CRP、ICAM-1、E-selectins在实验性牙周炎大鼠牙周组织中的表达及意义

目的:建立实验性大鼠牙周炎模型,检测C反应蛋白(C reaction protein,CRP)、细胞间粘附分子-1(intercellular adhersion molecule,ICAM-1)、E-选择素(E-selectins)在大鼠牙周组织中的表达及血清中的水平,探讨CRP、ICAM-1、E-selectins在牙周炎发展中的变化。方法:选取纯种6月龄雄性SD大鼠40只,随机分为4组,正常组(A组),4周牙周炎组(B组),8周牙周炎组(C组),12周牙周炎组(D组),每组10只。建立实验性牙周炎动物模型,按计划时间处死实验大鼠,分别检测牙周组织、血清中CRP、ICAM-1、E-selectins的变化。结果:实验性牙周炎随着时间的延长,牙周破坏程度的加深,牙周组织中和血清中的CRP、ICAM-1、E-selectins不断升高,可能进一步加重牙周组织的炎症。结论:CRP、ICAM-1、E-selectins作为牙周炎和心脑血管疾病的共同危险细胞因子,不仅随着牙周炎症的加重而变化,而且能够作为牙周炎导致心脑血管疾病危险因素的证据。     

OPG、RANKL在2型糖尿病伴牙周炎大鼠牙槽骨中的表达

目的:探讨骨保护素(OPG)和核因子κB受体活化因子(RANKL)在2型糖尿病伴牙周炎大鼠模型中的表达水平.方法:46只Wistar雄性大鼠,随机分为健康组10只;单纯牙周炎组12只;2型糖尿病组12只;2型糖尿病牙周炎组12只;分别建模,免疫组织化学方法检测牙槽骨OPG、RANKL蛋白表达.结果:与健康组相比,OPG在2型糖尿病组、单纯牙周炎组、2型糖尿病伴牙周炎组表达水平依次降低,RANKL的表达水平依次增强;OPG及RANKL的表达除2型糖尿病牙周炎组与单纯牙周炎组间比较无差异外,其余组间比较有统计学意义(P＜0.05).结论:炎症可能导致破骨细胞及免疫细胞中RANKL的上调和成骨细胞中OPG的下调.     

Eosinophil cationic protein and histamine production by neutrophils from patients with periodontitis

1 Background

Periodontitis develops through an inflammatory process caused by an infection at the microbial biofilm, followed by tissue destruction mediated by leukocytes, which cause clinically significant destruction of connective tissue and bone. Several elements derived from the bacteria cause the inflammatory response and the release of mediators involved in destruction of the periodontium. There are number of inflammatory mediators released by leukocytes, mainly neutrophils, upon bacterial challenge. Neutrophils produce and release eosinophil cationic protein (ECP) and histamine, two important inflammatory mediators; however, their role has not been characterized in periodontal inflammation. Thus, the purpose of this study is to investigate whether neutrophils from patients with periodontitis can produce ECP and histamine in response to lipopolysaccharides (LPSs).

2 Methods

ECP and histamine production in response to LPSs was analyzed by enzyme‐linked immunosorbent assay. Expression of the histidine decarboxylase and ECP was also analyzed by flow cytometry and fluorescence microscopy in neutrophils from patients with periodontitis in response to LPS.

3 Results

It was found that neutrophils from patients with periodontitis express higher levels of histidine decarboxylase and ECP than those from healthy volunteers, and they also release higher levels of histamine.

4 Conclusion

Findings described could represent new knowledge indicating neutrophils as a source of histamine and ECP in the progression of periodontitis.     

Association of interleukin-1 receptor antagonist gene polymorphisms with early onset periodontitis in Japanese

BACKGROUND/AIMS: Early onset periodontitis (EOP), newly 'aggressive periodontitis', is considered to have genetic basis, which have not been clearly defined. The interleukin-1 (IL-1) gene cluster polymorphism as one of genetic factors may influence the expression of several chronic inflammatory diseases. The aim of this study is to investigate the frequency of single nucleotide polymorphisms (SNPs) in the genes encoding IL-1alpha, IL-1beta and a variable number of tandem repeat (VNTR) polymorphisms in the IL-1 receptor antagonist gene (IL-1RN) in 47 generalized EOP (G-EOP) patients and 97 periodontally healthy controls. MATERIAL AND METHODS: All subjects were of Japanese descent and systemically healthy. They were identified according to established clinical criteria. SNPs in the IL-1alpha (+ 4845) and IL-1beta (- 511, + 3954) genes were analyzed by amplifying the polymorphic region using polymerase chain reaction (PCR), followed by restriction-enzyme digestion and agarose gel electrophoresis. IL-1RN (VNTR) polymorphisms were then detected by PCR amplification and fragment size analysis. RESULTS: There was no significant difference in the IL-alpha (+ 4845) and IL-1beta (- 511, + 3954) genotypes and allele frequencies between G-EOP patients and healthy controls. However, the frequency of IL-1RN (VNTR) polymorphic alleles was found to be significantly increased in G-EOP patients (chi2 test, P = 0.007; odds ratio = 3.40). Additionally, the carriage rate of IL-1RN (VNTR) polymorphisms was significantly higher in G-EOP patients than in healthy controls (chi2 test, P = 0.005; odds ratio = 3.81). CONCLUSION: These findings suggest that IL-1RN (VNTR) polymorphisms are associated with G-EOP in Japanese.     

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髓样细胞触发受体-1（triggering receptor expressed on myeloid cells-1,TREM-1）是新近发现的免疫球蛋白超家族的一种跨膜受体,表达于单核（巨噬）细胞等骨髓分化细胞,在疾病作用中研究现仍处于起步阶段。多项研究显示,TREM-1在感染性疾病的炎症触发和级联放大过程中扮演重要角色,而牙周炎是由牙茵斑生物膜引起的感染性疾病,TREM-1在其引起的免疫炎症反应中可能发挥重要作用。本文就TREM-1最新研究进展及其与牙周炎的关系进行综述。     

Toll样受体4在大鼠实验性牙髓炎和根尖周炎中的表达

目的:建立大鼠实验性牙髓炎及根尖周炎模型,了解TLR4在大鼠牙髓和根尖周组织的表达情况。方法:采用单纯髓腔开放法建立大鼠实验性牙髓炎及根尖周炎模型,进行组织学及X线片观察。免疫组化检测TLR4在牙髓炎中的表达,RT-PCR检测TLR4在根尖周炎中的表达。结果:组织学及X线观察证实成功复制大鼠牙髓炎及根尖周炎模型,免疫组化结果表明大鼠正常牙髓组织成牙本质细胞层TLR4阳性表达,牙髓内部血管内皮细胞亦见表达,牙髓成纤维细胞未见表达。炎症牙髓组织TLR4表达较正常组增强。RT-PCR检测发现正常和炎症根尖周组织均有TLR4mRNA表达,炎症组表达较正常组增强。结论:TLR4在牙髓组织和根尖周组织均有表达并参与了牙髓根尖周炎症的发生和发展。     

侵袭性牙周炎及慢性牙周炎患者血清Dickkopf-1水平的影响因素研究

目的 观察牙周炎与血清Dickkopf-1(DKK-1)水平的关系,探讨影响血清DKK-1水平的可能因素.方法 收集侵袭性牙周炎(aggressive periodontitis,AgP)、慢性牙周炎(chronic periodontitis,CP)患者各20例(分别为AgP组、CP组)及分别与AgP、CP患者年龄匹配的健康对照者各20名(分别为H1组、H2组),采用酶联免疫吸附测定法检测血清DKK-1水平.采用t检验比较各组血清中DKK-1水平的差异,并采用相关分析和多元线性回归方法分析血清DKK-1水平与牙周临床参数及年龄等因素的关系,检测水准为双侧α =0.05.结果 AgP组血清DKK-1水平[(12.36±3.19) μg/L]显著高于CP组[(8.90 ±2.73) μg/L] (P=0.001),但与其对照组H1组[(12.37±2.74) μg/L]相比差异无统计学意义(P=0.99),CP组与H2组[(8.85 ±2.56) μg/L]相比差异也无统计学意义(P =0.896);AgP与CP组血清DKK-1水平与牙周探诊深度、附着丧失、出血指数、探诊出血阳性位点百分比间均未见显著相关(AgP组r值分别为-0.029、-0.223、0.062、-0.412;CP组r值分别为-0.156、0.185、-0.379、0.051);总体样本血清DKK-1水平与年龄呈显著负相关(r=-0.453,P =0.000);多元线性回归分析显示年龄对血清DKK-1水平有影响(β=-0.391,t=-3.626,P=0.001).结论 未发现牙周炎对血清DKK-1水平产生影响;年龄是影响血清DKK-1水平的重要因素,血清DKK-1水平随年龄增加而降低.     

Etanercept作用下糖尿病Wistar大鼠牙周炎变化的实验研究

[摘要] 目的 观察肿瘤坏死因子α抑制剂Etanercept对实验性糖尿病Wistar大鼠牙周炎的影响。方法 12周龄Wistar大鼠45只,腹腔内一次性注射STZ诱导糖尿病。结扎糖尿病大鼠牙颈部建立牙周炎模型。将实验糖尿病大鼠分为糖尿病组,糖尿病牙周炎组和糖尿病牙周炎+Etanercept组。观察大鼠的牙龈指数、牙周袋深度、松动度。免疫组化观察IL-1β、IL-6在牙周组织中的表达。结果 糖尿病组大鼠牙龈指数、牙周袋深度、松动度与糖尿病牙周炎组和糖尿病牙周炎+Etanercept组有差别(P<0.05);糖尿病牙周炎组和糖尿病牙周炎+Etanercept组相比有差别(P<0.05)。糖尿病牙周炎组中IL-1β、IL-6表达高于糖尿病组(P<0.05),各时间段糖尿病牙周炎+Etanercept组牙周组织中IL-1β、IL-6表达低于糖尿病牙周炎组(P<0.05)。结论 肿瘤坏死因子α抑制剂Etanercept对实验性糖尿病Wistar大鼠牙周炎有抑制作用。     

Expression of transient receptor potential vanilloid receptor 1 and toll-like receptor 4 in aggressive periodontitis and in chronic periodontitis

Öztürk A, Y?ld?z L. Expression of transient receptor potential vanilloid receptor 1 and toll‐like receptor‐4 in aggressive periodontitis and in chronic periodontitis. J Periodont Res 2011; 46: 475–482. © 2011 John Wiley & Sons A/S Background and Objective: The objective of the present study was to evaluate the expression and the distribution of the transient receptor potential vanilloid receptor 1 (TRPV1) and of toll‐like receptor 4 (TLR4) in tissue samples from patients with periodontal disease (aggressive periodontitis and chronic periodontitis) and from healthy controls. Material and Methods: Ten tissue samples from each disease group (aggressive periodontitis and chronic periodontitis) and from healthy subjects were obtained during routine oral surgical procedures. Subgingival specimens were collected from sites with advanced loss of support (probing depth > 5 mm) and specimens from the corresponding healthy controls were obtained during tooth extraction for orthodontic reasons or following surgical extraction of an impacted third molar. The distribution of TRPV1 and TLR4 receptors in human gingival tissue was studied by immunohistochemistry. Results: Both TLR4 and TRPV1 were detected in gingival tissues from healthy subjects, and from patients with chronic periodontitis and aggressive periodontitis, particularly in gingival keratinocytes, fibroblasts, inflammatory cells and the endothelial lining of capillaries in connective tissues. Histologic examination of the samples from healthy controls disclosed that clinically healthy gingiva does not correspond to histologically healthy gingiva. Subsequently, these samples were redesignated as gingivitis samples. TRPV1 was down‐regulated in all cell types in samples obtained from patients with chronic periodontitis compared to samples obtained from patients with gingivitis, whereas TLR4 was down‐regulated only in the epithelium and in gingival fibroblasts. In contrast, the levels of these markers in patients with aggressive periodontitis were similar to those in healthy patients. Conclusion: Local expression of TRPV1 and TLR4 in gingival tissues may contribute to both physiological and pathological processes in the periodontium. Our data suggest that TRPV1 and TLR4 may play a role specifically in the pathophysiology of chronic periodontitis.     

Oral treatment with complement factor C5a receptor (CD88) antagonists inhibits experimental periodontitis in rats

Breivik T, Gundersen Y, Gjermo P, Taylor SM, Woodruff TM, Opstad PK. Oral treatment with complement factor C5a receptor (CD88) antagonists inhibits experimental periodontitis in rats. J Periodont Res 2011; 46: 643–647. © 2011 John Wiley & Sons A/S Background and Objective: The complement activation product 5a (C5a) is a potent mediator of the innate immune response to infection, and may thus also importantly determine the development of periodontitis. The present study was designed to explore the effect of several novel, potent and orally active C5a receptor (CD88) antagonists (C5aRAs) on the development of ligature‐induced periodontitis in an animal model. Material and Methods: Three different cyclic peptide C5aRAs, termed PMX205, PMX218 and PMX273, were investigated. Four groups of Wistar rats (n = 10 in each group) were used. Starting 3 d before induction of experimental periodontitis, rats either received one of the C5aRas (1–2 mg/kg) in the drinking water or received drinking water only. Periodontitis was assessed when the ligatures had been in place for 14 d. Results: Compared with control rats, PMX205‐ and PMX218‐treated rats had significantly reduced periodontal bone loss. Conclusion: The findings suggest that complement activation, and particularly C5a generation, may play a significant role in the development and progression of periodontitis. Blockade of the major C5a receptor, CD88, with specific inhibitors such as PMX205, may offer novel treatment options for periodontitis.     

Association of interleukin-1 gene polymorphisms with early-onset periodontitis

Background, aims: Early onset periodontal diseases (EOP) are a group of inflammatory disorders characterised by a rapid rate of periodontal tissue destruction, in young individuals who are otherwise healthy. There is now substantial evidence to suggest that genetic factors play a rôle in the pathogenesis of EOP but the precise nature of these factors remains unclear. Polymorphisms in cytokine genes which may underpin inter‐individual differences in cytokine synthesis and secretion have been associated with other diseases which have an inflammatory pathogenesis, including chronic adult periodontal disease (CAPD). Method: We therefore investigated the frequency of polymorphisms in the genes encoding interleukin‐1β (IL‐1β) and its receptor antagonist (IL‐1RA) in 70 EOP patients, including a subgroup of 21 localised EOP (L‐EOP) patients and 72 periodontally healthy controls. All subjects were of Caucasian heritage and systemically healthy. A single nucleotide polymorphism (SNP) in exon 5 of the IL‐1β gene (IL‐1β⁺³⁹⁵³) was analysed by amplifying the polymorphic region using PCR, followed by restriction digestion with Taq1 and gel electrophoresis. Results: The frequency of IL‐1β genotypes homozygous for allele 1 (corresponding to the presence of a restriction site) of the IL‐1β⁺³⁹⁵³ SNP was found to be significantly increased in EOP patients (χ² test, p=0.025). Upon stratification for smoking status a significant difference was found in the IL‐1β genotype distribution between EOP smokers compared to control smokers (F‐exact test, p=0.02), but not between EOP non‐smokers and control non‐smokers. The IL‐1β 1/1 genotype occurred at a higher frequency in EOP smokers (odds ratio=4.9) compared to control smokers. A variable number tandem repeat polymorphism (VNTR) in intron 2 of the IL‐1RA gene was analysed by amplifying the polymorphic region using PCR and fragment size analysis by gel electrophoresis. There was no evidence for an association of an IL‐1RA genotype with EOP. However the combination of IL‐1β allele 1 and IL‐1RA allele 1 (corresponding to 4 repeats) was associated with EOP (Clump, p=0.01). Conclusions: These findings suggest that an IL‐1β genotype in combination with smoking, and a combined IL‐1β and IL‐1RA genotype are risk factors for EOP and support a role for genetic and environmental factors in susceptibility to EOP.