核酸探针杂交技术用于检测伴放线放线杆菌

伴放线放线杆菌（Ａ．ｃ）是青少年牙周炎的主要致病菌，Ａ．ａ的传统检测方法费时费力。本文从酸分子杂交的原理出发，就核酸探针的种类及其杂交、探针的标记、临床应用、评价及使用酸探针杂交技术检测Ａ．ａ的前景做了介绍。     

核酸探针杂交技术用于检测伴放线放线杆菌

伴放线放线杆菌(A.a)是青少年牙周炎的主要致病菌,A.a的传统检测方法费时费力。本文从核酸分子杂交的原理出发,就核酸探针的种类及其杂交、探针的标记、临床应用、评价及使用核酸探针杂交技术检测A.a的前景做了介绍。     

伴放线放线杆菌在龈下菌斑和颊黏膜分布研究

目的:比较伴放线放线杆菌(actinobac illus actinomycetem com itans,A.a)在不同类型牙周炎患者龈下菌斑和颊黏膜中的分布。方法:通过聚合酶链反应(polym erase chain reaction,PCR)对侵袭性牙周炎患者(AgP)、慢性牙周炎患者(CP)、牙周健康者口腔龈下菌斑和颊黏膜中的A.a进行检测,分析该菌分别在两部位的相对含量。结果:AgP组菌斑和颊黏膜样本中A.a阳性检出率均为41.7%,分别高于CP组(菌斑16.7%、颊黏膜10.0%)和牙周健康组(菌斑和颊黏膜均为0%)。AgP组A.a在菌斑和颊黏膜的相对含量分别为38.5%和22.2%,高于CP组(菌斑19%、颊黏膜12.75%)。结论:A.a不仅存在于龈下菌斑中,也能够粘附于颊黏膜;A.a是AgP的主要优势菌也参与了CP的菌群组成。     

伴放线放线杆菌全染色体DNA探针的制备和鉴定

本研究的目的是制备伴放线放线杆菌全染色DNA探针,并评价其特异性和敏感性。抽提标准菌株Aa Y4 DNA,经~(32)P标记后制成探针,然后用斑点杂交法鉴定探针性质。结果Aa全染色体DNA探针的灵敏性为8×10~2纯菌或1ng同源DNA,探针与口腔常见菌的杂交显示仅与嗜沫嗜血杆菌有较弱的交叉反应。提示~(32)P标记Aa全染色体DNA探针的敏感性、特异性均较高,且方便实用,有临床推广应用价值。     

DNA探针鉴定伴放线放线杆菌及其与牙周炎临床参数的关系

目的 评估ＤＮＡ探针鉴定伴放线放线杆菌（Ａａ）的价值,探讨Ａａ与牙周炎临床表现间的相关性。方法 ３２例成人牙周炎患者５９个部位龈下菌斑经Ａａ选择性培养后,菌落分别用ＤＮＡ探针法及生化法鉴定,并根据探针法的检出率探讨Ａａ感染与牙周炎临床参数间的关系。结果 ＤＮＡ探针法与培养法有一致性（Ｐ〈０．０１,ｒ＝０．７８但探针法的检出率（２６／５９）明显高于培养法（１９／５９）（Ｐ〈０．０５）;Ａａ的检出率与     

伴放线放线杆菌外膜蛋白的研究进展

伴放线放线杆菌与牙周炎,特别是与局限性侵袭性牙周炎有着密切的关系.伴放线放线杆菌外膜蛋白作为其重要毒力因子,在牙周病的发病中起着重要的作用.本文就近年来有关伴放线放线杆菌外膜蛋白的结构特征、外膜蛋白的表现型、外膜蛋白与血清型、外膜蛋白的致病性等研究进展作一综述.     

伴放线放线杆菌菌落生长形态变化的观察

目的：观察伴放线放线杆菌（Actinobacillus actinomycetemcomitans,Aa)从粗糙型到光滑型的转变过程，认别Aa在实验室传代过程中出现的不同生长形态。方法：从牙周炎患者龈下菌班中分离出的原代菌株8株，应用固体及液体培养基连续传代，液体培养每次传代的同时接种固体培养基观察相应的菌落形态。结果：液体培养获得3株光滑型转变株。菌落的变化从粘附的小菌落到沉淀的大菌落到完全的均匀生长，转化过程大约需要7－8代。在这一过程中相应传至固体培养基上生长的Aa从粘附的半透明的小菌落变大、不透明并失去粘附的特性，又随着边缘的扩散变为扁平，透明度也增加；与此同进内部的星形结构逐渐变简单、变小，最后消失。固体培养未获得典型的转变株。结论：Aa从粗糙型到光滑型的转变是一个菌落湿度逐渐增加，体积逐渐增大，并逐渐失去内部结构的过程。这一过程至少可以看到半透明突起的粗糙型，不透明突起的光滑型和近乎透明的扁平光滑型3种菌落形态。     

伴放线放线杆菌高毒株与局限性青少年牙周炎相关性研究进展

伴放线放线杆菌与局限性青少年牙周炎的关系密切。该菌的多种毒力因子中，白细胞毒素的研究最深入。菌株编码白细胞毒素的操纵子中启动子区的结构存在差异，从而影响产生白细胞毒素的量，形成致病力强的高毒株，并与不同人群中局限性青少年牙周炎的发病情况相关。本文就伴放线放线杆菌与局限性青少年牙周为的相关性进行综述。     

伴放线共生放线杆菌表面相关物质的骨吸收活性研究

目的：通过对伴放线共生放线杆菌（Aa)表面相关物质（SAM）的提取,纯化,研究其潜在的骨吸收活性,以揭示SAM在牙周病变过程中的作用。方法：Aa国际标准菌株培养,SAM提取,过Sepharyl-S200层析柱和DEAE－Sephacel层析柱,所有的管用酚硫法测其碳水化合物含量,纯化后鉴定其骨吸收活性.结果：冻干的Aa(1.0g)产生0．1375g粗提物,通过Sepharyl-S200时出现单峰,通过DEAE－Sephacel出现两个峰,骨吸收实验证明SAM有骨吸收活性。结论：Aa的SAM在骨吸收方面具有极大潜能,提示SAM在牙周病骨损害上起到重要作用。     

Quantitative aspects of the subgingival distribution of Actinobacillus actinomycetemcomitans in a patient with localized juvenile periodontitis

Abstract The subgingival microflora in a patient with localized juvenile periodontitis was studied. Of the 97 sites investigated, 28 (29%) showed attachment loss. A correlation was found between the number of Actinobacillus actinomycetemcomitans cells and the clinical attachment level and probing pocket depth. Of the 97 test sites, 70 (73%) were positive for A. actinomycetemcomitans. Of the total number of A. actinomycetemcomitans cells isolated from this patient, more than 99% were found at sites with attachment loss, <1 % being present at sites without attachment loss. The mean percentage of A. actinomycetemcomitans was 21.2% at sites with attachment loss and 0.45% at sites without attachment loss. The distribution of Porphyromonas gingilis showed a symmetrical pattern, being present at the 1st molar and 2nd premolar sites in all quadrants and at the lower incisor sites. This species was absent at multiple sites showing overt attachment loss.     

Inhibition of Actinobacillus actinomycetemcomitans leukotoxicity by bacteria from the subgingival flora

Actinobacillus actinomycetemcomitans produces a pore-forming leukotoxin that lyses human polymorphonuclear leukocytes and monocytes. Certain proteolytic bacteria may coexist with A. actinomycetemcomitans in periodontal pockets. We aimed therefore to examine whether oral bacteria can modify the leukotoxicity of A. actinomycetemcomitans. A total of 55 strains representing 45 bacterial species of the subgingival flora were tested. Each strain was incubated with the highly toxic strain of A. actinomycetemcomitans HK 1519 and the leukotoxic activity of the suspension against human polymorphonuclear leukocytes was determined from the activity of the lactate dehydrogenase released upon lysis of the leukocytes. Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Prevotella melaninogenica and Prevotella loeschii inhibited the leukotoxicity of A. actinomycetemcomitans cells as well as the activity of leukotoxin purified from the same strain. The bacterial strains without the ability to block leukotoxic activity also failed to destroy pure leukotoxin even after 5 h of incubation. The proteolytic degradation of leukotoxin by P. gingivalis was mainly dependent on the activity of the enzymes R- and K-gingipains. P. intermedia and P. nigrescens also degraded the leukotoxin by enzymes. The results imply a role of the periodontal microflora in modifying the virulence of A. actinomycetemcomitans by destroying its leukotoxin.     

Effects of subgingival irrigation on A. actinomycetemcomitans

The effects of repeated subgingival irrigation on Actinobacillus actinomycetemcomitans was examined. 24 periodontal pockets harboring A. actinomycetemcomitans in 3 juvenile and 4 adult periodontitis patients were studied. The protocol included bi-weekly subgingival irrigation with hydrogen peroxide of the periodontal sites until the micro-organism was no longer detected by selective culture, or for 6 months. A. actinomycetemcomitans was gradually suppressed to below detection following the irrigation regime and could no longer be detected in 46% of the sites at completion of the irrigation protocol. The sites were microbiologically re-examined 5 months after cessation of the irrigation regime. A. actinomycetemcomitans re-occurred in only 2 of the sites from which it had originally been suppressed below detection. The results indicate: (1) that the irrigation regime tested has some potential to suppress A. actinomycetemcomitans in periodontal pockets; (2) that the effect of the irrigation protocol generally lasted for 5 months; (3) that the reduction rate of A. actinomycetemcomitans to below detectable levels seems related to the initial number of cultivable bacteria from the periodontal pocket.     

伴放线放线杆菌细胞膨胀致死毒素重组蛋白的表达

目的 通过分子克隆技术构建细胞膨胀致死毒素(cytolethal distending toxin,CDT)的重组表达载体,并诱导重组CDT蛋白的表达,以期为重组CDT蛋白的生物学功能研究奠定基础.方法 采用聚合酶链反应(PCR)扩增获得CDT的编码基因cdtABC,通过TA克隆和限制性酶切将cdtABC与目的载体pQE60连接,转化感受态大肠杆菌后诱导重组COT蛋白的表达,收集细菌总蛋白进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳和蛋白质印迹法鉴定.结果 pQE60-cdtABC转染的感受态大肠杆菌中均携带cdtABC基因,该片段与GenBank中的DNA一致性高达99%.细菌总蛋白中21 000、25 000、32 000左右的蛋白增多,蛋白质印迹法检测发现带有6-His标记的目的蛋白.结论 本研究成功构建了CDT的重组表达载体,并诱导重组CDT蛋白的表达.     

Simultaneous isolation of Actinobacillus actinomycetemcomitans from subgingival and extracrevicular locations of the mouth

Abstract In the present study, a total of 619 subgingival and extracrevicular samples from 66 early-onset periodontitis, 42 adult periodontitis/gingivitis and 36 treated Actinobacillus actinomycetemcomitans-associated periodontitis patients were selectively cultivated for presence of A. actinomycetemcomitans. The organism was recovered from 68% cases with early-onset periodontitis, 24% cases with adult periodontitis/gingivitis and 50% of treated patients. Associations between recovery from pooled subgingival plaque and samples from extracrevicular locations as well as between different extracrevicular samples, were not heterogeneous with regard to different groups with the exception for cheek/saliva comparisons (odds ratios: early-onset periodontitis 825; adult periodontitis 8.1; treated patients 117; 0.05<p<0.1). For associations between recovery of A. actinomycetemcomitans from pooled subgingival plaque/extracrevicular samples, Mantel-Haenszel's odds ratios of between 12.2 and 21.6 were calculated (p<0.0001). The organism was isolated from 17 cheek mucosa samples of 18 patients identified as still harboring the organism after therapy. Present results point to the considerable value of cheek mucosa samples especially in treated patients to diagnose persistent A. actinomycetemcomitans colonization of the oral cavity.     

伴放线放线杆菌超声粉碎滤液诱导外周血中多形核白细胞脱粒的体外研究

目的:研究伴放线放线杆菌(actinobac illus actinomycetem com itans,Aa)可溶性产物对体外培养的多形核白细胞(polymorphonuc lear leukocytes,PMNLs)的影响,探讨该菌在牙周炎发病过程中的作用。方法:采集健康青少年外周血,Percoll梯度离心法分离PMNLs,将Aa超声粉碎滤液分别加入PMNLs(实验组)与含抗毒血清的PMNLs(对照组)中,在1m in、5m in时电镜观察摄片,20m in、40m in和60m in时细胞学检测并记数。结果:实验组PMNLs在透射电子显微镜下可见明显的形态学变化,其颗粒伴部分胞液脱出细胞外,对照组细胞无明显变化;实验组脱粒的PMNLs百分数较对照组显著增多(P<0.01)。结论:Aa可溶性产物可诱导外周血PMNL中颗粒向细胞周边移动并脱出,降低宿主抵抗力,参与牙周炎的发生。     

臭氧水对伴放线放线杆菌的灭活效果观察

目的探讨臭氧水对牙周可疑致病菌伴放线放线杆菌（Aa）的灭活效果。方法采用悬液定量杀菌方法和和化学方法在实验室进行观察。用4、8、15mg／L的臭氧水分别对悬液中A。作用1、2、3min。结果当臭氧水浓度为4mg／L对悬液中An没有杀灭作用。当臭氧水浓度为8mg儿时,对悬液中An作用1min,杀灭率为57％,当浓度上升至15mg／L时,对悬液中Aa作用1min．杀灭率上升至98％．而延长杀菌时间至3min．杀菌率维持在97％～99％。结论在25℃的室温条件下．臭氧水浓度达到15mg／L．臭氧水温控制在15℃～18℃时对悬液中Aa有快速、有效的杀灭作用。     

放线共生放线杆菌表面相关物质对成骨样细胞Mc3T3—E1碱性磷酸酶和矿化能力的影响

目的：观察放线共生放线杆菌表面相关物质对Mc3T3-E1细胞碱性磷酸酶活性的影响。方法：酶动力学方法。结果：此物质明显抑制细胞的碱性磷酸酶活性,在100mg/L浓度7d时与对照组差别显著（P＜0．05）。结论：此物质在骨吸收过程中起重要作用。     

伴放线放线杆菌诱导人外周血淋巴细胞活化及凋亡的体外研究

目的研究伴放线放线杆菌诱导人外周血淋巴细胞活化及凋亡的作用。方法选取10名全身及牙周组织健康受试者,分离外周血淋巴细胞,在有／无伴放线放线杆菌情况下培养0—96h,用荧光探针（AnnexinV—FITC、PI、CD69-TC7）进行标记,并进行流式细胞仪检测。结果全淋巴细胞加伴放线放线杆菌组AnnexinV＋／PI-细胞百分数在48h、72h、96h分别为13．42±2．88、22．74±2．18、46．92±4．28,全淋巴细胞组AnnexinV＋／PI-细胞百分数在48h、72h、96h分别为8．46±2．53、6．36±2．36、9．36±2．67,2组间存在明显差异（P〈0．01）。CD69加淋巴细胞加伴放线放线杆菌组和CD69＋淋巴细胞组AnnexinV＋／PI-细胞百分数除48h外的4个时间点上都无明显差异（P〉0．05）。CD69＋淋巴细胞加伴放线放线杆菌组AnnexinV＋／PI-细胞百分数在各个时间点上都明显高于全淋巴细胞加伴放线放线杆菌组（P〈0．01）。结论伴放线放线杆菌能够诱导人外周血淋巴细胞活化,并且能够通过活化促进其凋亡。