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。     

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。     

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。     

伴放线放线杆菌菌毛研究进展

伴放线放线杆菌是侵袭性牙周炎的可疑致病菌，菌毛是其重要的致病因子。本文对伴放线放线杆菌菌毛的形态、相关基因和蛋白、基因表达的相关调控、致病作用以及免疫原性进行了综述。     

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炎的相关性进行综述。     

伴放线放线杆菌菌毛研究进展

伴放线放线杆菌是侵袭性牙周炎的可疑致病菌,菌毛是其重要的致病因子。本文对伴放线放线杆菌菌毛的形态、相关基因和蛋白、基因表达的相关调控、致病作用以及免疫原性进行了综述。     

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。     

伴放线放线杆菌菌毛融合蛋白基因的克隆

目的　探讨克隆伴放线放线杆菌菌毛融合蛋白基因ltb-fap的方法。方法　采用PCR方法扩增伴放线放线杆菌菌毛相关蛋白(Fap)和大肠杆菌不耐热性肠毒素B亚单位(Ltb)的融合蛋白基因ltb-fap,NcoⅠ/EcoRⅠ双酶切载体pET28a(+)及ltb-fap基因,连接形成重组质粒pETltb-fap,转化至大肠杆菌DH5α,扩增后提取质粒pETltb-fap进行PCR鉴定、酶切鉴定和序列分析。结果　本实验扩增的ltb基因约为303 bp,fap基因约为228 bp,融合基因约为 531 bp。重组质粒含外源基因片段约为531 bp,酶切鉴定可得到一条约531 bp带,DNA测序结果表明与GenBank中的fap基因序列有97·4%的同源性。结论　成功克隆出融合蛋白基因ltb-fap,为进一步进行蛋白表达、制备抗体奠定基础。     

伴放线放线杆菌产生的细胞致死膨胀毒素及其与牙周病的关系

伴放线放线杆菌能产生一种热不稳定蛋白——细胞致死膨胀毒素。该毒素主要由三个相邻的基因编码，分别为cdtA，cdtB，cdtC，对应的蛋白产物分别是CDTA，CDTB，CDTC，它们构成三聚体的全毒素。它能引起细胞体积膨胀，使细胞停滞在G2／M周期而不能进入分裂期，通过线粒体途径导致淋巴细胞凋亡，诱导细胞因子的合成和分泌。由于该毒素具有多种细胞毒性，在牙周炎的发生发展中可能具有重要的致病作用。     

Outer membrane proteins of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus studied by SDS-PAGE and immunoblotting

This investigation characterized and compared outer membrane proteins (OMP) of the closely related Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus by means of SDS-PAGE patterns and reactions on immunoblots with rabbit antiserum against A. actinomycetemcomitans FDC Y4. Reactions with serum from a patient with Papillon Lefévre syndrome (PLS), from whom periodontal wild strains of A. actinomycetemcomitans had been isolated, were also studied. OMP were purified with selective solubilization from lyophilized cells of 10 wild and 4 reference strains of A. actinomycetemcomitans and 4 reference strains of H. aphrophilus. OMP profiles from wild and reference strains of A. actinomycetemcomitans were similar while those from A. actinomycetemcomitans and H. aphrophilus differed. The most prominent difference was absence of a heat modifiable protein in H. aphrophilus strains. Immunoblotting revealed strong common antigens in most strains, including a heat modifiable protein with mol wt 34 kDa, as well as a 29 kDa and a 16.5 kDa protein. Treatment with pronase and sodium periodate confirmed the protein nature of the major OMP antigens.     

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目的 分析不同血清型伴放线放线杆菌粗糙型菌株外膜蛋白的差异。方法 于2009年8—9月，在青岛大学医学院选用3种不同血清型的伴放线放线杆菌粗糙型菌株D7S、SA716和SA1151，分别培养并收集菌细胞，采用超声破碎及超离心技术提取外膜蛋白，SDS-PAGE电泳方法分析其分子质量。结果 全细胞电泳显示，3个菌株蛋白带的分子质量在10～200 ku之间分布，3个菌株之间蛋白带分布未见明显差异;提取的外膜蛋白电泳显示，3个菌株有共同的6个蛋白带，分子质量分别为100、75、64、45、34、31 ku;SA1151和SA716存在共同的分子质量为29 ku的蛋白带;D7S和SA716存在共同的分子质量为27 ku的蛋白带;SA1151存在清晰的分子质量为110 ku的蛋白带。结论 不同血清型伴放线放线杆菌粗糙型菌株之间的外膜蛋白可能存在一定的差异。     

Laminin binding to a heat-modifiable outer membrane protein of Actinobacillus actinomycetemcomitans

The interaction of Actinobacillus actinomycetemcomitans with the basement membrane protein, laminin, was examined in a ¹²⁵I-labeled protein-binding assay. The binding of laminin increased by lowering the pH. The ability to bind laminin was decreased in cells at the stationary phase of growth and by the presence of blood in the culture medium. Laminin binding to this bacterium was saturable, and the affinity constant was 4.6 nM. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot (ligand blot) analysis of cell-envelope and outer membrane of A. actinomycetemcomitans displayed a ¹²⁵I-laminin-reactive protein band with a molecular weight of 29 k. The laminin-binding protein was the previously described outer membrane protein A of A. actinomycetemcomitans. It was identified by its heat-modifiable property, detergent-solubility profile and reactivity with outer membrane protein A-specific polyclonal antiserum. At acidic pH, ¹²⁵I laminin bound to several cell-envelope components of A. actinomycetemcomitans , but at neutral pH, laminin bound only to the heat-modifiable protein. Despite the existence of the laminin-binding protein, cells grown in blood-containing media did not bind laminin. Several mammalian proteins interfered with laminin-bacterial interaction, including lactoferrin, which binds to the same bacterial protein that inhibited and displaced the laminin-bacterial interaction.     

伴放线放线杆菌与牙周病相关细胞凋亡关系的研究

牙周病是口腔的常见病和多发病,但其具体机制至今尚未完全明了。大量研究证实,细胞凋亡在牙周病的发生和发展过程中起重要作用。伴放线放线杆菌是牙周炎主要致病菌之一,可产生多种毒力因子。本文就伴放线放线杆菌的各种毒力因子与细胞凋亡及伴放线放线杆菌与牙周组织内多种细胞凋亡的关系进行综述。     

Longitudinal human serum antibody responses to outer membrane antigens of Actinobacillus actinomycetemcomitans

We hypothesize that serum antibody responses to antigens of a periodontopathogen would vary temporally and that the specificity of these host antibodies would relate to infection and disease activity. To test the hypothesis, we obtained between 6 and 13 serum samples from Actinobacillus actinomycetemcomitans (Aa)-positive (serological and microbiological) periodontitis patients at time points between 18 and 42 months into the study, and evaluated specific antibody responses to outer membrane antigens (OMA) of Aa strain Y4. Sera from these patients detected 22 different OMA. Early-onset (EOP; n=7) and adult (AP; n=11) periodontitis patients responded to 35% and 41% of the OMA, respectively. 2 of 9 sera from healthy subjects detected no antigens and 7/9 sera detected 7% of the OMA (p<0.0001 versus EOP and AP). The frequency of antibody responses to the 17 kDa antigen were similar between diseased, infected patients and the uninfected, normal subjects, suggesting that it may be stimulated as a cross-reactive antigen. Antibody to the 28, 38, and 90 kDa antigens were significantly more common in diseased patients (>90%) versus normal subjects (p<0.01, p<0.002, and p<0.002, respectively) and were unique among diseased, infected patients, which may be indicative of infections with Aa. A 65 kDa antigen showed an increased frequency of reaction in the AP versus the EOP (p=0.01) patients, which exemplified a potential distinction in response to Aa between adult and early-onset disease classifications. Seventeen of 22 OMA could be detected in every sample from at least one patient. Longitudinal samples from seropositive EOP and AP reacted 80-100% of the time with the 17, 28, and 100 kDa antigens. Finally, five antigens of 15, 38, 58, 65, and 79 kDa were detected in 33-83% of the seropositive patients; however, antibody to these OMA reacted variably at different sampling points, suggesting some antigenic response diversity over time.     

细胞致死性扩张毒素和外膜蛋白的结构功能和致病机制

伴放线放线杆菌是青少年牙周病的主要致病菌,与侵袭性牙周炎密切相关。伴放线放线杆菌细胞致死性扩张毒素（CDT）和外膜蛋白（OMP）等毒力因子使其更易定植到宿主体内,破坏宿主的免疫调节,从而进一步引起牙周组织破坏和加速牙周病的进展。本文主要就CDT和OMP毒力因子目前的结构功能及致病机制作一综述,以期对其深入研究有所帮助。     

Prevalence of Actinobacillus actinomycetemcomitans in an ethnic adult Chinese population

AIM: The aim of this study was to determine the prevalence and the structure of the leukotoxin promoter region of Actinobacillus actinomycetemcomitans in an ethnic Chinese population. METHOD: Subgingival plaque samples were collected from 42 patients with moderate to advanced periodontitis and 50 periodontally healthy patients. A. actinomycetemcomitans was detected directly from the crude subgingival plaque by PCR using leukotoxin gene specific primers. The presence of A. actinomycetemcomitans was determined by a single 285 bp PCR amplicon. RESULTS: A. actinomycetemcomitans was found to be present in the subgingival plaque of 68 out of a total of 92 patients examined (74%). 29 out of the 42 periodontitis patients tested were carriers of A. actinomycetemcomitans (69%). Among the periodontally healthy patients studied, 39 out of 50 subjects possessed the bacteria (78%). PCR analysis of the promoter region of the ltx operon revealed that none of the 42 moderate to advanced periodontitis patients examined harboured A. actinomycetemcomitans strains with the JP2-like promoter of the ltx operon, known to enhance leukotoxin expression. 2 out of the 27 advanced periodontitis patients clinically diagnosed as suffering from rapidly progressive periodontitis were found to be carriers of the mildly toxic strain of A. actinomycetemcomitans with the characteristic 652-like promoter. CONCLUSIONS: The high prevalence of A. actinomycetemcomitans, regardless of whether the subgingival samples were analysed from patients with healthy or diseased periodontium suggests that this bacterial species is part of the normal oral flora of ethnic Chinese. Our preliminary results also suggested that subjects who harboured the mildly toxic strain of A. actinomycetemcomitans were potentially susceptible to aggressive forms of periodontitis.     

Structural proteins of the Actinobacillus actinomycetemcomitans bacteriophage øAa

?Aa is an A1 morphotype bacteriophage which infects certain strains of Actinobacillus actinomycetemcomitans. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of dissociated, purified phi Aa particles revealed 7 major structural proteins (P1-P7) ranging in size from 17.5 to 52.7 kilodaltons (Kd). Treatment of the intact phage particles with 67% dimethyl sulfoxide (DMSO) resulted in the separation of the virion head and tail subunits. Purification of the head subunits was accomplished by sucrose density gradient centrifugation of the DMSO-treated phage particles. The purified head subunits were composed of a single protein having an electrophoretic mobility which corresponded to a 39.5 Kd protein (P3) of the intact virus. Raising the pH of a purified phi Aa suspension to 12.7 disrupted the head subunits, as well as the tail tube and tail fibers, releasing intact contractile tail sheaths. The tail sheaths were collected by centrifugation. The purified tail sheaths were analyzed by SDS-PAGE and were found to be composed of two proteins (P1 and P2) having molecular weights of 52.7 and 41.2 Kd respectively. The location of each of the 4 remaining major structural proteins in the phi Aa virion remains to be determined.     

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.