白介素10对伴放线放线杆菌内毒素诱导兔肺巨噬细胞凋亡的影响

目的：探讨白介素10（IL-10）对伴放线放线杆菌内毒素（Aa—LPS）体外诱导兔肺巨噬细胞凋亡作用的影响。方法：经兔气管肺泡灌洗获得肺泡巨噬细胞,随机分为空白对照组、Aa—LPS组、Aa—LPS＋IL-10组。按实验分组加入Aa—LPS（1汕g／mL）、IL-lo（o．1p．g／mL）,24h后裂解细胞,荧光定量PCR法检测促凋亡基因bax、p53和caspase-3的表达。结果：Aa—LPS组bax、caspase-3的表达较空白对照组明显升高（P〈0．05）,p53的表达与空白对照组比较差异无统计学意义（P〉0．05）。Aa—LPS＋IL-10组bax、p53、caspase．3的表达较Aa—LPS组降低（P〈0．05）。结论：Aa—LPS体外对肺巨噬细胞有促凋亡作用,IL-10可抑制Aa—LPS的促凋亡作用,其机制可能与细胞凋亡的线粒体途径有关。     

伴放线放线杆菌血清型分析

目的 PCR法检测伴放线放线杆菌（Actinobacillus actinomy＇etemcomitans，Aa）临床分离菌株血清型，分析其与flp-1基因型的关系。方法用血清型特异性引物，通过普通PCR和多重PCR的方法对60株Aa临床分离菌株的血清型进行鉴定，并分析其与flp-1基因型的关系。结果 60株Aa临床分离菌株中血清型c型63，33％，e型23．33％，b型6.67％，a，f型各占3．33％，未检测到d型菌株；在24名被检测者中，15名检测到c型An菌株，3名检测到b型菌株，各有2名分别检测到a、e、f型菌株。fip-1基因型Ⅰ型菌株的血清型均为a型，40株Ⅱ型菌株中38株为c型，Ⅳ型菌株均为b型，11株Ⅴ型菌株中9株为e型，Ⅵ型菌株均为e型。结论 Aa血清型分布以c型为主，fip-1基因型与菌株血清型存在一定对应关系。     

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。     

伴放线放线杆菌粘附特性研究

目的分析伴放线放线杆菌的粘附特性及菌毛结构基因tip-1的遗传多样性对菌株粘附活动的影响。方法检测不同孵育条件下5种tip-1基因型临床分离菌株和光滑型菌株的粘附活动。结果临床分离菌株的粘附量随菌液浓度,孵育时间的增加而增加。tip-1基因型Ⅱ型菌株的粘附量高于其它4型菌株,光滑型菌株的粘附量低于临床分离菌株。生理温度下菌株粘附数高,低温下明显降低。厌氧条件和有氧条件下的粘附量无显著性差异。结论伴放线放线杆菌临床分离菌株的粘附存在时间和菌量依赖性,并要求一定新陈代谢活性,粘附效率在氧浓度改变时没有明显变化。伴放线放线杆菌表型影响菌株的粘附作用。不同tip-1基因型菌株粘附能力存在差异,Ⅱ型菌株粘附能力最强。     

伴放线放线杆菌磷酸胆碱的电泳分析

目的 比较3个磷酸胆碱阳性的伴放线放线杆菌菌株在蛋白酶K作用后电泳结果的变化,分析磷酸胆碱抗原在细菌中的附着结构.方法 将培养收集的伴放线放线杆菌破碎处理后,加入蛋白酶K水解,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE),考玛斯亮蓝染色显示蛋白条带的分布情况,免疫印迹法检测磷酸胆碱的分布情况.结果 伴放线放线杆菌的3个菌株SA716、SA1398、SA2791通过SDS-PAGE电泳,可见未经蛋白酶K处理的细菌悬液显示连续分布的蛋白条带,而蛋白酶K处理后,只显示1条蛋白条带,该条带在只有等量蛋白酶K的对照组也出现,而在未经蛋白酶K处理的细菌悬液中无该条带,可以确定这一条带是蛋白酶K,细菌蛋白都已经被分解.未经蛋白酶K处理的菌株通过SDS-PAGE电泳和磷酸胆碱免疫印迹检测,磷酸胆碱显示阳性结果,磷酸胆碱附着结构分子量大小约为9 kDa,而蛋白酶K处理后,显示阴性结果,磷酸胆碱信号消失.结论 伴放线放线杆菌磷酸胆碱信号在蛋白酶K处理后消失,提示该抗原的附着结构为蛋白成分.     

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.     

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。     

伴放线放线杆菌形态变化对白细胞毒素分泌影响的研究

目的观察伴放线放线杆菌形态变化对白细胞毒素分泌的影响。方法选择粗糙型和光滑型伴放线放线杆菌各8株,应用聚丙烯酰胺凝胶电泳,检测液体培养12、24、48、60、72h的菌体及培养上清液中116kDa大小白细胞毒素蛋白条带的情况,应用超滤法分离纯化培养上清液蛋白,应用台盼蓝染色排除法检测上清液蛋白白细胞毒素活性。结果粗糙型伴放线放线杆菌菌株液体培养12、24、48、60、72h菌体蛋白电泳均可见116kDa大小的蛋白条带,培养上清液蛋白电泳结果显示116kDa大小的蛋白条带均出现于培养24和48h;光滑型伴放线放线杆菌菌株液体培养12、24、48、60、72h菌体蛋白电泳结果均缺少116kDa大小的蛋白条带,培养上清液蛋白电泳结果显示116kDa大小的蛋白条带出现于培养12和24h;实验菌株培养上清液提取蛋白均具有白细胞毒素活性。结论伴放线放线杆菌粗糙型和光滑型菌株均可分泌具有直接杀灭人多形核白细胞活性的白细胞毒素,但粗糙型菌株分泌白细胞毒素的时间晚于光滑型。     

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。     

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。     

Involvement of caspases in apoptotic cell death of murine macrophages infected with Actinobacillus actinomycetemcomitans

Infection of murine macrophages in vitro with periodontopathic bacterium Actinobacillus actinomycetemcomitans induces apoptotic cell death. In this study, we investigated the involvement of caspases in apoptotic cell death of A. actinomycetemcomitans-infected macrophages. Two peptide inhibitors of caspases, benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone (Z-VAD-FMK) and benzyloxycarbonyl-Asp-Glu-Val-Asp (OMe)-fluoromethyl ketone (Z-DEVD-FMK), inhibited apoptotic cell death of murine macrophage cell line J774.1 infected with A. actinomycetemcomitans. During the process of apoptosis, interleukin-1beta (IL-1beta) was detected in the culture supernatants of J774.1 cells. IL-1beta secretion was blocked by the caspase-1 inhibitor, Z-VAD-FMK, indicating that caspase-1 is involved in not only the induction of apoptosis but also the IL-1beta secretion from A. actinomycetemcomitans-infected J774.1 cells. Immunoblot analysis revealed that the infection of A. actinomycetemcomitans to J774.1 cells induced the cleavage of retinoblastoma protein (Rb), suggesting that caspase-3 was activated by A. actinomycetemcomitans infection. The cytosol from A. actinomycetemcomitans-infected J774.1 cells induced Rb proteolysis in vitro, which was inhibited by the caspase-3 inhibitor, Z-DEVD-FMK. Furthermore, caspase-3-like activity was markedly increased in J774.1 cells infected with A.actinomycetemcomitans between 12 h and 24 h, which was subsequently inhibited by the addition of caspase-3 inhibitor, Z-DEVD-FMK. These findings indicate that caspase-3 induces apoptosis in J774.1 cells infected with A. actinomycetemcomitans. Taken together, these results suggest that caspase-1 and caspase-3 are involved in the induction of apoptosis in A. actinomycetemcomitans-infected macrophages.     

Actinobacillus actinomycetemcomitans lipopolysaccharide stimulates collagen phagocytosis by human gingival fibroblasts

Introduction:  Collagen phagocytosis by fibroblasts is involved in the intracellular pathway related to collagen breakdown in soft connective tissues. The possible role of lipopolysaccharide (LPS) in regulating this fibroblast function has not been elucidated so we investigated the effect of LPS from Actinobacillus actinomycetemcomitans , a periodontopathic bacterium, on collagen phagocytic activity in human gingival fibroblasts and associated regulatory mechanisms.
Methods:  LPS pretreatment stimulated binding of collagen-coated beads to cells and, subsequently, their internalization.
Results:  The LPS-activated collagen phagocytic process was enhanced in the presence of the soluble form of CD14 (sCD14) or LPS-binding protein (LBP), while the LPS/LBP treatment activated Akt and induced actin reorganization. Furthermore, these LPS/LBP-induced effects were partially suppressed by adding phosphatidyl-inositol-3 kinase (PI3K) inhibitors.
Conclusion:  These results suggest that A. actinomycetemcomitans LPS disturbs the homeostasis of collagen metabolism within gingival tissue by facilitating collagen phagocytosis by gingival fibroblasts, and serum sCD14 and LBP positively regulate the action of LPS. In addition, the PI3K/Akt signaling is thought to partially mediate the LPS/LBP-stimulated collagen phagocytic pathway, which may be dependent on actin cytoskeletal rearrangement.     

放线共生放线杆菌白细胞毒素水平检测

目的　检测放线共生放线杆菌 (Actinobacillusactinomycetemcomitans,Aa)临床分离菌株白细胞毒素水平 ,区分高毒株与低毒株。方法　应用聚合酶链反应 (polymerasechainreaction ,PCR)检测白细胞毒素操纵子启动子区域基因序列的差异。检测临床菌株 6 8株 ,其中b型 17株 ,c型 42株 ,a型9株。阳性对照高毒株为JP2 ,低毒株为ATCC43717等 5株Aa国际参考菌株 ,阴性对照为 12株异种菌国际参考菌株。结果　 6 8株临床分离菌株扩增片段均为 10 2 2bp ,JP2扩增片段为 492bp ,ATCC43717等 5株扩增片段均为 10 2 2bp ,12株异种菌参考菌株无扩增片段出现。结论　 6 8株临床分离菌株全部为低毒株     

In vitro antimicrobial susceptibility of different serotypes of Actinobacillus actinomycetemcomitans

In vitro susceptibility of Actinobacillus actinomycetemcomitans ( A.a .) serotypes to selected antimicrobial agents was investigated by the agar dilution method on supplemented Mueller-Hinton test medium. Eighty-three A.a . strains, 80 recent isolates from 40 periodontally healthy or diseased subjects, and three type strains were included in the study. Serotype a represented 20, serotype b 32, serotype c 17, and serotype e 7 and nontypable 4 of the tested strains. The most effective drugs against all A.a . serotypes in vitro were cefaclor, cefuroxime, tetracycline hydrochloride, doxycycline, trimethoprim-sulfamethoxazole (cotrimoxazole), and ciprofloxacin, which inhibited 100% of the strains at 4.0 μg/ ml, 4.0 μg/ml, 1.0 μg/ml, 2.0 μg/ml, 0.06 μg/ml, and 0.015 μg/ml, respectively. Serotypes a and e were more susceptible to cafaclor and cefuroxime than were serotypes b and c; 100% of the first two groups were inhibited at 2.0 μg/ml and 1.0 μg/ml. Ampicillin inhibited 92% of the tested strains at 1.0 μg/ml. Serotype b was always susceptible to ampicillin. Metronidazole exhibited the best activity against serotype a strains. The lowest minimal inhibitory concentration values for benzylpenicillin, ampicillin, erythromycin, doxycycline, and metronidazole were encountered among serotype b isolates. The results of the present study indicate minor differences in the in vitro antimicrobial susceptibility patterns of different A.a . serotypes, except to metronidazole. Also, the new oral cephalosporins and cotrimoxazole, rare antimicrobial agents in periodontology, showed promising efficacy against all A.a . strains.     

牙周炎患者唾液中伴放线放线杆菌的检出状况分析

目的 检测不同类型牙周炎患者唾液中的伴放线放线杆菌(Actinobacillusactinomycetemcomitans,Aa),探讨唾液和集合龈下菌斑中Aa检出率的差异以及唾液中Aa的存在状况与牙周临床指标的关系. 方法 收集50例侵袭性牙周炎(aggressive periodontitis,AgP)患者、48例慢性牙周炎(chronic periedontitis,CP)患者和25例非牙周炎者的非刺激性全唾液和集合龈下菌斑,应用聚合酶链反应(PcR)技术检测两种样本中的Aa. 结果 Aa在AgP患者唾液中的检出率(32%)显著高于非牙周炎者(4%)和CP患者(15%),差异均有统计学意义(P<0.01,P<0.05),同时Aa在AgP患者唾液中的检出率也显著高于集合龈下菌斑样本(16%),差异亦有统计学意义(P<0.05).年龄≤30岁是唾液中存在Aa的危险指征(OR=3.23,P<0.05);出血指数≥3的位点超过70%与唾液中存在Aa有关(OR=19.21,P<0.01). 结论 AgP患者唾液样本中Aa的检出率明显高于集合龈下菌斑样本,亦高于CP患者和非牙周炎者,提示Aa可能参与AgP的发生和发展.     

抗伴放线放线杆菌IgY的制备及抑菌试验研究

目的:观察伴放线放线杆菌诱导母鸡产生特异性IgY抗体情况,以及其抑制伴放线放线杆菌(A.a)和牙龈二氧化碳噬纤维菌(C.g)生长效果。方法:应用免疫接种法、水稀释法、盐析法、液体培养抑菌法、以及ELISA法,诱导、提取和纯化IgY抗体,取一定量抗体与细菌共同培养,测定抑制伴放线放线杆菌和牙龈二氧化碳噬纤维菌生长效果。结果:两步硫酸铵盐析沉淀的IgY抗体纯度达85.6%～90.3%;抗原结合效价为1∶32000;抗伴放线放线杆菌IgY抗体与牙龈二氧化碳噬纤维菌交叉免疫反应的抗原结合效价为1∶8000;当抗伴放线放线杆菌IgY抗体浓度在5.0、1.0、0.1g/L时,细菌浓度在5×108CFU/L培养24h其抑菌率分别为31.60%(P=0.004)、10.24%(P=0.024)、-3.30%,培养72h其抑菌率分别为64.20%(P=0.004)、53.21%(P=0.002)、11.20%。细菌浓度在1×108CFU/L培养24h其抑菌率分别为35.71%(P=0.004)、30.95%(P=0.012)、11.11%,培养72h其抑菌率分别为65.11%(P=0.005)、54.04%(P=0.002)、16.17%;5.0g/L的抗伴放线放线杆菌IgY与1×108CFU/L牙龈二氧化碳噬纤维菌培养24h其抑菌率为41.61%(P=0.005),培养72h抑菌率为86.99%(P=0.014)。结论:伴放线放线杆菌能够诱导母鸡产生高效价的特异性IgY抗体,该抗体在一定的浓度内有抑制伴放线放线杆菌和牙龈二氧化碳噬纤维菌生长的作用;伴放线放线杆菌与牙龈二氧化碳噬纤维菌存在着共同抗原。     

采用PCR方法鉴别伴放线放线杆菌的6种血清型

目的：探索采用PCR的方法对伴放线放线杆菌的不同菌株进行血清型分类。方法：根据伴放线放线杆菌不同血清型特异性多糖抗原基因序列设计6对不同的寡核苷酸引物，用这6对引物分别对所选择伴放线放线杆菌6种不同的血清型菌株各3株，共18株，其中参考菌株6株，系ATCC29523（血清型a），ATCC43718（血清型b），ATCC33384（血清型c），IDH781（血清型d），IDH1705（血清型e）以及CU1000（血清型f），其余12个菌株均为临床分离株的DNA进行PCR扩增分析。结果：每一对引物均针对相应的血清型产生特异性单一条带PCR产物，产物大小分别为428bp（a），298bp（b），559bp（c），690bp（d），211bp（e），232bp（f）。全部18个菌株均能够被准确识别，无交叉反应。结论：PCR方法可以快速准确地鉴别伴放线放线杆菌目前已知的全部6种血清型。     

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.     

Intra-oral colonization of macaque monkeys by Actinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans was acquired by captive Macaca fascicularis 3 to 6 months after birth, and all monkeys aged over 6 months harbored detectable levels. This microorganism was most frequently isolated from the gingival plaque of the incisor (and other) teeth compared with other oral sites. Strains were leukotoxic by bioassay and Western blot analysis. Antibodies in macaque serum contained neutralized the leukotoxin of a human A. actinomycetemcomitans strain. High titres of maternal neutralizing anti-leukotoxin antibodies were detected in neonates; the titre then fell rapidly so that by 6 months the antibody titer was zero. Antileukotoxin antibody production was detected after 6 months of age, rapidly reaching a high level within 2 years after birth. The presence of leukotoxic strains of A. actinomycetemcomitans in the gingival region did not appear to be correlated with an increase in susceptibility to periodontal disease.