细菌直接PCR和双引物策略鉴定16S rRNA基因技术的建立

目的探索一种基于16S rRNA基因的快速鉴定细菌方法,为临床诊断治疗及耐药菌的分子遗传分析提供科学依据。方法对卫生部室间质评菌株和临床病人标本分离培养纯菌落,用双蒸水稀释菌落,然后直接以菌液为模板优化反应体系PCR扩增16S rRNA基因片段,再测序扩增片段。将测序结果在细菌Ribosomal数据库中进行同源性比对,根据序列同源性鉴定病原细菌。结果本实验鉴定的卫生部质评菌株结果与卫生部报告结果一致。本实验能够一次性鉴定出临床病人标本分离的菌株。结论本研究优化了一种免纯化细菌核酸直接PCR鉴定细菌16S rRNA基因的方法。本研究建立的基于病原细菌16S rRNA基因鉴定方法可用于细菌的快速诊断。     

口腔和胃幽门螺杆菌临床菌株的耐酸耐盐特性及同源性分析

目的检测口腔和胃幽门螺旋杆菌(H.pylori,HP)分离株的形态、耐酸耐盐特性,分析口腔和胃HP菌株的同源性。方法从同一胃溃疡患者的唾液和胃病变组织分离鉴定HP菌株,检测相同浓度的菌株在pH值为7.0、6.5、6.0、5.5、5.0、4.5和NaCl浓度为0%、0.5%、1.0%、1.5%、2.0%、2.5%的哥伦比亚培养基上培养后的菌落数量。结果革兰染色镜检观察到口腔和胃HP菌株均为弧形,S形或海鸥展翅状的革兰阴性杆菌;耐酸、耐盐试验结果表明,二者均表现出良好的耐酸耐盐特性,但胃HP分离株明显优于口腔分离株。结论口腔和胃H.pylori菌株在形态上具有高度一致性,而在耐酸、耐盐特性上表现出一定的异质性。     

从前列腺液中分离到结核硬脂酸棒状杆菌

目的 对分离自慢性前列腺炎患者前列腺液中的一株棒状样细菌进行分类学鉴定,探讨其种系发生及生物学地位.方法 利用形态、生理和生化特性等表型性状初步进行细菌鉴定,通过细胞壁化学成分分析、16S rRNA基因测序及基因比对明确其种系来源.结果 分离、培养出革兰染色阳性的棒状样杆菌;生化反应不活泼;细胞壁二氨基酸为内消旋二氨基庚二酸(meso-DAP),全细胞糖含阿拉伯糖、半乳糖和甘露糖,化学型为胞壁Ⅳ型.GenBank数据库序列比对显示,该菌株与结核硬脂酸棒状杆菌(C tuberculostearicum)菌株ATCC35692的16S rDNA序列相似度最高,为99.4%(仅存在8个核苷酸差异).结论 从表型性状和种系发生研究可确定该菌株为结核硬脂酸棒状杆菌,本研究与模式株相比亦存在一定差异,初步考虑该分离株可能足该菌种的一个新业型.该菌分离自前列腺液,在国内外尚属首例.     

抗分枝杆菌海洋微生物的分离与鉴定

目的 从海洋微生物中发现具有抗分枝杆菌活性的细菌,并对其进行鉴定.方法 从印度洋热带地区的海底沉积物中分离海洋微生物.通过纸片法活性测定,发现了一株具有抗耻垢分枝杆菌活性的细菌.光学显微下观察细菌形态和菌落.测定细菌的理化特性,提取其基因组,荧光法测定基因组的Tm值,扩增其16S rDNA并进行测序.将序列提交NCBI...     

网孔过滤法分离纯化肌源性干细胞

探讨乳大鼠肌源性干细胞的体外培养及纯化分离方法.采用Ⅺ型胶原酶和胰蛋白酶消化乳大鼠骨骼肌,网孔过滤纯化肌源性干细胞后,通过体外原代和传代培养,经生长曲线、细胞融合率等指标研究肌源性干细胞的增殖和分化能力,免疫细胞化学染色法进行鉴定.结果表明:通过上述方法分离得到小圆形或短梭形的细胞,免疫细胞化学染色desmin及CD34为阳性.说明网孔过滤法可提高肌源性干细胞的纯度.     

成人食管上皮细胞的体外培养及生物学特性

目的 培养成人正常食管上皮细胞,建立能够体外长期培养的食管上皮细胞系,为上皮细胞的体外研究提供实验材料.方法 取食管癌患者正常食管上皮,用0.25%Dispase酶和0.25%胰蛋白酶/0.02?TA消化获取成人食管上皮细胞,使用无血清角化细胞培养液培养,通过细胞形态学观察和角蛋白、上皮膜抗原(EMA)免疫组织化学染色鉴定细胞.结果 原代培养8d后,细胞汇合成片呈铺路石样生长,细胞角蛋白、上皮膜抗原表达阳性,可连续传代.结论 为体外分离培养成人正常食管上皮细胞建立了方便可行的方法.     

改进的大鼠原代Kupffer细胞分离与鉴定方法

目的 改进原分离方法,探索出一种稳定性强、经济实用、高纯度的大鼠原代Kupffer细胞(Kupffer cells,KC)的分离方法.方法 (1)原位胶原酶灌注法;(2)Percoll液不连续的密度梯度离心以分离Kupffer细胞;(3)选择性贴壁纯化Kupffer细胞;(4)Kupffer细胞吞噬墨汁实验、CD68免疫荧光染色鉴定.结果 Kupffer细胞得率≥(7～8)×107/肝,纯度≥98.0%,Kupffer细胞吞噬活性强.结论 此方法稳定性强、经济实用、KC纯度高及性能优良,可满足PRC、Western blot等分子生物学所需要的原代细胞数.     

犬骨骼肌成肌细胞体外分离、纯化及培养方法改良探索

目的:探讨犬骨骼肌成肌细胞(SkMs)体外分离、纯化及培养方法的改良并研究其生物学特性.方法:采用机械分离结合Ⅱ型胶原酶、中性蛋白酶双酶一步消化法分离犬骨骼肌成肌细胞,经差速贴壁法纯化后,在骨骼肌细胞生长培养基(SKGM)中进行原代和传代培养,免疫细胞化学染色鉴定.结果:改良后的培养方法适于获取犬骨骼肌成肌细胞,SKGM培养基适于犬骨骼肌成肌细胞的体外培养.SkMs在细胞密集或低血清分化培养基作用下可融合成肌管.结蛋白(desmin)单克隆抗体(mAb)细胞化学染色鉴定SkMs呈阳性,纯度在90%以上.结论:通过改良后的双酶一步消化法获得的SkMs在合适的培养条件下能够增殖、分化并保持其生物学特性,为其在基因治疗和组织工程中的应用奠定了基础.     

从血液中分离出一株山夫顿堡沙门菌

20 0 2年 9月 ,从 1例高热患儿的血液中分离出山夫顿堡沙门菌 (S .senften berg)。患者 ,女 ,13个月。腹疼、腹泻 ,体温 39.5℃ ,实验室检查 :血WBC 2 .5×10 9 L。粪便稀 ,WBC 2～ 6 HP ,RBC 1～ 3 HP ;血培养 2次均分离出沙门菌属的细菌。2次血培养均用生物双相培养瓶(梅里埃公司 )产品 ,35℃ 2 4h瓶内液体作者单位 :2 5 2 60 0山东省临清市人民医院检验科通讯作者 :张广玲混浊 ,固体面可见小菌落 ,即取瓶内液体和琼脂面菌落 ,转种血平板 ,35℃ 2 4h生长出灰白色、湿润、光滑、不溶血小菌落。革兰染色为阴性小杆菌。该菌生化反应 …     

牙龈卟啉单胞菌prtH基因分布与多态性研究

目的　分析牙龈卟啉单胞菌 (P .gingivalis)prtH基因的遗传多态性 ,从而了解细菌变异与其对牙周致病作用的相关性。方法　以PCR技术从牙周炎患者口腔中分离的P .gingivalis进行prtH基因扩增 ;采用斑点杂交法 ,以生物素标记prtH基因为探针 ,与以引物B进行PCR扩增为阳性的P .gingivalis基因组DNA杂交 ;对PCR产物进行AluⅠ限制性酶切和DNA测序分析。结果　以prtH基因A、B引物对 14 0株临床分离株进行PCR扩增 ,分别在 76例 (引物A)和 117例 (引物B)出现阳性扩增产物 ,阳性率分别为 5 4 .2 %、84 %。对 34例引物B扩增阳性的P .gingivalisDNA进行点杂交检测 ,阳性吻合率为 82 .4 %。与GenBank中菌株ATCC332 77(U15 2 82 )和W83(L2 74 83)的prtH基因序列相比 ,5株P .gingivalisPCR扩增产物的测序存在着缺失和点突变。结论　在慢性牙周炎患者临床P .gingivalis分离菌株中存在着prtH基因的遗传多态性 ,本实验建立了具有高度敏感性和特异性的PCR检测方法     

牙周基础治疗对糖尿病患者血清和龈沟液中肿瘤坏死因子-α的影响

目的 观察牙周基础治疗对2型糖尿病伴牙周炎患者血清及龈沟液中肿瘤坏死因子-α(TNF-α)的浓度、临床牙周状态、血糖控制的影响.方法 选取2型糖尿病伴牙周炎患者60例,随机分做牙周基础治疗组(观察组)和不做牙周基础治疗组(对照组),每组各30人.分别在治疗前、治疗后1个月和3个月记录所有患者牙周临床指数:探诊深度(PD),附着丧失(AL)及菌斑指数(PLI),并检测糖化血红蛋白(HbAlc)及血清及龈沟液中TNF-α的含量.结果 观察组中PD、PLI和龈沟液中TNF-α含量在治疗后1个月和3个月时均显著降低(P＜0.05),AL和血清中HbAlc及TNF-α含量仅在治疗后3个月显著降低(P＜0.05).对照组的PD,AL,PLI,HbAlc,龈沟液及血清中TNF-α的差异无统计学意义(P＞0.05).结论 牙周基础治疗有助于2型糖尿病伴牙周炎患者的血糖控制,牙周状态改变和血清及龈沟液中TNF-α含量下降.     

牙周炎患者龈沟液IL-10、IL-18和IFN-γ测定的临床意义

目的：测定牙周炎患者龈沟液IL-10、IL-18及IFN-γ浓度，探讨三种细胞因子含量的变化及其意义。方法：IL-10、IL-18及IFN-γ三种细胞因子均采用放射免疫分析。结果：牙周炎患者治疗前IL-18及IFN-γ两项龈沟液细胞因子的浓度极显著高于正常对照组（P均〈0.01）；IL-10水平略低于正常对照组，但无明显统计学意义（P〉0.05）；治疗一个月后，IL-18及IFN-γ两项龈沟液细胞因子的浓度显著下降，但仍显著高于正常对照组（P均〈0.05）；IL-10含量则升高极显著（P〈0.01）。相关分析数据显示，牙周炎组IL-18及IFN-γ两项龈沟液细胞因子的浓度与牙周袋深度、附着丧失呈显著正相关（r=0.2617、0.2802，P均〈0.05）。而IL-10浓度与牙周袋深度、附着丧失程度呈显著负相关（r=-0.1743，P〈0.05）。结论：牙周炎患者龈沟液IL-10、IL-18及IFN-γ三项细胞因子水平发生了显著变化，表明牙周炎患者体内存在免疫功能调节紊乱。     

Activation of neutrophil collagenase in periodontitis

Neutrophil collagenase (matrix metalloproteinase 8 [MMP-8]) is an important mediator of tissue destruction in inflammatory diseases. Studies of anaerobic periodontal infections have shown that active MMP-8 in gingival crevicular fluid is associated with the degradation of periodontal tissues in progressive periodontitis whereas the latent enzyme is predominant in gingivitis. Since the activation of MMP-8 appears to be a crucial step in periodontitis, we have examined the activation of MMP-8 in gingival crevicular fluid samples by using a soluble biotinylated collagen substrate. Analysis of gingival crevicular fluid in periodontitis, gingivitis, and controls revealed sixfold (P < 0.001)-higher levels of active collagenase in periodontitis (n = 12) samples compared to gingivitis (n = 17) samples, which exhibited low levels of activity, while controls (n = 25) showed no activity. After gingival crevicular fluid was collected, no further activation of latent collagenase occurred in vitro. Although both MMP-1 and MMP-8, but not MMP-13, could be detected by immunoblots, blocking antibodies to MMP-1 showed that collagenase activity was largely contributed by MMP-8, which was localized to the matrix of diseased tissues. The MMP-8 in gingival crevicular fluid migrated primarily as a 60-kDa form with smaller amounts of a 78-kDa species, whereas MMP-8 isolated from peripheral neutrophils migrated at 70 and 89 kDa, corresponding to active and latent forms of the enzyme, respectively. Most of the MMP-8 in the 60- and 70-kDa bands selectively bound to tissue inhibitor of metalloproteinase 2 and collagen, indicating that most, but not all, of the enzyme in these bands was in an activated form. However, the amounts of the 78- and 60-kDa forms from gingival crevicular fluid in different samples did not correlate (r2 = 0.028) with the latent and active enzyme measured by collagenase assay. Collectively, these studies have identified distinct forms of latent and active MMP-8 in gingival crevicular fluid that appear to result from a unique activation mechanism that occurs in periodontitis. The complexity of MMP-8 activation is further indicated by the presence of latent, activated, and superactivated forms of MMP-8 in the 60- and 70-kDa bands obtained from gingival crevicular fluid and neutrophil samples, respectively.     

Partial characterization of an interleukin-1-like factor in human gingival crevicular fluid from patients with chronic inflammatory periodontal disease.

A significant level of interleukin-1-(IL-1)-like activity was detected in gingival crevicular fluid obtained from sites in patients with chronic inflammatory periodontal disease, confirming a previous report of IL-1-like activity detected in human gingival crevicular fluid from patients with chronic inflammatory periodontitis (J. A. Charon, T. A. Luger, S. E. Mergenhagen, and J. J. Oppenheim, Infect. Immun. 38:1190-1195, 1982). In the present study, we sought to investigate whether this IL-1-like activity belonged to IL-1 alpha or IL-1 beta and to characterize some of the biochemical properties of this factor. Polyclonal antibodies against recombinant human IL-1 alpha or IL-1 beta (rIL-1 alpha or rIL-1 beta) have been used for serological comparison of the IL-1-like factor. IL-1-like activity was completely neutralized by anti-human rIL-1 alpha antiserum, but not by anti-human rIL-1 beta antiserum. On gel filtration with a high-pressure liquid chromatographic Superose 12 column, IL-1-like activity was separated into two peaks, one with a molecular weight of about 43,000 and the other with a molecular weight of less than 17,000. The majority of the IL-1-like factor with a low molecular weight in human gingival crevicular fluid migrated at a molecular weight of about 17,000 under the reducing conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specificity of the IL-1-like factor was further confirmed by an immunochemical method (Western blotting [immunoblotting]) by using anti-human rIL-1 alpha monoclonal antibodies. On isoelectric chromatography with a high-pressure liquid chromatographic Mono P column, the pI of this IL-1-like factor was between pH 4.9 and 5.2. These results suggest that the IL-1-like factor in human gingival crevicular fluid from diseased sites in patients with chronic inflammatory periodontitis consists predominantly of IL-1 alpha.     

Immunoglobulin G response to subgingival gram-negative bacteria in human subjects

Serum and gingival crevicular fluid from normal healthy adults and patients with periodontitis were screened for immunoglobulin G antibodies to antigens from Bacteroides gingivalis 381, Bacteroides intermedius 24, Bacteroides loescheii ATCC 15930, Fusobacterium nucleatum ATCC 25586, Eikenella corrodens 1073, Actinobacillus actinomycetemcomitans ATCC 29522, and Capnocytophaga sp. strain M-12. Immunoglobulin G antibody titers to the antigens were measured by an enzyme-linked immunosorbent assay. The antibody levels to B. gingivalis in serum and gingival crevicular fluid were significantly higher in the samples from patients with periodontitis than in samples from healthy individuals. Although there were individual differences within patient groups, a positive correlation (P less than 0.01) was found between the serum immunoglobulin G levels to B. gingivalis and the development of periodontitis. The antibodies to F. nucleatum (P less than 0.05), E. corrodens (P less than 0.05), and A. actinomycetemcomitans were slightly higher in patients with periodontitis than in normal subjects. There were no remarkable differences between the two groups in titers to B. intermedius, B. loescheii, and Capnocytophaga sp.     

Highly specific protease-based approach for detection of porphyromonas gingivalis in diagnosis of periodontitis

Porphyromonas gingivalis is associated with the development of periodontitis. Here we describe the development of a highly specific protease-based diagnostic method for the detection of P. gingivalis in gingival crevicular fluid. Screening of a proteolytic peptide substrate library, including fluorogenic dipeptides that contain d-amino acids, led to the discovery of five P. gingivalis-specific substrates. Due to the presence of lysine and arginine residues in these substrates, it was hypothesized that the cleavage was mediated by the gingipains, a group of P. gingivalis-specific proteases. This hypothesis was confirmed by the observation that P. gingivalis gingipain knockout strains demonstrated clearly impaired substrate cleavage efficacy. Further, proteolytic activity on the substrates was increased by the addition of the gingipain stimulators dithiothreitol and l-cysteine and decreased by the inhibitors leupeptin and N-ethylmaleimide. Screening of saliva and gingival crevicular fluid of periodontitis patients and healthy controls showed the potential of the substrates to diagnose the presence of P. gingivalis proteases. By using paper points, a sensitivity of approximately 10(5) CFU/ml was achieved. P. gingivalis-reactive substrates fully composed of l-amino acids and Bz-l-Arg-NHPhNO(2) showed a relatively low specificity (44 to 85%). However, the five P. gingivalis-specific substrates that each contained a single d-amino acid showed high specificity (96 to 100%). This observation underlines the importance of the presence of d-amino acids in substrates used for the detection of bacterial proteases. We envisage that these substrates may improve the specificity of the current enzyme-based diagnosis of periodontitis associated with P. gingivalis.     

Crevicular Fluid and Serum Concentrations of Progranulin and High Sensitivity CRP in Chronic Periodontitis and Type 2 Diabetes

Introduction. This study was designed to correlate the serum and gingival crevicular fluid (GCF) levels of progranulin (PGRN) and high sensitivity C-reactive protein (hs CRP) in chronic periodontitis and type 2 diabetes mellitus (DM). Design. PGRN and hs CRP levels were estimated in 3 groups: healthy, chronic periodontitis, and type 2 DM with chronic periodontitis. Results. The mean PGRN and hs CRP concentrations in serum and GCF were the highest for group 3 followed by group 2 and the least in group 1. Conclusion. PGRN and hs CRP may be biomarkers of the inflammatory response in type 2 DM and chronic periodontitis.     

Modification of cystatin C activity by bacterial proteinases and neutrophil elastase in periodontitis.

AIM: To study the interaction between the human cysteine proteinase inhibitor, cystatin C, and proteinases of periodontitis associated bacteria. METHODS: Gingival crevicular fluid samples were collected from discrete periodontitis sites and their cystatin C content was estimated by enzyme linked immunosorbent assay (ELISA). The interaction between cystatin C and proteolytic enzymes from cultured strains of the gingival bacteria Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans was studied by measuring inhibition of enzyme activity against peptidyl substrates, by detection of break down patterns of solid phase coupled and soluble cystatin C, and by N-terminal sequence analysis of cystatin C products resulting from the interactions. RESULTS: Gingival crevicular fluid contained cystatin C at a concentration of approximately 15 nM. Cystatin C did not inhibit the principal thiol stimulated proteinase activity of P gingivalis. Instead, strains of P gingivalis and P intermedia, but not A actinomycetemcomitans, released cystatin C modifying proteinases. Extracts of five P gingivalis and five P intermedia strains all hydrolysed bonds in the N-terminal region of cystatin C at physiological pH values. The modified cystatin C resulting from incubation with one P gingivalis strain was isolated and found to lack the eight most N-terminal residues. The affinity of the modified inhibitor for cathepsin B was 20-fold lower (Ki 5 nM) than that of full length cystatin C. A 50 kDa thiol stimulated proteinase, gingipain R, was isolated from P gingivalis and shown to be responsible for the Arg8-bond hydrolysis in cystatin C. The cathepsin B inhibitory activity of cystatin C incubated with gingival crevicular fluid was rapidly abolished after Val10-bond cleavage by elastase from exudate neutrophils, but cleavage at the gingipain specific Arg8-bond was also demonstrated. CONCLUSIONS: The physiological control of cathepsin B activity is impeded in periodontitis, owing to the release of proteinases from infecting P gingivalis and neutrophils, with a contribution to the tissue destruction seen in periodontitis as a probable consequence.     

Direct detection of Prevotella intermedia and P. nigrescens in suppurative oral infection by amplification of 16S rRNA gene.

A specific 16S rDNA PCR and subsequent hybridisation reaction was designed to discriminate between strains of Prevotella intermedia (n = 15) and P. nigrescens (n = 15). This technique was then used to detect the presence of these two bacterial species in acute suppurative oral infection. A total of 36 pus samples aspirated from 26 peri-apical abscesses, three root canals, three periodontal abscesses, two cases of refractory periodontitis, one cyst and one haematoma was examined. A portion of the pus sample was processed by PCR and the remainder of the specimen was subjected to routine culture. The PCR-based technique gave an identical pattern of detection of P. intermedia or P. nigrescens to that obtained by culture for 30 of the 36 specimens. Either P. intermedia or P. nigrescens was present in 14 samples and neither species was detected in 16 samples. In the remaining six samples the PCR method indicated the presence of one (n = 3) or both (n = 3) of the Prevotella species but neither or only one species was isolated by culture. It is concluded that the presence of P. intermedia and P. nigrescens in pus can be detected rapidly and specifically by direct PCR amplification of 16S rDNA. P. nigrescens was detected more frequently than P. intermedia in suppurative peri-apical infection both by culture and PCR.     

Periodontitis vaccine decreases local prostaglandin E2 levels in a primate model

Interleukin-1beta, tumor necrosis factor alpha, prostaglandin E2 (PGE2), and Porphyromonas gingivalis-specific immunoglobulin G levels in gingival crevicular fluid were measured in primates immunized with a P. gingivalis vaccine followed by ligature-induced periodontitis. Only PGE2 levels were dramatically suppressed (P < 0.0001) in immunized animals versus controls. A significant correlation (P < 0.027) was also found between PGE2 levels and decreased bone loss scores. This study presents the first evidence of a potential mechanism involved in periodontitis vaccine-induced suppression of bone loss in a nonhuman primate model and offers insight into the role of PGE2 in periodontal destruction.