混合牙列双生子儿童口腔微生物组群结构的变性梯度凝胶电泳分析

目的通过变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)分析混合牙列期同卵双生子(monozygotic twins,MZ)和异卵双生子(dizygotic twins,DZ)口腔微生物群组结构的差异,同时比较双生子儿童中有龋及无龋儿童口腔微生物群组结构差异。方法同卵双生子7对,异卵双生子3对,其中有龋者8个,无龋者12个。提取唾液DNA后,使用通用引物扩增细菌的16 s rRNA V2区,PCR-DGGE电泳后进行数据分析。结果同卵双生子与异卵双生子均表现出明显的聚类分布,证明亲缘之间口腔微生物组群结构更为相似,但在同卵双生子与异卵双生子之间差异无统计学意义。在有龋儿童和无龋儿童之间比较,发现有龋组平均条带数(11.88±4.05)条,无龋组平均条带数(14.31±5.71)条,有龋组条带数较少,但两者之间差异无统计学意义(P>0.05)。结论对于口腔微生物群组的构成,环境的影响可能大于遗传的影响。在有龋儿童中,微生物种类有减少。     

2型糖尿病患者肠道菌群两种分子指纹图谱分析研究

目的 利用重复共有序列(ERIC)和变性梯度凝胶电泳(DGGE)两种分子指纹图谱技术对2型糖尿病患者肠道菌群结构特征分析,探讨2型糖尿病与肠道菌群的相关性及对两种方法的评价。方法 收集8例健康人和7例2型糖尿病患者粪便样本,提取粪便菌群总DNA,采用ERIC-PCR和DGGE-PCR分子指纹图谱技术对两组人群肠道菌群结构分析,比较其多样性、相似性等生态学特征。结果 与健康对照组比较,2型糖尿病患者肠道菌群图谱条带和Shannon-Wiener多样性指数降低,但无统计学意义; 组内相似性降低差异有统计学意义(P＜0.05),菌群结构发生变化; 两种指纹图谱技术均能直观反映肠道菌群结构特征,ERIC方法简便,反映菌群多样性良好,但是实验影响因素较多,不可切胶测序; DGGE能较好反映菌群多样性、相似性等生态学特征,而且可选择条带切胶测序。结论 2型糖尿病患者肠道菌群组成结构发生改变,糖尿病的发生与肠道菌群有一定相关性; ERIC和DGGE是研究肠道菌群分辨效率高、重复性好的指纹图谱技术,DGGE并可进行切胶测序比对鉴定细菌,二者可结合使用。     

口腔干燥时唾液的水解酶活性

本文研究了舍格林综合征有口腔干燥症状患者唾液的性质及酶的活性。对21例有明显口腔干燥的舍格林综合征病人和31名健康人(对照组)作咀嚼橡皮试验,测定了唾液分泌量、可溶性蛋白含量、脱落上皮细胞和白细胞计数、唾液的硷性和酸性磷酸酶活性、pH3.5和7.5时的酸性及中性蛋白酶活性。结果与讨论:口腔干燥患者的口粘膜干燥、充     

中国健康人血清游离表皮生长因子受体外显子18-22序列分析

目的 分析中国健康人血清游离DNA中表皮生长因子受体(EGFR)外显子18～22基因序列是否存在突变,为监测EGFR酪氨酸激酶选择性抑制荆(EGFR TKIs)的临床疗效提供正常参考数据.方法 采用高灵敏度DNA提取方法结合高保真巢式PCR和基因测序技术对42例中国健康人外周血血清游离DNA中EGFR TKIs作用靶点所在结构域即EGFR外显子18～22的基因序列和氨基酸序列进行分析.结果 从外周血血清游离DNA中均扩增出EGFR外显予18～22的基因片段,片段大小(分别为400,374,404,408争419 bp)与预期设计片段完全相符.健康人外周血游离DNA中存在EGFR基因突变,突变频率出现最多的是EGFR外显子20.41名健康人样本中有7例样本出现1处相同位点的沉默点突变(4838499 G→A),其氨基酸序列(Glu)没有发生改变;EGFR外显子18也出现点突变,34名健康人样本中有1名健康者样本出现3处点突变,其中2处点突变(4830989 G→C;4831025 G→C)导致氨基酸序列发生改变(Glu→Gln;Ala-Pro),另外1处点突变(4831000 A→C)为沉默突变,其氨基酸序列(Thr)没有发生变化;EGFR外显子19、外显子21～22没有出现基因突变.结论 通过巢式PCR结合基因测序可以分析外周血游离DNA中EGFR基因序列,健康人外周血游离EGFR外显子18和20存在基因点突变,其中外显子18的点突变导致了氨基酸序列改变.     

口腔扁平苔藓患者唾液白介素17的检测

目的:检测口腔扁平苔藓患者(oral lichen planus,OLP)治疗前后唾液中白介素17(IL-17)的水平,研究唾液IL-17与OLP发病以及病情变化的关系.方法:分别收集OLP患者治疗前后和健康对照组的唾液样本,用ELISA方法检测唾液中IL-17的表达水平.结果:OLP患者治疗前后及健康对照组的唾液样本中均未检出IL-17.结论:唾液中IL-17浓度较低,需进一步完善唾液保存方法和提高检测技术的灵敏度来检测唾液中IL-17 表达水平.     

外周血检测miR-21方法的建立及在支气管哮喘中的应用

目的设计茎环结构引物,建立外周血检测微小RNA-21(miR-21)的检测方法,并初步应用于支气管哮喘患者外周血单个核细胞(PBMC)样本的检测。方法设计miR-21的茎环结构反转录引物和聚合酶链反应(PCR)扩增引物,以U6为内参照,利用SYBR green实时荧光PCR检测20例支气管哮喘患者和20名健康人的外周血miR-21的表达水平,并分析2组间的差异。结果 SYBR green实时荧光PCR检测U6和miR-21含量的熔解曲线单一,PCR产物特异。在外周血中20例哮喘患者的miR-21水平明显高于20名健康人(P<0.001)。结论本实验成功建立了SYBR green荧光定量PCR检测外周血miR-21表达水平的技术平台,为miR-21在支气管哮喘发生中分子生物学机制研究建立了基础。     

血清及唾液中干扰素γ和白细胞介素10水平与不同类型的口腔扁平苔藓(英文)

背景:许多细胞因子在唾液和血清中都能检测出,并且在口腔黏膜疾病的诊断,预后的监控和治疗方面更具有临床意义。目的:比较不同类型口腔扁平苔藓患者血清及唾液中的白细胞介素10和干扰素γ的水平,探索唾液作为生物学样本代替血液来研究口腔扁平苔藓中干扰素γ和白细胞介素10的可行性。方法:收集2014年1至7月在佳木斯大学附属口腔医院牙周黏膜病科就诊的口腔扁平苔藓患者45例,根据患者疾病类型分为糜烂型(糜烂组)15例,充血红斑型(充血红斑组)15例,网纹型(网纹组)15例。另取15名就诊于佳木斯大学附属口腔医院体检科身体健康者作为对照组。采用ELISA法分别检测4组对象血清和唾液中干扰素γ和白细胞介素10的表达水平。结果与结论:与正常组相比,口腔扁平苔藓患者血清和唾液中干扰素γ水平较低(P<0.01),且糜烂组、充血红斑组和网纹组患者血清和唾液中干扰素γ水平差异有显著性意义。与正常组相比,糜烂组和充血红斑组患者血清和唾液中白细胞介素10水平较高(P<0.01或P<0.05);与网纹组相比,糜烂组和充血红斑组患者血清和唾液中白细胞介素10水平较高。提示口腔扁平苔藓患者血清和唾液中干扰素γ和白细胞介素10水平高度相关,可以通过检查唾液替代血液来研究口腔扁平苔藓中干扰素γ和白细胞介素10的水平。     

揉按颊车穴与叩齿对于健康人唾液分泌的影响研究

目的 探讨颊车穴揉按与叩齿法对健康人唾液流率以及口腔湿润主观评分的影响,为临床口腔干燥症患者的中医护理提供前期研究基础。方法 采用随机对照试验,纳入135名符合标准受试者。采用信封法,随机分为颊车穴揉按组、叩齿组以及空白组,每组45名。分别记录受试者揉按穴位后、叩齿后以及未干预情况下后的唾液流率和口腔湿润主观评分。结果 穴位揉按组、叩齿组和空白组的唾液流率差异有统计学意义（P<0.001）,叩齿组最高。颊车穴揉按组、叩齿组口腔湿润主观评分高于空白组,差异有统计学意义（P<0.001）。结论 颊车穴揉按和叩齿法均能有效刺激唾液分泌,有效滋润口腔黏膜。     

肺癌患者唾液中细菌构成分析

目的探讨唾液中细菌构成与肺癌的关系。方法收集鳞癌和腺癌以及非肿瘤对照组唾液标本,每组10例。对唾液中大量的16s rDNA进行测序分析。结果经定量PCR(qPCR)证实,5个细菌集群在癌症和对照组样本间有显著差异。结论 16s测序和PCR相结合的研究显示,有两个细菌属(二氧化碳噬纤维菌属和韦永氏球菌属)水平癌症组要显著高于非癌症组。     

骨髓移植受者唾液免疫成分的定量测定

骨髓移植受者常出现口腔及唾液腺并发症,如粘膜炎症,细菌、病毒、真菌感染,口腔干燥症及龋齿等。唾液中分泌型免疫球蛋白(主要为sIgA、IgG)是口腔粘膜抵御微生物及病毒入侵的主要成分。为了骨髓移植抵御受者唾液免疫球蛋白的含量变化,作者对42例患者进行     

Detection of Mycoplasma pneumoniae P1 subtype variations by denaturing gradient gel electrophoresis

There were several methods to detect p1 gene variations in Mycoplasma pneumoniae. In this study polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) assay was performed to establish a rapid and precise detection method for identifying M. pneumoniae p1 gene variations. We detected p1 gene variations in 109 M. pneumoniae clinical isolates from Shanghai, China, which were collected from 2009 to 2011 by DGGE, and compared this method with the PCR-based restriction fragment length polymorphism assay and sequencing. By PCR-DGGE method, among the 109 M. pneumoniae isolates, 101 (92.7%) isolates were classified into type I, and 8 (7.3%) were classified into type II. Seven (6.9%) type I variations and 8 (100%) type II variations were identified. The match rate of p1 gene variation detected by DGGE reached 100% when compared to DNA sequencing and was more sensitive than restriction fragment length polymorphism. One new type II variant, designated as V2d, was found in this study. The sequence of the new variant was characterized. Our results indicated that PCR-DGGE is a rapid and reliable bio-technique for direct detection of p1 gene variations.     

Salivary cortisol for the evaluation of Cushing's syndrome

Cortisol concentrations were measured in matched plasma and salivary samples from 8 healthy controls, 8 patients with Cushing's syndrome and 4 patients suspected of having spontaneous hypercortisolism. In healthy subjects, the circadian rhythm in salivary cortisol paralleled that in plasma. Absence of the diurnal rhythm in Cushing's syndrome was seen in saliva as well as in plasma. After ACTH stimulation, mean peak cortisol in saliva showed a 3-fold increase while in plasma there was a 2.5-fold increment above baseline. Cushing's syndrome, due to pituitary or adrenal adenoma was diagnosed equally well by measuring the cortisol response to cosyntropin in either plasma or saliva. Finally, the low- and high-dose dexamethasone suppression test was reflected equally well in both plasma and saliva. In patients suspected of having Cushing's syndrome dynamic tests can be performed in both plasma and saliva. However, in some samples, the salivary cortisol measurement appears advantageous over plasma cortisol determination.     

Salivary IgA concentration is influenced by the saliva collection method.

Measurement of salivary IgA is useful for the non-invasive assessment of secretory immunity, especially in children and infants. In our study, the influence of three commonly used methods ("spitting", "suction", "Salivette") of saliva collection on the yield of salivary IgA concentration was analysed in 54 samples of salivary secretion collected from six healthy children according to a cross over protocol. Nephelometrically determined IgA concentrations were significantly lower in saliva collected by the Salivette device (mean +/- SEM: 23 +/- 7 mg/l) than in saliva collected by the suction (46 +/- 8 mg/l) or spitting method (48 +/- 8 mg/l). Salivary flow assessed by the spitting method was inversely correlated with salivary IgA concentration. We conclude that salivary IgA assessment is influenced by the saliva collection method, and that studies dealing with this topic should accurately describe the methods used for collecting saliva so that data may be properly compared.     

维吾尔医学异常黑胆质体液结肠癌患者肠道菌群的分布情况研究

目的通过先进的分子生物学方法分析维吾尔医学异常黑胆质结肠癌患者肠道菌群分布情况及多样性。方法对结肠癌患者进行维吾尔医学体液分型并挑取其中异常黑胆质患者,采集患者粪便样品,提取细菌总DNA并设计一对通用引物扩增16SrDNA的V6~V8可变区进行变形梯度凝胶电泳(DGGE),从DGGE指纹图谱中比较各个结肠癌患者肠道菌群分布情况。结果通过试验得到了维吾尔族异常黑胆质结肠癌肠患者肠道菌群结构特征的DNA指纹图谱,从指纹图谱上选择一些特异性条带切下来回收,测序,测序出来的序列在基因库进行比对做出进化树了解菌群之间的亲缘性。结论异常黑胆质结肠癌患者肠道菌群基因序列的亲缘性结果显示肠道菌群的多样性明显少其中肠道优势菌比例低。     

Detection by denaturing gradient gel electrophoresis of pncA mutations associated with pyrazinamide resistance in Mycobacterium tuberculosis isolates from the United States-Mexico border region

Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with pyrazinamide (PZA) resistance in the pncA gene of Mycobacterium tuberculosis. DGGE scans for mutations across large regions of DNA and rivals sequencing in its ability to detect DNA alterations. Specific mutations can often be recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint. Five PCR target fragments were designed to scan for DNA alterations across 600 bp of pncA in 181 M. tuberculosis isolates from patients residing in the U.S-Mexico border states of Texas and Tamaulipas, respectively. A region of pncA was observed with a high GC content and a melting temperature approaching 90 degrees C that was initially refractory to denaturation, and a DGGE target fragment was specifically designed to detect mutations in this region. DGGE detected pncA mutations in 82 of 83 PZA-resistant isolates. By contrast, only 1 of 98 PZA-susceptible isolates harbored a detectable DNA alteration. The pncA gene was sequenced from 41 isolates, and 32 DNA alterations in 32 PZA-resistant isolates were identified, including 11 new mutations. DGGE also detected nine isolates whose susceptibility to PZA appeared to be incorrect, and DNA sequencing confirmed these apparent errors in drug susceptibility testing. These results demonstrate the power and usefulness of DGGE in detecting mutations associated with PZA resistance in M. tuberculosis.     

垂直变性梯度凝胶电泳在大肠癌APEX基因变异检测中的应用

目的探讨变性梯度凝胶电泳(denaturinggradientgelelectrophoresis,DGGE)在大肠癌APEX基因单核苷酸多态性(singlenucleotidepolymorphism,SNP)或基因突变筛选中的应用价值。方法对散发性大肠癌APEX基因5个外显子进行DGGE检测及DNA测序,分析DGGE带型和测序结果之间的关系。结果根据DGGE原理及实验观察结果,发现无论何种碱基改变,在DGGE胶上通常只可能呈现4种带型中的一种,本研究发现其中的3种。杂合子的特征是在变性胶的泳道上出现不同水平的4条带,纯合子呈单一条带;带有错配的DNA分子在凝胶上的位置总是落后于没有错配的分子;当碱基改变为A/T→G/C时,突变DNA分子泳动较快,其条带位于野生型分子之前,当碱基改变为G/C→A/T时,结果相反。结论对于一已知的SNP,利用DGGE带型可以判断SNP的基因型和等位基因频率而免去进一步测序工作;对突变检测而言,DGGE可预测碱基改变的类型,有利于对测序结果判读的准确性。     

Detection of rpoB mutations associated with rifampin resistance in Mycobacterium tuberculosis using denaturing gradient gel electrophoresis

Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with rifampin (RIF) resistance in the rpoB gene of Mycobacterium tuberculosis. DGGE scans for mutations across large regions of DNA and is comparable to DNA sequencing in detecting DNA alterations. Specific mutations are often recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint. Five DGGE primer sets that scanned for DNA alterations across 775 bp of rpoB were developed. These primer sets were used to scan rpoB for DNA alterations in 296 M. tuberculosis patient isolates from the United States-Mexico border states of Texas and Tamaulipas. The most useful primer set scanned for mutations in the rifampin resistance-determining region (RRDR) and detected mutations in 95% of the RIF-resistant isolates compared to 2% of RIF-susceptible isolates. Thirty-four different alterations were observed within the RRDR by DGGE. In addition, isolates harboring mixtures of DNA within rpoB were readily detected by DGGE. A second PCR primer set was used to detect the V146A mutation in 5 to 7% of RIF-resistant isolates. A third primer set was used to detect mutations in 3% of RIF-resistant isolates, some of which also harbored mutations in the RRDR. Only 1 of 153 RIF-resistant isolates did not have a detectable rpoB mutation as determined by DGGE and DNA sequencing. These results demonstrate the power and usefulness of DGGE in detecting mutations associated with drug resistance in M. tuberculosis.     

Recovery of pefloxacin in saliva and feces and its action on oral and fecal floras of healthy volunteers.

Pefloxacin, a new fluoroquinolone, was given to 10 volunteers in single 400-mg oral doses repeated at 12-h intervals during 7 days. Serum, saliva, and feces samples were collected before and at appropriate intervals after the initiation of treatment. Drug concentrations were determined by bioassay. Qualitative and quantitative analyses of the saliva and fecal floras were performed. Mean concentrations in saliva (3.46 micrograms/ml on day 1 and 7.54 micrograms/ml on day 7) were closely related to levels in serum. High concentrations of pefloxacin were found in the feces (645 micrograms/g on day 8). No modification of oral flora was observed. In the fecal flora, members of the family Enterobacteriaceae were eliminated between days 2 and 8. The alterations in streptococci and anaerobic flora were not significant; Bacteroides fragilis was more resistant to pefloxacin after treatment. Clostridium difficile was not detected, and there was no overgrowth by yeasts. No side effects were observed.     

Correlation between concentrations of 8-oxo-7,8-dihydro-2′-deoxyguanosine in urine,plasma and saliva measured by on-line solid-phase extraction LC-MS/MS

Background8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) is the most frequently measured biomarker of oxidative stress. Chromatographic-based methods for 8-oxodGuo in urine are well established; however, the 8-oxodGuo measurement in plasma and saliva has been problematic.MethodsWe firstly and successfully applied an on-line solid-phase extraction (SPE) LC-MS/MS following manual SPE pretreatment to quantify the 8-oxodGuo both in plasma and saliva. Urine, plasma and saliva specimens were simultaneously collected from 50 healthy adults and measured for 8-oxodGuo.ResultsMean baseline levels of 8-oxodGuo in plasma and saliva were 21.7 ± 9.2 and 5.1 ± 2.6 pg/ml, respectively, being far lower than that in urine (6.2 ± 4.8 ng/ml). The 8-oxodGuo levels obtained in this study for plasma and saliva were, however, up to several hundred times lower than those reported by commercial ELISA kit in the literature. Furthermore, the 8-oxodGuo levels in plasma and saliva were significantly correlated with the 8-oxodGuo levels in urine (Spearman correlation coefficients, r = 0.33, P = 0.02 for plasma and r = 0.56, P = 0.0015 for saliva). 8-OxodGuo in plasma was also correlated with the 8-oxodGuo in saliva (r = 0.52, P = 0.0041).ConclusionsSignificantly correlations were observed between plasma, saliva and urine, giving the possibility of using other body fluids in addition to urine for assessing whole body oxidative stress.