变异链球菌F-ATPase亚基β基因多态性及表达研究

目的研究变异链球菌（Streptococcus燃，简称变链菌）临床分离株耐酸因子F-AT—Pase亚基B结构基因uncD的遗传多态性和mRNA表达水平差异，探讨其与细菌耐酸力的关系。方法实验株包括18株高耐酸和20株低耐酸变链菌临床株，以特异引物从细菌组DNA扩增uncD，行限制性内切酶长度多态性分析（RFLP）和测序比较；应用半定量RT-PCR两步法和凝胶成像系统定量软件，对20株不同基因型和不同耐酸力变链菌uncD基因的mRNA表达水平进行评价和比较。结果AluI—RFLP产生A、B基因型，测序证实导致多态出现的基因变异不在uncD的功能区和催化结构域；这两种基因型在不同耐酸力菌株的分布不同（P〈0．05），高耐酸性菌株中A型基因uncD的检出高于低耐酸力菌株。不同耐酸力菌株uncD的mRNA表达水平不同（P〈0．05），但A、B两基因型菌株uncD的mRNA表达差异没有统计学意义（P〉0．05）。结论F-ATPase的β亚基基因具有明显的遗传多态性，基因型和mRNA表达水平的多态性与菌株的耐酸力有一定的关系，虽然β亚基基因功能区高度保守，但在酸耐受适应中，仍表现为F—ATPase较活跃上调的亚基基因。     

血清BLyS和APRIL水平与非霍奇金氏病关联性的初步研究

目的：研究非霍奇金氏病（NHL）患者血清B淋巴细胞刺激因子（BLyS）和增殖诱导配体（APRIL）蛋白水平，并探讨其与血清乳酸脱氢酶（LDH）的关系。方法：应用酶联免疫吸附试验（ELISA）法检测LDH升高患者组（10例）和LDH正常NHL患者组（12例）NHL患者血清BLyS和APRIL水平，以健康志愿者（23例）作为对照；同时将血清BLyS和APRIL水平与患者血清乳酸脱氢酶（LDH）进行相关性分析。结果：NHL患者组血清BLyS水平显著低于健康对照组（P〈0.05）；NHL患者血清APRIL水平与健康对照组无显著性差异（P〉0.05），但LDH升高患者组血清APRIL水平明显高于LDH正常患者组（P〈0.05）；NHL患者组和LDH升高患者组血清APRIL水平与LDH正相关（r=0.923、0.799，P〈0.05）。结论：NHL患者血清BLyS、APRIL水平与健康对照组差异，提示BLyS和APRIL可能与NHL的预后及恶性程度有关。     

差异显示技术对变异链球菌生物膜基因表达差异的分析

目的 对生物膜和浮游状态下的变异链球菌细胞进行比较研究,采用限制性酶切差异显示技术比较分析两者的基因表达谱.方法 分别收集变异链球菌浮游菌细胞与贴壁的生物膜细胞,分离纯化总RNA,对于逆转录获得的cDNA进行限制性酶切差异显示PCR技术（RFDD-PCR）比较分析两者的表达谱,对于获得的差异表达片段通过克隆测序并于BLAST比对获得这些片段的基因信息,进而推断其功能.结果 通过对差异片段的分析,确证其中4条片段分属结构基.fruA、SMU.438c、SMU.751与adhB/C.结论 限制性酶切差异显示技术可用于分析生物膜形成过程中基因表达的差异.     

LDH及sTfR在成人急性溶血性贫血中的意义

目的 探讨血清乳酸脱氢酶(lactic dehydrogenase,LDH)及可溶性血清转铁蛋白受体(soluable transferritin receptor,sTfR)在无慢性贫血史成人急性溶血性贫血(acute hemolutic anemia,AHA)诊断中的意义.方法 无慢性溶血性疾病史的32例成人急性溶贫患者采用酶标比色法测定血清LDH水平,ELISA方法检测血清sTfR水平并与正常体检者进行比较分析.结果 急性溶贫患者血清sTfR水平为49.3±13.1 nmol/L,对照组为15.5 ±2.1 nmol/L,两组比较有统计学差异(P＜0.05);急性溶贫患者血清LDH水平为531.1 ±111.2 IU/L,对照组为105.5 ±42.1 IU/L,两组比较有统计学差异(P＜0.05);不同程度AHA患者血清LDH及sTfR不同.结论 血清LDH、sTfR活性可作为无慢性溶血性疾病成人AHA诊断筛选.     

纳米铁对小鼠血清乳酸脱氢酶及其同工酶的影响

目的 探讨大剂量纳米粒径铁粉经口染毒对小鼠血清乳酸脱氢酶（LDH）及其同工酶的影响.方法 40只CD小鼠随机均分（雌雄各半）为对照（Control）组、微米粒径铁粉（Micro-Fe）组、微米粒径氧化铁粉（Micro-Fe2O3）组和纳米粒径铁粉（Nano-Fe）组.将以上材料制成水溶液,以羧甲醛纤维素为辅剂,以5 g/Kg体重剂量一次经口灌胃,14天后处死,分别测定脏器系数,取血分离血清,测定LDH及其同工酶、α-HBDH活性变化.结果 Micro-Fe、Micro-Fe2O3和Nano-Fe组小鼠肝脏系数明显高于对照组（P＜0.05）,血清LDH活性明显低于对照组（P＜0.05）,4组小鼠血清α-HBDH活性间差异无显著性（P＞0.05）.Nano-Fe组小鼠血清LDH3明显低于Micro-Fe组,但与Control组和Micro-Fe2O3组相比差异不显著;Nano-Fe组小鼠血清LDH4和LDH5明显高于对照组（P＜0.05）.结论 大剂量铁摄取可能对小鼠血清LDH具有一定抑制效应,Nano-Fe对血清LDH同工酶的影响与Micro-Fe和Micro-Fe2O3有所不同,血清LDH同工酶分析可能比血清LDH能更好地反映Nano-Fe对生物效应的影响.     

B淋巴细胞刺激因子及其受体与多发性骨髓瘤关联性的临床实验诊断研究

目的:探讨B细胞激活因子(BLyS) 及其受体在多发性骨髓瘤(MM)发病中的作用机制.方法:用酶联免疫吸附测定法(ELISA)和实时荧光定量聚合酶链反应 (RFQ-PCR),对19例MM患者和30名健康人血清BLyS含量、外周血单个核细胞(PBMCs) 中BLyS及其受体mRNA 含量进行检测;并比较MM患者血清BLyS含量与外周血BLyS及其受体基因表达水平和免疫球蛋白(IgG、IgA、IgM)浓度、乳酸脱氢酶(LDH)、轻链(kap、lam)的相关性.结果:MM患者血清 BLyS含量[(7.95±4.93) g/L 明显高于健康对照组[(2.70±1.09) g/L,P<0.01 ,并与LDH浓度呈正相关性(r=0.734,P<0.001);97.4% (18/19),84.2% (16/19)的MM患者PBMCs中BLyS、BCMA mRNA表达水平较正常人明显增高,并且也与LDH的含量呈正相关;而47.32% (9/19)患者TACI mRNA表达水平降低.结论:MM患者的血清BLyS蛋白浓度和PBMCs中BLyS及其受体基因表达水平有异常改变,说明BLyS及其受体可能参与MM的发病;BLyS及其受体表达水平也许可作为预后的一个指标.     

乳酸脱氢酶在前列腺增生与前列腺癌中的分析

目的 探讨乳酸脱氢酶在前列腺增生与前列腺癌患者血清中的表达情况,在前列腺穿刺活检或手术前的诊断价值.方法 回顾性分析2018年1月至2021年3月就诊于首都医科大学附属北京世纪坛医院进行前列腺手术或穿刺活检患者临床资料,按照纳入及排除标准,最终选择262例患者,其中前列腺癌89例,前列腺增生患者173例.以病理结果为诊断标准,使用SPSS 21.0软件进行数据分析处理,比较前列腺癌与前列腺增生组间乳酸脱氢酶表达水平,并分析疾病诊断风险因素,绘制受试者工作曲线(receiver operating characteristic curve,ROC),分析术前乳酸脱氢酶对于前列腺癌疾病预测的诊断价值.结果 乳酸脱氢酶在前列腺癌血清中表达水平显著高于前列腺增生患者,差异具有统计学意义(P<0.05).单变量Logistic回归研究发现术前血清乳酸脱氢酶(P=0.010)是预测前列腺癌的独立危险因素之一.将乳酸脱氢酶用于前列腺癌诊断,其曲线下面积为0.610,约登指数最高时乳酸脱氢酶截止值为173.5 U/L时,特异性为72.3％,灵敏度为50.6％.结论 血清乳酸脱氢酶对于前列腺癌有一定诊断价值,可以作为前列腺疾病辅助诊断指标之一.     

蔗糖浓度对口腔变形链球菌gbpC基因表达的影响

目的 观察不同浓度蔗糖环境对变形链球菌葡聚糖结合蛋白C编码基因gbpC表达的影响.方法 变形链球菌分别在0.5%、1.0%、5.0%蔗糖条件下培养,提取总RNA,逆转录成cDNA,利用TaqMan实时荧光定量PCR技术检测不同环境条件下变形链球菌gbpC基因的表达.结果 当蔗糖浓度从0.5%升高至1.0%时变形链球菌gbpC基因表达明显上调(P<0.05),而5.0%蔗糖较1.0%蔗糖条件下gbpC表达无明显改变(P>0.05);黏附较强菌株其gbpC基因表达高于黏附较弱菌株,特别是在0.5%和1.0%蔗糖环境中差异具统计学意义(P<0.05).结论 蔗糖量从0.5%增至1.0%可促进变形链球菌gbpC基因表达,这可能是蔗糖促进变形链球菌黏附的机制之一;gbpC基因的表达量可能与变形链球菌黏附力相关.     

急性白血病患者血清LDH、α-HBDH检测的临床价值

急性白血病（acute leukemia,AL）是临床较常见的恶性肿瘤,不仅表现外周血及骨髓的异常,而且存在酶学的异常表达[1]。本文通过测定AL患者化疗前、化疗后完全缓解（CR）、未缓解（NR）、治疗后复发（RC）时血清乳酸脱氢酶（LDH）及α-羟丁酸脱氢酶（α-HBDH）水平变化,探讨其在AL病程进展及预后中的临床价值。     

子宫内膜异位症TGF-β_1及其基因的表达及意义

目的研究子宫内膜异位症中转化生长因子β1（TGF-β1）及其基因表达,探讨TGF-β1在子宫内膜异位症中的作用机制。方法 54例子宫内膜异位症异位内膜、在位内膜,50例对照组正常子宫内膜中,采用免疫组化方法检测TGF-β1的表达,SYBR Green实时定量聚合酶链反应（RQ-PCR）检测TGF-β1 mRNA的表达量。结果 TGF-β1在子宫内膜异位症异位内膜组中的蛋白表达显著高于在位内膜组及对照组（P〈0.05、P〈0.05）;TGF-β1在子宫内膜异位症在位内膜组中的表达与对照组中的表达无显著性差异（P〉0.05）;TGF-β1mRNA在子宫内膜异位症异位内膜中的表达量显著高于在位内膜组和对照组（P〈0.05、P〈0.05）,TGF-β1mRNA在位内膜组的表达量与对照组中的表达量无显著性差异（P〉0.05）。结论 TGF-β1可能参与子宫内膜异位症的发病机制,影响其发生发展。     

Identification of unique bacterial gene segments from Streptococcus mutans with potential relevance to dental caries by subtraction DNA hybridization

Using DNA subtractive hybridization, 49 unique gene segments were identified from a strain of Streptococcus mutans that was isolated from a patient with severe early childhood caries (S-ECC). Further hybridization with DNA from other S. mutans strains isolated from both caries-active and caries-free subjects yielded five unique sequences of DNA common to strains associated with S-ECC.     

不同龋敏感儿童牙菌斑内微生物群落的分子生态学特点

背景：玻璃离子水门汀充填的儿童牙体仍易发生继发龋现象,与充填材料表面牙菌斑内复杂微生物群落具有密切关系,而传统微生物学方法无法获知牙菌斑微生物的重要信息。 目的：利用先进的现代分子生态学技术解析不同龋敏感儿童玻璃离子水门汀表面牙菌斑内微生物群落结构与重要致龋微生物的数量水平。 方法：选择3-5岁儿童24名,按乳牙龋失补牙面指数不同分为无龋组、中龋组和高龋组,每组8名。采集各组儿童全口牙面菌斑,利用变性梯度凝胶电泳进行微生物群落多样性分析与微生物种群鉴定,利用荧光原位杂交考察致龋微生物Streptococcus spp.的数量分布,利用实时定量PCR考察重要致龋菌Streptococcus mutans占总菌的相对数量。 结果与结论：无龋组牙菌斑内微生物群落的多样性显著高于中龋组和高龋组(P < 0.05),中龋组和高龋组中大量富集的某些微生物可能在龋病发展过程中起重要作用。3组样本共检出16个微生物菌属,Streptococcus spp.和Actinomyces spp.可能是高龋组中重要的致龋微生物。Streptococcus spp.和Streptococcus mutans在高龋组中所占比例显著高于无龋组和中龋组(P < 0.01)。综合来看,研究采用的分子生态学技术可以较好反映牙菌斑内与致龋过程密切相关的复杂微生物群落。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

Microbial risk indicators of early childhood caries

The aim of this study was to use molecular identification methods, such as 16S RNA gene sequence and reverse-capture checkerboard hybridization, for identification of the bacteria associated with dental caries and with dental health in a subset of 204 twins aged 1.5 to 7 years old. A total of 448 plaque samples (118 collected from caries-free subjects and 330 from caries-active subjects) were used for analysis. We compared the bacteria found in biofilms of children exhibiting severe dental caries, with different degrees of lesion severity, with those found in biofilms of caries-free children. A panel of 82 bacterial species was selected, and a PCR-based reverse-capture checkerboard method was used for detection. A simple univariate test was used to determine the overabundance and underabundance of bacterial species in the diseased and in the healthy groups. Features identified with this univariate test were used to construct a probabilistic disease prediction model. Furthermore, a method for the analysis of global patterns of gene expression was performed to permit simultaneous analysis of the abundance of significant species by allowing cross-bacterial comparisons of abundance profiles between caries-active and caries-free subjects. Our results suggested that global patterns of microbial abundance in this population are very distinctive. The top bacterial species found to be overabundant in the caries-active group were Actinomyces sp. strain B19SC, Streptococcus mutans, and Lactobacillus spp., which exhibited an inverse relationship to beneficial bacterial species, such as Streptococcus parasanguinis, Abiotrophia defectiva, Streptococcus mitis, Streptococcus oralis, and Streptococcus sanguinis.     

Characterization of preparations enriched for Streptococcus mutans fimbriae: salivary immunoglobulin A antibodies in caries-free and caries-active subjects.

The ability of bacteria to adhere to salivary pellicle-coated enamel tooth surfaces is a critical step in oral bacterial colonization. Oral bacteria adhere to receptors of host origin in salivary pellicle. Streptococcus mutans has been identified as the major etiological agent of human dental caries and composes a significant proportion of the oral streptococci in carious lesions. Bacterial fimbriae are small (100 to 300 nm) hairlike appendages emanating from the cell surface. Preparations enriched for S. mutans fimbriae were isolated by a shearing technique and alternating high- and low-speed centrifugations. A representative fimbrial preparation had two distinct double bands comprising four proteins of approximately 100 to 200 kDa and one faint band at 40 kDa on reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis/immunoblots and had demonstrable glucosyltransferase activity. Rabbit antisera raised against the preparation specifically stained the fuzzy coat of S. mutans, demonstrating short fimbria-like structures protruding 100 to 200 nm from the cell surface. Controls without antifimbria antibody did not exhibit this staining. There were significantly higher (P < or = 0.05) levels of salivary immunoglobulin A, but not serum immunoglobulin G, antibodies to the enriched S. mutans fimbria preparation by enzyme-linked immunosorbent assay from caries-free subjects than from caries-active subjects. The results suggest that S. mutans fimbriae may be an important adherence factor to which caries-free subjects mount a protective salivary immune response.     

Cariogenicity of Streptococcus mutans V403 glucosyltransferase and fructosyltransferase mutants constructed by allelic exchange.

Streptococcus mutans produces several enzymes which metabolize sucrose. Three glucosyltransferase genes (gtfB, gtfC, and gtfD) and a single fructosyltransferase gene (ftf) encode enzymes which are important in formation of exopolysaccharides. Mutants of S. mutans V403 carrying single and multiple mutations of the gtfB, gtfC, gtfD, and ftf genes recently have been constructed by allelic exchange in our laboratory. Using selected strains from this panel of mutants, we examined the importance of water-insoluble glucan, water-soluble glucan, and fructan production in cariogenicity while controlling for the effects of strain and species variability. Genetic and biochemical characterization of mutants and assays of glucosyltransferase and fructosyltransferase activities were performed to ensure that the phenotypes of strains coincided with deficiencies predicted by genotype. The young gnotobiotic rat model of cariogenicity was used to assess virulence of the wild-type strain and isogenic mutants. Mutant strains were less virulent than the wild type in almost every location examined for caries on tooth surfaces and level of involvement of lesions (depth and severity). Inactivation of either gtfB and gtfC or ftf dramatically reduced virulence; the subsequent inactivation of gtfD did not enhance the effect of reduced virulence.     

Virulence of a spaP mutant of Streptococcus mutans in a gnotobiotic rat model

Streptococcus mutans, the principal etiologic agent of dental caries in humans, possesses a variety of virulence traits that enable it to establish itself in the oral cavity and initiate disease. A 185-kDa cell surface-localized protein known variously as antigen I/II, antigen B, PAc, and P1 has been postulated to be a virulence factor in S. mutans. We showed previously that P1 expression is necessary for in vitro adherence of S. mutans to salivary agglutinin-coated hydroxyapatite as well as for fluid-phase aggregation. Since adherence of the organism is a necessary first step toward colonization of the tooth surface, we sought to determine what effect deletion of the gene for P1, spaP, has on the colonization and subsequent cariogenicity of this organism in vivo. Germ-free Fischer rats fed a diet containing 5% sucrose were infected with either S. mutans NG8 or an NG8-derived spaP mutant strain, PC3370, which had been constructed by allelic exchange mutagenesis. At 1-week intervals for 6 weeks after infection, total organisms recovered from mandibles were enumerated. At week 6, caries lesions also were scored. A significantly lower number of enamel and dentinal carious lesions was observed for the mutant-infected rats, although there was no difference between parent and mutant in the number of organisms recovered from teeth through 6 weeks postinfection. Coinfection of animals with both parent and mutant strains resulted in an increasing predominance of the mutant strain being recovered over time, suggesting that P1 is not a necessary prerequisite for colonization. These data do, however, suggest a role for P1 in the virulence of S. mutans, as reflected by a decrease in the cariogenicity of bacteria lacking this surface protein.     

Lactate dehydrogenase from Streptococcus mutans: purification, characterization, and crossed antigenicity with lactate dehydrogenases from Lactobacillus casei, Actinomyces viscosus, and Streptococcus sanguis.

A cytoplasmic fructose-1,6-diphosphate-dependent lactate dehydrogenase (LDH; EC 1.1.1.27) from Streptococcus mutans OMZ175 was purified to homogeneity as judged by sodium dodecyl sulfate-gel electrophoresis. The purification consisted of ammonium sulfate precipitation of the cytoplasmic fraction, DEAE-Sephacel and Blue-Sepharose CL.6B chromatography, and Sephacryl S200 gel permeation. The catalytic activity of the purified enzyme required the presence of fructose-1,6-diphosphate with a broad optimum between pH 5 and 6.2. The concentration of fructose-1,6-diphosphate required for half-maximal velocity was around 0.02 mM and was affected by the pyruvate concentration. The enzyme seemed to have at least two binding sites for the activator which interact in a cooperative manner. Increasing concentrations of fructose-1,6-diphosphate up to 2 mM enhanced the relative affinity of the enzyme for pyruvate and modified the pyruvate saturation curve from sigmoidal to hyperbolic. The enzyme activity showed also a sigmoidal response to NADH, exhibiting two binding sites for the cofactor with a Hill coefficient of about 1.9. The molecular weight of the native enzyme was 150,000 as determined by gel permeation on Sephacryl S200. Monomers (38,000 daltons) and dimers (85,000 daltons) were observed by sodium dodecyl sulfate-gel electrophoresis; the latter form was dissociated after reduction with 2-mercaptoethanol, and the enzyme could be considered a tetramer. Antibodies obtained against the purified S. mutans OMZ175 LDH cross-reacted with the sodium dodecyl sulfate-dissociated forms of LDHs from different S. mutans serotypes, Streptococcus sanguis OMZ9, Lactobacillus casei ATCC 4646, and Actinomyces viscosus NY 1. A competitive enzyme-linked immunosorbent assay allowed us to detect a very close relationship between the native states of L-LDHs from S. mutans serotypes and S. sanguis. Cross-reactions were also observed with the LDHs from A. viscosus and L. casei, the latter being the least related. A very weak immunological relationship was obtained between the L-LDH from S. mutans OMZ175 and the D-LDH from Lactobacillus leichmannii, whereas no cross-reaction could be detected with mammal LDHs.     

Regulation of expression of Streptococcus mutans genes important to virulence.

Studies were initiated to investigate the regulation of Streptococcus mutans genes which are believed to be important to virulence. Operon fusions were constructed between S. mutans gene regulatory regions and a promoterless chloramphenicol acetyltransferase gene (cat) found on the plasmid pMH109. Specifically, fusions were generated between cat and the S. mutans genes encoding fructosyltransferase (ftf) and the glucosyltransferase B/C (gtfB/C) operon. Constructs were confirmed by restriction enzyme analysis, and the fusions were subcloned into the integration vehicle pVA891. Following generation of multimeric DNA, recombinant plasmids were introduced into the s. mutans genome by Campbell-type insertion, resulting in single-copy operon fusions. Chloramphenicol acetyltransferase specific activities were used to monitor the expression of the S. mutans gtfB/C operon and ftf determinants. The expression of these genes is increased by the presence of sucrose and is followed by a rapid decline in expression over time. Additionally, expression of the gtfB/C operon is increased in S. mutans cells bound to artificial tooth pellicles.     

An 87-kilodalton glucan-binding protein of Streptococcus sobrinus B13.

An 87-kDa glucan-binding protein (GBP) of Streptococcus sobrinus B13 (serotype d) was isolated and purified from extracellular culture supernatant by using affinity chromatography on Sephadex G-50 and elution with a guanidine HCl gradient. Western blot (immunoblot) analysis showed it to be antigenically related, but not completely identical, to the 74-kDa GBP of Streptococcus mutans Ingbritt. The 87-kDa GBP has no glucosyltransferase activity. A possible role for this GBP in the cariogenicity of S. sobrinus B13 is suggested.     

Evidence that L-(+)-lactate dehydrogenase deficiency is lethal in Streptococcus mutans.

In order to construct an effector strain for the replacement therapy of dental caries, we wished to combine the properties of low-level acid production and high-level colonization potential in a strain of Streptococcus mutans. To this end, we made a deletion in the lactate dehydrogenase (LDH) gene cloned from the bacteriocin-producing S. mutans strain JH1000. However, we were unable to substitute the mutant for the wild-type allele by transformation with linear DNA fragments. The mutated gene, carried on a suicide vector, was shown by Southern analysis to integrate into the JH1000 chromosome to yield transformants carrying both the wild-type gene and mutated LDH gene. Three spontaneous self-recombinants of one heterodiploid strain were isolated by screening 1,500 colonies for a loss of the tetracycline resistance encoded by the gene used to mark the LDH deletion. In all three cases, Southern analysis showed that a loss of tetracycline resistance was accompanied by a loss of the mutated LDH gene, resulting in restoration of the wild-type genotype. However, screening the same number of colonies for self-recombinants that did not make lactic acid during anaerobic growth in Todd-Hewitt broth failed to identify clones in which the wild-type allele was lost. A second, simpler screening of more than 80,000 colonies grown aerobically on glucose tetrazolium medium to identify low-level-acid-producing colonies was also unsuccessful. These results are interpreted as indicating that LDH deficiency is lethal in S. mutans under the cultivation conditions used in these experiments. The physiological bases for this hypothesis are described.