PIC-BE诱导K562/ADM细胞凋亡及逆转其MDR的研究

β榄香烯吗素（ＰＩＣ－ＢＥ）是抗癌新药β榄香烯的水溶性衍生物．采用人红白血病的多药耐药性（ＭＤＲ）细胞株Ｋ５６２／ＡＤＭ作为实验模型,观察ＰＩＣ－ＢＥ对Ｋ５６２／ＡＤＭ细胞的生长抑制和凋亡诱导作用,并进而研究其对该细胞ＭＤＲ的可能影响．结果显示：（１）Ｋ５６２／ＡＤＭ细胞对ＡＤＭ具有明显的抗性,与Ｋ５６２细胞相比,抗性倍数约为４０倍,而两者对ＰＩＣ－ＢＥ的ＩＣ５０接近,无显著差异;（２）ＰＩＣ－ＢＥ（１０．０～３０．０μｇ／ｍｌ）对Ｋ５６２／ＡＤＭ细胞具有明显的生长抑制和凋亡诱导作用,两种作用的强度在一定的范围内均具药物浓度和作用时间依赖性;（３）低毒剂量ＰＩＣ－ＢＥ（１０．０μｇ／ｍｌ）与ＡＤＭ（４．０μｇ／ｍｌ）联合应用,可显著增强ＡＤＭ对该细胞的生长抑制和凋亡诱导作用,升高细胞内ＡＤＭ的浓度,降低该细胞对ＡＤＭ的ＩＣ５０,使该细胞对ＡＤＭ的抗性有数倍逆转．上述结果提示,ＰＩＣ－ＢＥ不仅是一种有效的广谱抗肿瘤剂,而且也是一种有效的ＭＤＲ逆转剂     

耐甲氧苯青霉素葡萄球菌mecA基因的检测

耐甲氧苯青霉素金黄色葡萄球菌（ＭＲＳＡ）和表皮葡萄球菌（ＭＲＳＥ）引起的医院感染已成为当今世界范围内的严重问题。青霉素结合蛋白２ａ（ＰＢＰ２ａ）是引起葡萄球菌耐甲氧苯青霉素的主要原因,它是由耐药基因ｍｅｃＡ编码的。本文主要综述近年来国外关于ｍｅｃＡ基因检测方法的研究进展,尤其是多聚酶链反应（ＰＣＲ）技术的应用。     

t-PA cDNA的克隆及其在毕赤酵母中的表达

应用ＰＣＲ方法,在ｔＰＡｃＤＮＡ５′端引入合适的限制酶位点和酵母分泌信号肽,与酵母表达载体ｐＰＩＣ９重组,构建表达质粒ｐＳＴＥＹ,利用ＬｉＣｌ转化法转化酵母菌ＹＳ１０８,在ＭＭ和ＭＤ平板上筛选表型,ＰＣＲ筛选ｔＰＡｃＤＮＡ与酵母染色体整合而形成的阳性克隆,阳性克隆经甲醇诱导表达后,用ＳＤＳＰＡＧＥ证明表达产物的分子量为６ｋＤａ左右,用酪蛋白板溶圈法测定ｔＰＡ的活性。Ｍｕｔｓ表型菌表达产物活性最高为２５００ＩＵ／ｍｌ,Ｗｅｓｔｅｒｎｂｌｏｔ证实表达产物具有天然ｔＰＡ分子的免疫原性。     

重组尿激酶型纤溶酶原激活物受体在酵母系统中的表达及鉴定

构建截断型可溶性尿激酶型纤溶酶原激活物受体（ｕ－ＰＡＲ）的原核及酵母表达质粒并分别在大肠杆菌和Ｐｉｃｈｉａｐａｓｔｏｒｉｓ酵母中高效表达．利用ＰＣＲ扩增截断型可溶性ｕ－ＰＡＲｃＤＮＡ片段,并分别连入酵母及原核表达载体中,构建成表达质粒．后者经诱导表达的蛋白被用于免疫家兔,获得抗ｕ－ＰＡＲ的抗血清,用于对酵母表达产物的鉴定．前者在甲醇的诱导下表达,产物分泌至培养液中．结果表明,原核表达的ｕ－ＰＡＲ蛋白免疫家兔后获得高效价的抗血清,利用此抗血清所作Ｗｅｓｔｅｒｎｂｌｏｔ证实酵母表达蛋白具有ｕ－ＰＡＲ的免疫原性,表观分子量４０ｋＤ左右．Ｍｕｔ－表型在第５ｄ为最高（２．５μｇ／ｍｌ）,Ｍｕｔ＋表型在第４ｄ为最高（７．５μｇ／ｍｌ）．因此,构建的可溶性ｕ－ＰＡＲ表达质粒在Ｐｉｃｈｉａｐａｓｔｏｒｉｓ酵母细胞获得表达,为进一步竞争拮抗受体功能的研究奠定了基础．     

用PCR扩增和克隆马立克氏病病毒糖蛋白D基因

用ＰＣＲ技术,从ＧＡ株马立克氏病病毒（ＭＤＶ）感染的成纤维细胞（ＧＥＦ）基因组ＤＮＡ中扩增出ＭＤＶ糖蛋白Ｄ（ｇＤ）抗原基因片段的约１３００ｂｐ编码序列,将该ｐｃＲ扩增的产物于ＥｃｏＲＩ和Ｋｐｎｌ位点克隆到ｐＵＣ１８质粒载体中,在以ｄｉｇｏｘｉｇｅｎｉｎ（ｄｉｇ）标记的ｇＤＰＣＲ产物作为探针,进行原位杂交初步筛选到阳性重组质粒克隆,再根据酶切分析筛选到含ＭＤＶｇＤ基因的重组质粒ｐ１８ＭｇＤ。将ｐ１８ＭｇＤ质粒ＤＮＡ用ｄｉｇ标记后,在Ｓｏｕｔｈｅｒｎｂｌｏｔ中,该探针能识别ＭＤＶ基因组ＤＮＡ的ＢａｍＨＩ－Ａ克隆中的Ａ片段ＤＮＡ。酶切位点分析表明,该ｇＤ克隆也和已发表的ＭＤＶ的ＲＢＩＢ株ｇＤ一样,不含有ＥｃｏＲⅠ、ＨｉｎｄⅢ、ＰｓｔⅠ、ＳｍａⅠ、ｐｖｕⅡ等酶切位点。证明该重组质粒是ＭＤＶｇＤ克隆。     

野生型p53基因对内皮细胞细胞间粘附分子-1表达的影响

细胞间粘附分子－１（ＩＣＡＭ－１）是介导白细胞与内皮细胞粘附的重要粘附分子．为研究野生型ｐ５３基因对内皮细胞ＩＣＡＭ－１表达的影响,分别采用流式细胞术和ＲＴ－ＰＣＲ／ＨＰＬＣ方法测定ＩＣＡＭ－１蛋白及ｍＲＮＡ水平．静息状态的内皮细胞表面结构性地表达有少量的ＩＣＡＭ－１,在肿瘤坏死因子α（ＴＮＦα,１０～１０００Ｕ／ｍｌ）诱导下,其表达呈剂量依赖性增加．将ｐ５３基因导入内皮细胞,则显著抑制ＴＮＦα诱导的内皮细胞表面ＩＣＡＭ－１的表达．ｐ５３基因的导入对静息状态内皮细胞表面结构性表达的ＩＣＡＭ－１影响较小．ｐ５３基因主要通过降低ＩＣＡＭ－１的ｍＲＮＡ水平而抑制内皮细胞表面ＩＣＡＭ－１的表达,但对蛋白的抑制程度小于对ｍＲＮＡ的抑制程度．提示：ｐ５３基因对内皮细胞ＩＣＡＭ－１表达的影响除转录水平控制外,还存在转录后水平的调控     

茶儿茶素氧化产物体外清除·OH自由基作用的研究

本文采用２－脱氧－Ｄ－核糖（ＤＲ）法产生．ＯＨ,测定了不同浓度的茶儿素氧化产物Ａ及Ｃ对．ＯＨ的清除作用。结果表明,在一定的浓度范围内,氧化产物Ａ及Ｃ均有很强的清除．ＯＨ的作用且最佳清除浓度为２００μｇ／ｍｌ。在茶多酚、氧化产物Ａ及Ｃ三种物质中,以产物Ｃ的清除作用最强,其ＩＣ５０值为７．３μｇ／ｍｌ,其次是Ａ,ＩＣ５０为１０．１μｇ／ｍｌ,最后是茶多酚,ＩＣ５０值为７０μｇ／ｍｌ。     

小鼠腺病毒单克隆抗体的研制及初步应用

应用杂交瘤技术获得４株分泌抗小鼠腺病毒（ＭｕｒｉｎｅＡｄｅｎｏｖｉｒｕｓＭＡｄ）单克隆抗体细胞株,并对其特性进行分析。经鉴定,它们所分泌的抗体类型均为ＩｇＭ,腹水效价为１０－３～１０－６。相对亲和力分别为０．１μｇ／ｍｌ（Ａ９）、０．６５μｇ／ｍｌ（Ｂｌ）、１２．５μｇ／ｍｌ（Ｇ４）和２３μｇ／ｍｌ（Ｄ４）。与其他１０种鼠源性病毒均无交叉反应,表明ＭｃＡｂ具有良好的特异性。单抗标记ＦＩＴＣ后用于人用鼠源性单抗制品及各种传代细胞和原代细胞中ＭＡｄ检测,获得良好的实验结果。     

金黄色葡萄球菌对甲氧西林耐药的分子机制

耐甲氧西林金黄色葡萄球菌是医院内和社区感染的重要现菌,所致感染治疗困难,因此成为各国学者关注与研究的热点。本文就近几年国内外在ＭＲＳＡ耐药分子机制的研究中取得的成果,如ｍｅｃＡ基因以及其他与ＭＲＳＡ对甲西林抗性表达相关基因的定位,结构及功能等作一介绍。     

利用PCR方法鉴定甲醇酵母转化子的表型

本文以含外源基因的甲醇酵母表达载体ｐＰＩＣ９／Ｅ２Ｆ－１－ＤＢ质粒ＤＮＡ电转化甲醇酵母ＧＳ１１５,小规模抽提所得转化子的总ＤＮＡ,并以其为模板,以乙醇氧化酶（ＡＯＸ１）５’端序列和３’端的ＴＴ序列为引物,进行ＰＣＲ反应。ＰＣＲ产物走０．８％Ａｇａｒｏｓｅ电泳,通过对ＰＣＲ产物的电泳带型分析,鉴定出甲醇酵母转化子的表型,即Ｍｕｔ＾＋或Ｍｕｔ＾ｓ。这一鉴定结果为转化子的诱导表达产物的ＳＤＳ－ＰＡＧＥ图     

Methicillin resistance in staphylococci isolated from subclinical mastitis in sheep

One hundred ovine milk samples were subjected to bacteriological analysis to detect staphylococci. Twenty-four staphylococcal strains isolated were characterised for methicillin resistance with disk diffusion test (DDT) after incubation at 24 and 48 h, oxacillin agar screen test, Minimal Inhibitory Concentration (MIC), nitrocefin test for beta-lactamase production and PCR for the mecA gene. Nine staphylococcal strains resulted resistant in DDT; some differences in the halo diameter at double incubation period were noted; eight of these strains were resistant in MIC test; just one strain was positive to oxacillin agar screen test. All strains were mecA negative by PCR and positive by nitrocefin test. On the basis of these results methicillin-resistant strains can be classified as beta-lactamase hyperproducers.     

mecA基因PCR扩增法检测耐甲氧西林金黄色葡萄球菌

目的 应用mecA基因PCR扩增法检测耐甲氧西林金黄色葡萄球菌（methicillin resistant staphylococcus aureus,MRSA）。方法 临床分离的70株金黄色葡萄球菌,应用mecA基因PCR扩增法鉴定MRSA,并与苯唑西林纸片扩散法进行比较。结果 70株金黄色葡萄球菌用PCR扩增法和纸片扩散法有6株鉴定有差异,4株。mecA基因阳性而纸片扩散法鉴定为敏感,1株mecA基因阳性纸片扩散法鉴定为临界耐药,1株mecA基因阴性却表现为苯唑西林耐药,2种方法符合率为91．43％。结论 mecA基因PCR扩增法可以准确、快速判定MRSA,特别是对隐匿型或低水平耐药菌株的检出有重要的价值。     

Quantitative disk diffusion as a convenient method for determining minimum inhibitory concentrations of oxacillin for staphylococci strains

In this study the susceptibility of 58 coagulase-negative staphylococci (CoNS) strains and 58 Staphylococcus aureus strains to oxacillin was evaluated by a novel method called quantitative disk diffusion (DD) method. The results obtained were compared to phenotypic methods as agar dilution (AD) for oxacillin, disk diffusion (DD) for cefoxitin, and related to the presence of the mecA gene detected by PCR. Minimum inhibitory concentrations (MIC) determined by the quantitative DD method were equivalent to MICs determined in the AD method for S. aureus (Student's t test, p=0.99) and CoNS (Student's t test, p=0.97). Incongruent results between PCR mecA gene determinations and the quantitative DD method were obtained in 8 strains (5 S. aureus and 3 CoNS) where the mecA gene expression was blocked. However, oxacillin resistance was detected by the proposed method even in staphylococci strains showing low-level or heterogeneous resistance to the antibiotic while other phenotypic methods failed. The single quantitative DD method is not expensive, it can be performed in any laboratory and permits accurate identification of oxacillin resistant staphylococci.     

Multiplex PCR detection of the antibiotic resistance genes in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> strains isolated from auricular infections

Thirty-five Staphylococcus aureus strains from auricular infections were isolated. The identification of strains was confirmed by Api ID 32 Staph strips, the antibiotic susceptibility test was performed using ATB Staph kit. PCR assay was used to detect the oxacillin resistance gene (mecA) and the erythromycin genes (ermA, ermB, ermC, msrA and mef). The susceptibility profile of all strains revealed a low resistance level to oxacillin and erythromycin. The PCR results show that 60 % of the strains are mecA positive. The frequency of erythromycin genes was: ermA (+) 22.8 %, ermB (+) 45.7, ermC (+) 17.1, msrA (+) 28.6. The mef gene was not detected in any strain. No correlations between genotypic and phenotypic methods for the determination of oxacillin and erythromycin resistance was found. However, multiplex PCR technique was shown to be a fast, practical and economic technique for the detection of methicillin-and erythromycin-resistant staphylococci.     

Comparison of methods for the detection of methicillin resistance in Staphylococcus aureus isolates from food products

AIMS: To compare several methods for detection of methicillin resistance in Staphylococcus aureus isolates from food. METHODS AND RESULTS: Two hundred S. aureus isolates from food of animal origin were screened for methicillin resistance by a PCR assay specific for the mecA gene, an oxacillin agar screen test and a cefoxitin disk diffusion test. Six out of 200 strains (3%) were found to be methicillin-resistant Staphylococcus aureus (MRSA) by PCR. The oxacillin agar screen test detected only one of the MRSA isolates (sensitivity of 16.7%) and mischaracterized three additional strains as MRSA (specificity of 98.45%). None of the MRSA strains was detected by the cefoxitin test (sensitivity of 0%), while 15 methicillin-susceptible S. aureus (MSSA) strains were misclassified as resistant (specificity of 92.3%). Fifteen MSSA strains displayed a beta-lactamase hyperproducer-like phenotype. The six MRSA (mecA-positive) strains resembled the characteristics of heteroresistant strains. CONCLUSIONS: As MRSA of animal origin may display atypical phenotypes, PCR appears to be more reliable for detection of methicillin resistance in animal strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The study stresses the need for implementing the methods of screening S. aureus from food of animal origin for methicillin resistance.     

Molecular analysis of methicillin-resistant staphylococci isolated from hospital patients and in the out-patient clinic

In this study, a molecular analysis of the methicillin-resistant coagulase-negative staphylococci strains was performed. The obtained results of the biochemical and drug resistant pattern investigations were insufficient to assess the relationship between the strains. Therefore genotyping by the restriction fragment length polymorphism analysis (PCR-RFLP) method was performed. Analyzed strains characterized presence of the mecA gene-PCR products. The PCR products were digested with DraI and TasI, and the fragments separated by agarose gel electrophoresis. Typing of the methicillin-resistant gene using PCR-RFLP showed that all MRCNS strains possess an identical restriction pattern of the mecA gene. This identical restriction pattern of the mecA gene in investigated strains may suggest an easy transfer of this gene between different staphylococci species and lead to the spreading of methicillin-resistant among hospital strains. Furthermore performing the comparison of different phenotype and genotype methods has shown that the PCR-RFLP method is quick and reliable, enabling the detection and estimation of the relationship between MRCNS strains.     

The use of oxacillin and cefoxitin disk-diffusion method and PCR for the identification methicillin-resistant Staphylococcus epidermidis

Methicillin-resistant S. epidermidis are recognized as one of the most important nosocomial infections. Because of the different expression level of mecA gene which is under regulatory genes control, detection of methicillin resistance by phenotypic methods may leads to false negative or false positive results. The aim of this study was to estimate effectiveness of MRSE strains identification using oxacillin (1 microg) and cefoxitin (30 microg) disk-diffusion method in comparison with PCR, considered as a "gold standard". The analysis of 120 strains isolated from clinical materials of patients of the University Hospital at the L. Rydygier Collegium Medicum in Bydgoszcz, University of Nicolaus Copernicus in Toruń indicated high degree of correlation between phenotypic methods with taken disks. Results consistency of detecting methicillin resistance between oxacillin disk diffusion method and PCR concerned 95% strains. In case of cefoxitin 4,2% S. epidermidis strains detected phenotypically as MSSE were mecA-positive. Our results show that disk-diffusion method with disks mentioned above is characterized by comparable specificity and sensitivity amounted 90,8% and 100% for oxacillin and 92,3% and 100% for cefoxitin respectively.     

MRSA中mecA及femB基因的检测与耐药相关性

研究femB、mecA基因在耐甲氧西林金黄色葡萄球菌(MRSA)中的表达与耐药的关系.运用PCR对MRSA的femB、mecA基因进行检测,MRSA耐药检测采用头孢西丁纸片法.40 株金黄色葡萄球菌(下简称金葡菌)通过头孢西丁纸片法,检出 30 株耐头孢西丁的菌株,通过PCR检测这 40 株金葡菌mecA基因,30 株MRSA全部为阳性, femB基因在 30 株MRSA中全部表达,而甲氧西林敏感的金黄色葡萄球菌(MSSA)的未表达.结果可见,PCR能快速准确地鉴定MRSA, mecA基因是MRSA的耐药基因,femB基因是MRSA的耐药相关基因.     

耐甲氧西林凝固酶阴性葡萄球菌中多种抗生素耐药基因的观察

探讨耐甲氧西林凝固酶阴性葡萄球菌(methicillin-resistant coagulase negative staphylococci,MRCNS)中多种抗生素耐药基因的分布情况,采取Kirby Bauer标准纸片扩散法(K-B纸片扩散法)对MRCNS开展药敏实验,并通过PCR检测菌种携带的抗生素耐药基因。药敏实验结果显示,96株MRCNS对庆大霉素、红霉素、莫匹罗星以及四环素的耐药性各有差异,但对氨苄西林的耐药性却达到100%,未发现对万古霉素具有耐药性的菌株;PCR检测结果显示,9种耐药基因merA、ermB、ermC、msrA、tetM、tetK、aac(6’)-aph(2")、aph(3’)-III、mupA的阳性检出率各有差异,没有扩增出vanA、ermA、vanB以及ant(6’)-I基因,但都扩增出mecA基因。结果表明,MRCNS可以同时携带多种抗生素耐药基因,属于一种多重耐药菌。     

Evaluation of oxacillin and cefoxitin disks for detection of resistance in coagulase negative staphylococci

Coagulase-negative Staphylococcus spp. was considered nonpathogenic until the emergence of multiresistance and the demonstration of their participation as infectious agents. In Brazil, oxacillin resistance may be present in over 80% of isolates, and the Clinical and Laboratory Standards Institute standardized a disk-diffusion method to predict this resistance in Staphylococcus. The aim of this study was to evaluate the variability among commercial disks of oxacillin (1 microg) and cefoxitin (30 microg) widely used in clinical laboratories of microbiology, compared with mecA gene and minimum inhibitory concentration (MIC) of oxacillin. The use of oxacillin and cefoxitin disks simultaneously allowed the detection of important differences, particularly, in less frequent species such as S. cohnii, S. haemolyticus, S. saprophyticus, and S. sciuri. Disks of cefoxitin of the brand 2 displayed good correlation with the mecA gene (98.7%) and oxacillin MIC (97.8%), while major discrepancies were observed using disks of brand 1. One of the critical points in the diffusion disk test is the quality of the disks: the use of better quality disks associated with molecular methods lead to better results to define the best antibiotic therapy.