恶性疟原虫特异性示踪物——乳酸脱氢酶的研究进展及其应用前景

建立一种快速而又实用的疟疾诊断方法一直是疟疾免疫学研究的主要目标之一。在所有研究中,循环抗原的检测是最有前途的方法。由干疟原虫抗原具有高度复杂性,致使研究人员在抗原的分析、提纯中遇到了困难。发现一种疟原虫特有的抗原成分,作为原虫存在与否的指示物,必将具有重大意义。MAKIER等的研究发现,恶性疟原虫乳酸脱氢酶(Lactate dehydrogenase of Plasmodium falciparum,LDH-p),作为疟原虫特殊形式的循环抗原,与人红细胞和其它许多微物质LDH具有显著不同的物理、生化特性,易于区分。LDH-p的检测可望成为检测原虫血症的首选方法。本文从LDH的提纯、LDH-p与LDH-r红细胞乳酸脱氢酶的分离、鉴别及其临床应用现状和前景等方面进行了综述和展望,旨在推动疟疾快速诊断研究的发展。     

复合PCR系统检测间日疟原虫的应用研究

应用一种简易、快速的复合 PCR扩增系统检测间日疟原虫 (P.v)及混合感染。方法 :以间日疟原虫和恶性疟原虫 (P.f)小亚单位核糖体核糖核酸基因 (SSUr DNA)特定片段为靶基因 ,设计并合成引物 ,建立复合 PCR扩增系统。采用煮沸法快速制备 DNA模板研究该系统的敏感性和特异性 ,并用于临床血样的检测。结果 :从间日疟原虫和恶性疟原虫感染血样中分别扩增出分子量大小为 70 5bp和 575bp的特定扩增带。而食蟹猴疟原虫、诺氏疟原虫及正常人血样均无扩增带出现。单独检测 P.v敏感度达到 0 .1 5× 1 0 -5(约 7个原虫 /μl全血 ) ,在 P.f存在的情况下检测 P.v敏感度为 0 .1 5× 1 0 -4 。检测 1 32例疟区门诊病人冻存血标本 ,1 0 4份与镜检法结果相同。并发现 1 4份混合感染和 5份为镜检虫种鉴别失误。结论 :该系统敏感性高 ,特异性强 ,操作简单 ,并可在一次扩增中同时检出间日疟原虫和恶性疟原虫 ,具有一定的推广应用价值。     

妊高症孕妇乳酸及乳酸脱氢酶含量的变化

目的 探讨妊娠期高血压患者体内乳酸及乳酸脱氢酶含量的变化.方法 取被确诊为妊娠期高血压病的住院产妇60例作为研究组.取同期住院年龄和孕周有可比性的正常待产妇40例作为对照组,早晨空腹采血测定乳酸、乳酸脱氢酶及相关生化指标的含量.结果 重度妊高症其乳酸和乳酸脱氢酶的检测结果均明显高于对照组(P<0.05).重度妊高症组与轻度妊高症组相比较差异有统计学显著性意义(P<0.05),含量的多少与妊高症的严重程度呈相关性.结论 乳酸可作为妊高症生化监测指标,尤其是与乳酸脱氢酶的联合检测对临床治疗和预后有指导意义.     

多重巢式PCR检测恶性疟和间日疟原虫

目的建立鉴别诊断恶性疟原虫(P.f)和间日疟原虫(P.v)的多重巢式PCR法。方法针对P.f、P.v 18S rRNA基因设计外引物和内引物,优化引物浓度与退火温度,建立可扩增出两种疟原虫基因片段的多重巢式PCR,并检测54例疑似疟疾临床标本,以镜检法为金标准评价敏感性和特异性等指标。结果该方法可扩增出162 bp(P.f)和112 bp(P.v)基因片段,并能检出混合感染。该方法检测P.f,敏感性为87.50%、特异性为63.33%;检测P.v,敏感性为69.23%、特异性为68.29%。结论所建立的多重巢式PCR方法能可靠诊断疟疾并鉴别虫种,敏感性高,在混合感染的诊断方面具有优越性。     

间日疟原虫的血液浓缩检查法

血液涂片检查疟原虫是临床诊断疟疾的依据,通常采用厚血膜法和薄血膜法。笔者从红斑狼疮细胞的检验方法中得到启发,采用血液离心分层方法使感染有疟原虫的红细胞浓集,既保持了疟原虫在红细胞中的形态完整,又提高了疟原虫的检出率。现报告如下。     

葡萄糖脱氢酶融合表达载体的构建

目的为方便葡萄糖脱氢酶的纯化,构建葡萄糖脱氢酶融合表达载体。方法通过聚合酶链反应(PCR)从枯草杆菌基因组中扩增葡萄糖脱氢酶基因,与源序列比较分析后连接到融合表达载体pET22b,导入JM109诱导表达,测定葡萄糖脱氢酶酶液比活力。结果构建的葡萄糖脱氢酶融合表达载体中目的基因序列与枯草杆菌来源的该基因序列相同,且粗提酶液比活力高达375 U/mg。结论运用分子生物学技术获得葡萄糖脱氢酶基因,成功构建了葡萄糖脱氢酶的融合表达载体,为酶的后续纯化工作提供了条件。     

间日疟原虫可溶性抗原对树突状细胞成熟的影响

目的观察间日疟原虫可溶性抗原(P.vAg)对树突状细胞(DC)成熟分化的影响。方法以间日疟患者感染红细胞获得P.vAg,体外刺激人单核来源的未成熟DC。采用流式细胞术分析不同浓度的P.vAg(0.1、1.0、10.0μg/mL)作用下再经脂多糖(LPS)诱导后DC成熟相关分子CD83、CD86、主要组织相容性抗原分子(HLA-DR)的表达变化。结果 3组P.vAg刺激的DC再经LPS诱导后表达CD83、CD86和HLA-DR阳性的百分率均高于对照组。3个剂量组CD83、CD86表达量均明显升高,差异有统计学意义(P<0.01或P<0.05),P.vAg10.0μg/mL组HLA-DR表达量明显升高,差异有统计学意义(P<0.05)。结论 P.vAg能上调DC部分成熟性表型的表达,表明P.vAg具有促进DC成熟的作用。     

血液病患者血清乳酸脱氢酶水平变化及临床意义

我们对 4 5例血液病患者进行了血清乳酸脱氢酶(ＬＤＨ)的检测 ,旨在探讨其变化特点及临床意义。一、材料和方法1.检测对象　我院确诊并住院治疗的 4 5例血液病患者 ,2 3例白血病患者中急性淋巴细胞性白血病 (ＡＬＬ) 9例、急性粒细胞性白血病 (ＡＭＬ) 11例、慢性粒细胞性白血病(ＣＭＬ) 3例 ;自身免疫性溶血性贫血 (ＡＩＨＡ) 5例 ;巨幼红细胞性贫血 (ＭＡ) 6例 ;再生障碍性贫血 (ＡＡ) 11例。年龄12～ 5 9岁。对照组 (ＮＣ)为健康体检者 ,年龄、性别与病例组相匹配。病例组于治疗前或初 (1周内 )取静脉血检测ＬＤＨ活力水平 ,除ＡＡ组外多…     

血液病患者血清乳酸脱氢酶水平变化及临床意义

我们对45例血液病患者进行了血清乳酸脱氢酶(LDH)的检测,旨在探讨其变化特点及临床意义.     

血清α-羟丁酸脱氢酶与乳酸脱氢酶在白血病患者中表达的临床意义

目的研究血清α 羟丁酸脱氢酶 (α HBDH)与乳酸脱氢酶 (LDH)的浓度变化在急性白血病患者病情变化、疗效、预后中的监测作用。方法用全自动生化分析仪检测了 6 2例初发 /复发急性白血病患者治疗前后血清α 羟丁酸脱氢酶(α HBDH)与乳酸脱氢酶 (LDH)浓度。结果急性白血病患者初发或复发时血清LDH及α HBDH明显增高 ,与正常对照组比较有显著性差异 (P <0 .0 1) ;治疗后达完全缓解与部分缓解者浓度下降 ,治疗前后血清LDH及α HBDH比较有显著性差异 (P <0 .0 0 1) ;复发时血清LDH与α HBDH值较复发前有明显上升 (P <0 .0 1) ;外周血白细胞数与血清LDH含量呈正相关 (r=0 .4 2 ,P <0 .0 1) ;外周血白细胞数与血清α HBDH含量呈正相关 (r =0 .4 0 ,P <0 .0 1)。结论动态检查血清LDH及α HBDH的变化对白血病患者的病情变化、疗效、预后有监测作用。     

Management of relapsing Plasmodium vivax malaria

Introduction: Relapses are important contributors to illness and morbidity in Plasmodium vivax and P. ovale infections. Relapse prevention (radical cure) with primaquine is required for optimal management, control and ultimately elimination of Plasmodium vivax malaria. A review was conducted with publications in English, French, Portuguese and Spanish using the search terms ‘P. vivax’ and ‘relapse’.

Areas covered: Hypnozoites causing relapses may be activated weeks or months after initial infection. Incidence and temporal patterns of relapse varies geographically. Relapses derive from parasites either genetically similar or different from the primary infection indicating that some derive from previous infections. Malaria illness itself may activate relapse. Primaquine is the only widely available treatment for radical cure. However, it is often not given because of uncertainty over the risks of primaquine induced haemolysis when G6PD deficiency testing is unavailable. Recommended dosing of primaquine for radical cure in East Asia and Oceania is 0.5 mg base/kg/day and elsewhere is 0.25 mg base/kg/day. Alternative treatments are under investigation.

Expert commentary: Geographic heterogeneity in relapse patterns and chloroquine susceptibility of P. vivax, and G6PD deficiency epidemiology mean that radical treatment should be given much more than it is today. G6PD testing should be made widely available so primaquine can be given more safely.     

乳酸清除率和乳酸脱氢酶对急性肺损伤预后的评估

目的评估早期乳酸清除率和乳酸脱氢酶(LDH)与急性肺损伤(ALI)患者预后的关系。方法回顾性分析我院2007年5月至2009年5月收治的63例ALI病例的临床资料;按有无并发多脏器功能不全综合征(MODS)和是否28d内死亡分为两组,比较两组间早期乳酸清除率和LDH水平;按早期乳酸清除率和LDH均值为界分为高低两组,比较两组间的病死率和MODS发生率。结果各组间性别、年龄等比较,差异无统计学意义(P均0.05);但MODS组和死亡组早期乳酸清除率分别低于非MODS组和存活组,MODS组和死亡组LDH值分别高于非MODS组和和存活组,差异均有统计学意义(P0.05)。低早期乳酸清除率组和高LDH组死亡率和MODS发生率分别高于高早期乳酸清除率组和低LDH组,差异有统计学意义(P均0.05)。结论早期血乳酸清除率和LDH对ALI患者预后的评估有较大价值。     

High specificity of semi-nested multiplex PCR using dried blood spots on DNA Banking Card in comparison with frozen liquid blood for detection of Plasmodium falciparum and Plasmodium vivax

Background: Venipuncture sampling in test tubes for detecting malaria parasites using PCR assays possesses a number of limitations such as reluctance of patients, some difficulties in transportation of blood samples and freezing them for long time. To overcome the mentioned limitations, some approaches have been employed by a number of authors. This study was proposed to compare between DNA Banking Card (DBC) filter papers containing dried finger‐prick blood and venipunctured frozen liquid blood. Methods: A total of 75 specimens was prepared from the equal enrolled individuals using three blood storage approaches; making Geimsa‐stained thin and thick smears from each individual to determine the malaria‐positive or ‐negative specimens, spotting two to three drops of finger‐prick blood onto the DBC filter paper, and collecting a 2‐ml venous blood sample into EDTA‐contained test tube from each individual. A semi‐nested Multiplex PCRtechnique with DNA extracted from the two latter sets of specimens was used for plasmodia diagnosis. Results: DNA samples isolated from dried blood spotted on the DBC filter papers resulted in 32 (42.7%) positive and 43 (57.3%) negative cases comparable with the results outcome of frozen liquid blood with 35 (46.7%) positive and 40 (53.3%) negative cases. Statistical analysis revealed higher sensitivity for SnM‐PCR using DNA from liquid blood with 100% vs. dried blood spotted on DBC with 97% but higher specificity for the DBC with 100% vs. liquid blood with 95.2%. Conclusions: Based on the results obtained from this study to overcome the problems of venipuncture frozen liquid blood sampling, replacement of a reliable filter paper for preserving finger‐prick blood samples is a trustable and useful facilitator particularly in remote malaria‐endemic areas. J. Clin. Lab. Anal. 25:185–190, 2011. © 2011 Wiley‐Liss, Inc.     

人红细胞乳酸脱氢酶的提纯及其性质研究

目的 研制乳酸脱氢酶的标准品，从人红细胞中提取了乳酸脱氢酶（LD）并对其特性进行了研究。方法 用经过改进的方法，包括完全溶解红细胞，羟磷灰石处理，硫酸铵沉淀，CM-Sephadex、C50、SephadexG100和5’-AMP-Sepharose亲和层析。结果 该提制品LD比活性达163.0KU/g蛋白，几乎不含其它杂酶（测不到ALT、AST、CK、GGT、ChE、ALP、Amy活性），聚丙烯酰胺凝胶电泳（自然凝胶系统——酶染色显示LD1、LD2区带，蛋白质染色显示与酶相应的区带。37℃条件下，正向反应中，对底物L-乳酸和NAD的米氏常数（Km)分别为1 。040和0.192mmol/LO；逆向反应中，对底物丙酮酸和NADH的Km分别为0.119和0.039mmol/L。结论 提纯的LD其催化性质和人混合血清KLD非常相似。本研究为今后进一步研制LD测定用参考品建立了基础。     

不同检测系统LDH测定结果的可比性研究

目的探讨同一临床实验室不同检测系统间乳酸脱氢酶(LDH)测定结果是否只有可比性,为血清酶测定的标准化提供实验数据。方法参考NCCLS的EP9-A文件,以日立7170生化分析仪、罗氏原装试剂、c.f.a.s校准品和质控品组成的检测系统(已通过ISO/IEC17025实验室认可)为比较方法,检测系统1-4为实验方法,用患者新鲜血清对LDH进行检测,计算试验方法(Y)和比较方法(X)之间的相对偏差(SE%),以CLIA’88规定的室间质量评价允许误差范围的1/2为标准,判断不同检测系统的可比性。结果LDH测定结果各系统的误差临床均可以接受。结论各检测系统测定LDH结果具有可比性。当同一实验室同一检验项目存在2个以上的检测系统时,应进行方法比对和偏差评估,以保证检验结果的可比性。     

毛细管电泳法分离血清中乳酸脱氢酶同工酶

目的利用还原型辅酶I（NADH）在340nm波长处特异性吸收峰，建立用毛细管区带电泳在线检测人血清中乳酸脱氢酶（LDH）同工酶的方法。方法实验中采用未涂层毛细管，长60cm，内径75μm，pH9．4（23℃）二氨基二甲基1，3丙二醇（AM2P）缓冲液含20mmol/L氧化型辅酶I（NAD^＋）、51，6mmol/L乳酸锂。结果在25min内完成电泳分离，乳酸脱氢酶同工酶1（LDH1）、乳酸脱氢酶同工酶5（LDH5）纯品各自均可得到较好峰形，而且LDH1、LDH2纯品的混合物亦可得到完全分离，在分析正常人血清与肝癌患者血清时，其结果与美国Helena公司REP全自动琼脂糖电泳结果一致，取得满意效果。结论该法快速、低耗，具有较好的推广应用价值。     

An improved spectrophotometric assay of pyruvate dehydrogenase in lactate dehydrogenase contaminated mitochondrial preparations from human skeletal muscle

In mitochondria-enriched preparations of human skeletal muscle, the measurement of pyruvate dehydrogenase activity, as determined by conventional spectrophotometric assay of NADH accumulation, is underestimated due to the oxidizing activity of the contaminating lactate dehydrogenase. Using a model reaction system consisting of varying mixtures of purified lactate and pyruvate dehydrogenases, we found that the presence of oxamate, a competitive inhibitor of the lactate dehydrogenase, allowed the measurement of a linear rate of pyruvate dehydrogenase activity without interference from lactate dehydrogenase. In the presence of 25 mM oxamate, this holds true up to a ratio of 30:1 for lactate to pyruvate dehydrogenases, respectively. A similar result was obtained when using human skeletal muscle mitochondria contaminated by lactate dehydrogenase. Rates of pyruvate dehydrogenase activity ranging from 50 to 120 nmol/min/mg protein could be routinely measured in such mitochondrial fractions. We concluded that the use of oxamate allows a spectrophotometric assay for pyruvate dehydrogenase activity to be utilized when screening for pyruvate dehydrogenase deficiency in mitochondria-enriched preparations of human skeletal muscle.