人巨细胞病毒糖蛋白gp52的原核表达及初步应用

目的 探讨以表达人巨细胞病毒（ＨＣＭＶ）中抗原性较强的蛋白片断代替全病毒抗原建立ＨＣＭＶ ＩｇＭ检测试剂盒的可行性。方法 以边反应扩增ＨＣＭＶ聚合酶辅助糖蛋白（ｇｐ５２）的有效抗原基因序列，导入带有高效转录启动子ｐＴｒｃ和６个串联组氨酸的原核表达载体ｐＴｒｃＨｉｓ，在大肠杆菌中表达，经金属鏊和亲和层析（ＩＭＡＣ）法纯化后，用酶联免疫吸附法检测新生儿血清，并与全病毒抗原检测进行比较。结果 重组ｇｐ５     

应用双抗原夹心法检测抗—HCV抗体

建立检测抗－ＨＣＶ抗体的双抗原夹心酶联免疫吸附测定方法。用基因工程手段，在大肠杆菌中表达ＨＣＶ抗原表位与大肠杆菌噬菌体ＭＳ２ ＤＮＡ聚合酶的融合蛋白，纯化后作为包被抗原，可溶性表达的ＨＣＶ抗原表位和霍乱毒Ｂ亚基的融合蛋白经辣根过氧化物酶标记后作为酶结合物，建立双抗原夹心ＥＬＩＳＡ。     

应用双抗原夹心法检测抗－HCV抗体

目的建立检测抗－ＨＣＶ抗体的双抗原夹心酶联免疫吸附测定（ＥＬＩＳＡ）方法。方法用基因工程手段，在大肠杆菌中表达ＨＣＶ抗原表位与大肠杆菌噬菌体ＭＳ２ＤＮＡ聚合酶（ＭＳ２－Ｐｏｌ）的融合蛋白，纯化后作为包被抗原，可溶性表达的ＨＣＶ抗原表位与霍乱毒素Ｂ亚基（ＣＴＢ）的融合蛋白经辣根过氧化物酶标记后作为酶结合物，建立双抗原夹心ＥＬＩＳＡ。结果ＭＳ２－Ｐｏｌ融合蛋白在大肠杆菌中得到高效表达并进行了纯化，所建立的双抗原夹心ＥＬＩＳＡ系统用于检测１４份ＨＣＶ阳性血清样品，抗－ＨＣＶ抗体检出率１００％；检测２１份ＨＣＶ阴性血清，没有假阳性结果。ＣＴＢ抗体不干扰反应。结论双抗原夹心法与采用二抗的ＥＬＩＳＡ系统具有相近的灵敏度和更好的特异性。可以避免由于二抗干扰带来的非特异性。     

捕获ELISA检测人巨细胞病毒IgM抗体

人巨细胞病毒 (ＨＣＭＶ)属疱疹病毒科 ,其感染极为普遍 ,在免疫抑制患者及孕妇中常可导致严重后果。本实验采用重组ＨＣＭＶ (ｒｈＣＭＶ) ｇｐ5 2蛋白[1] 作抗原和辣根过氧化物酶(ＨＲＰ)标记的鼠抗 ｇｐ5 2单克隆抗体[2 ] ,建立了捕获ＥＬＩＳＡ ,检测血清中的ＨＣＭＶＩｇＭ抗体。现将结果报告如下。1　材料与方法1 1　抗原浓度的确定　ｒｈＣＭＶ ｇｐ5 2蛋白[1] 纯度达 95 %。在捕获ＥＬＩＳＡ检测程序中加入不同浓度的ｇｐ5 2抗原 ,每个浓度重复 3孔 ,将同一抗原浓度的 3个阳性血清Ａ值分别除以 3份阴性血清的平均Ａ值 ,求得Ｐ/Ｎ值…     

巨细胞病毒即刻早期基因mRNA检测方法的建立

目的建立快速简便的逆转录聚合酶链反应（ＲＴＰＣＲ）方法检测人巨细胞病毒（ＨＣＭＶ）即刻早期（ＩＥ）基因ｍＲＮＡ。方法设计合成跨越ＨＣＭＶＩＥ基因内含子区的一对引物，分别用ＲＴＰＣＲ和ＰＣＲ技术扩增ＨＣＭＶｍＲＮＡ和ＤＮＡ，用Ｓｏｕｔｈｅｒｎ杂交鉴定阳性产物。结果ＨＣＭＶＩＥ基因ｍＲＮＡ表达在感染后６小时即可出现，并持续到感染后至少９６小时，ＲＴＰＣＲ检测的最低敏感性为１００ｆｇ总ＲＮＡ，其他病毒和细胞ＲＮＡ不能被扩增。结论ＲＴＰＣＲ技术检测ＨＣＭＶｍＲＮＡ敏感、特异，有望应用于临床作为ＨＣＭＶ活动性感染的早期诊断方法     

以抗原半乳糖苷酶融合蛋白为酶结合物检测丙型肝炎抗体

目的 为了降低检测丙型肝炎（丙肝）病毒（ＨＣＶ）抗体的假阳性,建立基于双抗原夹心法的酶联免疫吸附测定（ＥＬＩＳＡ）系统。方法 利用基因工程手段,在大肠杆菌中表达ＨＣＶ嵌合抗原（含核心、Ｃ３３ｃ,ＮＳ４及ＮＳ５抗原）与大肠杆菌β－半乳糖苷酶的融合蛋白,以该融合蛋白为酶结合进行ＥＬＴＳＡ。结果 构建了重组质粒,并在大肠杆菌中表达了融合蛋白,融合蛋白具有ＨＣＶ抗原的抗原性,并且保持了的酶活性。以重组融合     

以抗原半乳糖苷酶融合蛋白为酶结合物检测丙型肝炎抗体

目的　为了降低检测丙型肝炎（ 丙肝） 病毒（ＨＣＶ） 抗体的假阳性,建立基于双抗原夹心法的酶联免疫吸附测定（ＥＬＩＳＡ） 系统。方法　利用基因工程手段,在大肠杆菌中表达ＨＣＶ 嵌合抗原（ 含核心、Ｃ３３ｃ ,ＮＳ４ 及ＮＳ５ 抗原） 与大肠杆菌β半乳糖苷酶的融合蛋白,以该融合蛋白为酶结合物进行ＥＬＴＳＡ。结果　构建了重组质粒,并在大肠杆菌中表达了融合蛋白,融合蛋白具有ＨＣＶ 抗原的抗原性,并且保持了的酶活性。以重组融合蛋白为酶结合物,用于检测抗ＨＣＶ 抗体时,阳性检出率和阴性特异性均接近使用辣根过氧化物酶系统的水平。灵敏度接近以辣根过氧化物酶为标记酶的ＥＬＩＳＡ 系统。结论　重组ＨＣＶ 抗原β半乳糖苷酶融合蛋白可以用作酶结合物,应用于ＨＣＶ 感染的诊断。     

免疫酶法检测鼻咽癌病人血清IgA/gp125抗体

建立了用表达Ｅｐｓｔｅｉｎ－Ｂａｒｒ病毒壳抗原ｇｐ１２５的重组痘苗病毒感染细胞为抗原，检测ＩｇＡ／ｇｐ１２５抗体的免疫酶法，并检测了鼻咽癌病人血清其它肿瘤病人和正常人的ＩｇＡ／ｇｐ１２５抗体，其灵敏度远远高于ＩｇＡ／ＥＡ，接近于８ｇＡ／ＭＡ和ＩｇＡ／ＶＣＡ，特异性高于ＩｇＡ／ＭＡ和ＩｇＡ／ＣＶＡ，接近于ＩｇＡ／ＥＡ。     

套式聚合酶链反应检测巨细胞病毒的初步研究

目的建立套式聚合酶链反应（ＰＣＲ）加限制性酶切分析方法检测人巨细胞病毒（ＨＣＭＶ）。方法在ＨＣＭＶ直接早期蛋白ＥｃｏＲＩＪＤＮＡ片段内，自行设计两对引物，建立套式ＰＣＲ检测ＨＣＭＶＤＮＡ，同时结合病毒分离检测临床标本。结果２３例新生儿肝炎综合征患儿中，１０例病毒分离及套式ＰＣＲ均阳性；１例病毒分离阴性，但套式ＰＣＲ阳性。对５８例妊娠早期孕妇血标本进行检测，套式ＰＣＲ阳性率为９％，病毒分离阳性率则为７％。结论套式ＰＣＲ加限制性酶切分析是一种临床检测ＨＣＭＶ的快速有效手段，值得临床推广。     

丙型肝炎病毒不同区抗原酶免疫试剂研究

目的　探讨丙型肝炎病毒（ＨＣＶ） 不同区抗原的反应性,为建立抗ＨＣＶ 酶免疫测定（ＥＩＡ） 确认试剂提供依据。方法　利用基因工程技术,重组所表达的ＨＣＶ 不同区抗原片段（ ＨＣＶＣ、ＮＳ３ 、ＮＳ４ 、ＮＳ５） ,建立了ＨＣＶ 单片段抗原ＥＩＡ 检测方法,检测了７４７ 份四川地区献血员血清。对其中３７３ 份血清,用２ 个厂生产的经批检合格的抗ＨＣＶＥＩＡ 试剂进行检测比较,对个别结果不符的样品进一步进行逆转录聚合酶链反应（ＲＴ－ ＰＣＲ） 分析。结果　献血员中抗ＨＣＶＣ 等４ 种抗体阳性检出率分别为４ ．６８ ％ （３５／７４７） 、５ ．２ ％ （３９／７４７） 、１ ．１ ％ （８／７４７） 、１ ．２ ％ （９／７４７） ,２ 家试剂阳性检出率均为４ ％（１５／３７３） 。结论　ＨＣＶ 不同区抗原片段的抗体检出率差异较大,ＮＳ３ 检出率最高,其次为Ｃ 区、ＮＳ５ 、ＮＳ４ 。说明抗ＨＣＶＥＩＡ 检测试剂以ＨＣＶ ＮＳ３ 区和Ｃ 区为主要抗原片段。     

Determination of creatine kinase isoenzyme MB activity in serum using immunological inhibition of creatine kinase M subunit activity. Activity kinetics and diagnostic significance in myocardial infarction.

This is a new method for the determination of creatine kinase isoenzyme MB activity in serum. The method uses direct activity measurement of creatine kinase B subunit activity after blocking of CK-M subunit activity by inhibiting antibodies. The test takes no longer than 15 min. The method yields an intra-serial C.V. of 2.0-12.9%, and a C.V. from day to day of 5.5%. The detection limit is 3.4 U/l creatine kinase MB. In the 95 cases with proven myocardial infarction several types of creatine kinase MB activity kinetics could be determined. The percentage of creatine kinase MB of peak CK-total is 6-25%, with a mean of 11.1%. The amount of creatine kinase MB with respect to total CK activity after reinfarction is higher than the amount after initial infarction.     

Dissociable correlates of two classes of retrieval processing in prefrontal cortex

Although substantial evidence suggests that the prefrontal cortex (PFC) implements processes that are critical for accurate episodic memory judgments, the specific roles of different PFC subregions remain unclear. Here, we used event-related functional magnetic resonance imaging to distinguish between prefrontal activity related to operations that (1) influence processing of retrieval cues based on current task demands, or (2) are involved in monitoring the outputs of retrieval. Fourteen participants studied auditory words spoken by a male or female speaker and completed memory tests in which the stimuli were unstudied foil words and studied words spoken by either the same speaker at study, or the alternate speaker. On "general" test trials, participants were to determine whether each word was studied, regardless of the voice of the speaker, whereas on "specific" test trials, participants were to additionally distinguish between studied words that were spoken in the same voice or a different voice at study. Thus, on specific test trials, participants were explicitly required to attend to voice information in order to evaluate each test item. Anterior (right BA 10), dorsolateral prefrontal (right BA 46), and inferior frontal (bilateral BA 47/12) regions were more active during specific than during general trials. Activation in anterior and dorsolateral PFC was enhanced during specific test trials even in response to unstudied items, suggesting that activation in these regions was related to the differential processing of retrieval cues in the two tasks. In contrast, differences between specific and general test trials in inferior frontal regions (bilateral BA 47/12) were seen only for studied items, suggesting a role for these regions in post-retrieval monitoring processes. Results from this study are consistent with the idea that different PFC subregions implement distinct, but complementary processes that collectively support accurate episodic memory judgments.     

俯卧位通气对高海拔地区肺复张治疗无效急性呼吸窘迫综合征患者氧合的影响

目的 探讨俯卧位通气对高海拔地区肺复张术(RM)治疗无效急性呼吸窘迫综合征(ARDS)患者的治疗作用.方法 从海拔2260m的地区医院筛选RM治疗无效的41例ARDS患者[平均氧合指数( PaO2/FiO2)较RM前升高＜20％视为RM无效],依不同病因分为肺内源性ARDS组(ARDSp组)和肺外源性ARDS组(ARDSexp组),每组再按信封法随机分为俯卧位组和仰卧位组,即ARDSp俯卧位组(11例)、ARDSp仰卧位组(9例)、ARDSexp俯卧位组(10例)、ARDSexp仰卧位组(11例).在通气前及通气1、2、3、4h监测动脉血氧分压( PaO2)、PaO2/FiO2、静态顺应性(Cst)、气道阻力(Raw)的变化.结果 通气lh时,ARDSexp俯卧位组PaO2/FiO2( mm Hg,l mm Hg=0.133 kPa)即较通气前显著升高(157.4±40.6比129.3±48.7,P＜0.05),并随通气时间延长呈持续增高趋势,4h达峰值(219.1 ±41.1);且ARDSexp俯卧位组通气3h内PaO2/FiO2较其他3组显著增高,另3组间则差异无统计学意义.ARDSp俯卧位组、ARDSexp俯卧位组通气4h时PaO2/FiO2均较相应仰卧位组显著增高(208.8±39.7比127.4±47.1,219.1±41.1比124.9±50.8,均P＜0.05).4组通气前后Cst无显著改变,各组间差异也无统计学意义.ARDSp俯卧位组通气4h时Raw(cmH2O·L-1·s-1)较通气前显著降低(6.8±1.7比10.7±1.8,P＜0.05),且明显低于其他3组;其他3组各时间点Raw组内及组间比较差异均无统计学意义.结论 俯卧位通气作为ARDS机械通气重要策略之一,可以改善RM无效高原ARDS患者的氧合,为抢救患者赢得宝贵的时间.     

Digital imaging among medical facilities

The Department of Veterans Affairs (VA) in the USA operates a network of 172 medical centres which all utilize a hospital information system (HIS) which has been developed and is currently maintained by the VA. During the past several years, an image management and communication module has been developed, installed and clinically utilized at the Washington DC and Maryland VA Medical Centres. This image management and communication system, referred to as the decentralized hospital computer program (DHCP) imaging system, is fully integrated with a commercial picture archiving and communication system (PACS). The system is utilized to capture, archive, and display all images generated within the hospital including radiology, nuclear medicine, pathology, endoscopy, bronchoscopy, and dermatology, intraoperative photographs, ECG data, and a limited number of paper documents. The ultimate goal of the project is to have all patient text and image data available at any clinical workstation to any authorized user anywhere within the network of medical centres. Clinical requirements for an imaging workstation include ease of use, rapid and reliable access to the complete set of patient information, and images which are of acceptable quality to meet the requirements of the user and the subspecialty. Patient confidentiality and data security must be safeguarded at all times. Integration of the images with the remainder of the patient's database was found to be critical to the success of the project. The experience at the Washington and Maryland facilities suggests that an imaging system that is successfully integrated with a hospital information system can provide substantial clinical and economic benefits both within and among medical centres. Clinical acceptance and utilization of the system has been excellent, particularly in diagnostic radiology where DHCP Imaging has been interfaced to a commercial PAC system. Based upon this initial experience, the VA has begun to deploy the system throughout its large network of medical centres.     

Myocardial elastography at both high temporal and spatial resolution for the detection of infarcts

Myocardial elastography is a novel method for noninvasively assessing regional myocardial function, with the advantages of high spatial and temporal resolution and high signal-to-noise ratio (SNR). In this paper, in-vivo experiments were performed in anesthetized normal and infarcted mice (one day after left anterior descending coronary artery [LAD] ligation) using a high-resolution (30 MHz) ultrasound system (Vevo 770, VisualSonics Inc., Toronto, ON, Canada). Radiofrequency (RF) signals of the left ventricle (LV) in longitudinal (long-axis) view and the associated electrocardiogram (ECG) were simultaneously acquired. Using a retrospective ECG gating technique, 2-D full field-of-view RF frames were acquired at an extremely high frame rate (8 kHz) that resulted in high-quality incremental displacement and strain estimation of the myocardium. The incremental results were further accumulated to obtain the cumulative displacements and strains. Two-dimensional and M-mode displacement images and strain images (elastograms), as well as displacement and strain profiles as a function of time, were compared between normal and infarcted mice. Incremental results clearly depicted cardiac events including LV contraction, LV relaxation and isovolumetric phases in both normal and infarcted mice, and also evidently indicated reduced motion and deformation in the infarcted myocardium. The elastograms indicated that the infarcted regions underwent thinning during systole rather than thickening, as in the normal case. The cumulative elastograms were found to have higher elastographic SNR (SNR(e)) than the incremental elastograms (e.g., 10.6 vs. 4.7 in a normal myocardium, and 6.0 vs. 2.4 in an infarcted myocardium). Finally, preliminary statistical results from nine normal (m = 9) and seven infarcted (n = 7) mice indicated the capability of the cumulative strain in differentiating infracted from normal myocardia. In conclusion, myocardial elastography could provide regional strain information at simultaneously high temporal (>/=0.125 ms) and spatial ( approximately 55 microm) resolution as well as high precision ( approximately 0.05 microm displacement). This technique was thus capable of accurately characterizing normal myocardial function throughout an entire cardiac cycle, at the same high resolution, and detecting and localizing myocardial infarction in vivo.     

Clinical Pharmacokinetics of Morphine

Morphine, the most widely used mu-opioid analgesic for acute and chronic pain, is the standard against which new analgesics are measured. A thorough understanding of the pharmacokinetics of morphine is required in order to safely and effectively use this analgesic in a wide variety of patients with different levels of organ function. A MEDLINE search was conducted to identify literature published between 1966 and January 2002 relevant to the pharmacokinetics of morphine. These publications were reviewed and the literature summarized regarding unique and clinically important elements of morphine disposition relative to its parenteral administration (including intravenous, intramuscular, subcutaneous, epidural and intrathecal administration), absorption profile (immediate release, controlled release, and sublingual/buccal, and rectal administration), distribution, and its metabolism/ excretion. Special populations, including infants, elderly, and those with renal/liver failure, have a unique morphine pharmacokinetic profile that must be taken into account in order to maximize analgesic efficacy and reduce the risk of adverse events.