基于L1蛋白的HPV血清学实验研究

目的 探讨基于基因重组HPV16 L1蛋白的血清学实验在人乳头瘤病毒16(HPV16)感染诊断中的应用,为HPV感染及宫颈癌的临床诊断提供实验依据.方法 选择63例宫颈癌患者,用PCR方法检测宫颈癌组织的HPV 型别;并采用重组原核表达系统制备HPV16 L1蛋白用ELISA方法检测其血清HPV16 L1抗体,同时以74份尖锐湿疣患者血清及120份健康体检者血清作为对照.结果 宫颈癌HPV通用型DNA阳性率为50.8%(32/63),HPV16 DNA阳性率为46%(29/63),HPV18 DNA阳性率为3.2%(2/63).宫颈癌组血清HPV16 L1抗体阳性率为44.4%(28/63),较健康对照组阳性率1.67%(2/120)和尖锐湿疣组阳性率10.8%(8/74)差异具有统计学显著性意义(P＜0.01).HPV16 DNA检测法与HPV16 L1 ELISA血清抗体检测法两者间呈中度相关(K= 0.524,P＜0.05).结论 宫颈癌以HPV16感染为多见,HPV16 L1血清ELISA 抗体检测可用作HPV16感染的血清学诊断和宫颈癌的免疫学研究.     

人乳头瘤病毒11型E7蛋白在原核细胞的表达及其诊断尖锐湿疣的价值

目的 研究人乳头瘤病毒(HPV)11型E7蛋白在原核细胞的表达及其在尖锐湿疣(CA)血清学诊断中的应用价值.方法 用PCR从CA患者组织中扩增HPV11 E7全长基因,构建重组质粒pET32a(+)/HPV11 E7,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导后表达,用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳和蛋白质印迹法(WB)分析鉴定;经镍螯合亲和层析胶体(Ni-NTA Agarose)纯化HPV11 E7融合蛋白后作抗原,用间接ELISA法对93例CA患者、43例宫颈癌患者和58名健康对照者血清标本进行IgG抗体检测.结果 HPV11 E7融合蛋白在原核表达系统中呈较高效表达(浓度为40μg/ml).CA组、宫颈癌组和健康对照组血清IgG抗体均值分别为1.545±0.131、0.586±0.155和0.674±0.150,阳性率分别为76.3%(71/93)、11.6%(5/43)和5.2%(3/58);CA组血清IgG抗体均值及阳性率均高于宫颈癌组和对照组(均P＜0.01).结论 HPV11 E7融合蛋白具较强的抗原性,用于CA血清学诊断试剂的研制具有临床应用价值.     

HPV 16型L2全长原核融合蛋白的交叉反应特性研究

目的 研究HPV 16型L2全长蛋白在HPV6、11、16和18型别间的交叉反应特性.方法 收集108例尖锐湿疣患者、156例宫颈癌患者和100名健康对照者的血清标本,采用PCR法从宫颈癌患者组织DNA中扩增HPV16 L2全长基因,将其克隆至原核表达载体PGEX-4T-1中,构建重组质粒PGEX-4T-1-HPV16 L2,将鉴定正确的重组质粒转化至E.coli BL21(DE3)中,经IPTG诱导表达,SDS-PAGE和WB鉴定;进一步通过WB检测HPV16 L2全长融合蛋白与HPV6、11、16和18型DNA检测阳性的患者血清的特异性结合能力.以镍螯合亲和层析柱(Nj-NTA Agarose)纯化HPV16 L2融合蛋白作为包被抗原,采用间接ELISA法检测3组标本中的HPV血清特异性IgG抗体.结果 成功构建了重组质粒PGEX-4T-1-HPV16 L2,含有HPV16 L2的融合蛋白在原核表达体系中呈较高效表达,表达量为27.2%;SDS-PAGE和WB检测结果显示在约相对分子质量82 000处可见特异性阳性信号条带;以HPV6、11、16和18 DNA阳性患者血清为一抗的WB结果显示,HPV16 L2全长融合蛋白在相对分子质量82 000处均出现特异性阳性信号条带.ELISA结果显示,尖锐湿疣组、宫颈癌组和健康对照组血清抗体A值分别为0.825±0.409、0.808±0.376、0.128±0.15,阳性率分别为92.6%、92.3%和6.0%.3组间血清抗体A值及阳性率差异均具有统计学意义(H=207.292,x2=251.846,P均＜0.01),而尖锐湿疣组与宫颈癌组之间的血清抗体均值(0.825和0.808)差异无统计学意义(H=0,P＞0.05).结论 HPV16 L2全长原核融合蛋白具有较强的抗原性,与HPV6、11、16和18型别间具有交叉反应,可进一步用于HPV感染及其相关肿瘤ELISA血清学检测试剂的通用型靶抗原研究.     

尖锐湿疣患者IL—2和IFN—r水平观察

目的对人乳头瘤病毒(HPV)感染所致尖锐湿疣患者的IL-2和IFN-r水平进行观察,探讨HPV感染对机体细胞因子的影响。方法该研究调查了30例经临床诊断的尖锐湿疣患者,并经HPV6.II型PCR检测为阳性,其中随机抽取了10例进行组织病理学诊断,诊断结果均为尖锐湿疣。30例患者为CA患者组,选择30例正常健康者作为对照组,采用夹心酶联免疫吸附法(ELISA)试剂盒对30例CA患者和30例对照组受试者的血清中白介素-2(IL-2),r干扰素(IPN-r)的含量进行检测。结果CA患者与正常对照组对比较血清中IL-2(17.75±7.52pg/ml,P<0.01)和IFN-r(27.89±9.1pg/ml,P<0.05)的含量显著性下降。结论HPV感染导致尖锐湿疣患者血清中IL-2,IFN-r含量显著性下降,引起细胞免疫功能下降,因此,在临床上可以考虑用白介素-2和r干扰素治疗尖锐湿疣。     

HPV阳性宫颈癌和CIN患者血浆miR-150,SCCA及CA125的表达及临床意义

目的 探讨血浆miR-150,鳞状细胞癌抗原(SCCA)及糖类抗原125(CA125)在人乳头瘤病毒(HPV)阳性宫颈癌患者中的表达及临床意义。方法采用RT-PCR检测108例HPV阳性宫颈癌患者(宫颈癌组),40例HPV阳性宫颈上皮内瘤变(CIN)患者(CIN组)和40例HPV单纯阳性/HPV阳性子宫良性病变者(对照组)血浆miR-150,SCCA及CA125水平,分析miR-150表达与宫颈癌临床病理特征的关系。应用ROC曲线评价miR-150,SCCA及CA125在HPV阳性宫颈癌患者中的早期诊断价值,多元Logistic回归模型分析三项指标与HPV阳性宫颈癌的关系。结果 宫颈癌组血浆miR-150,SCCA及CA125表达水平均明显高于CIN组和对照组[miR-150(2^-ΔΔCt):10.28±4.16,4.72±1.18和1.83±0.64; SCCA(ng/ml):9.86±2.14,3.18±0.85和1.35±0.34; CA125(U/ml):46.26±11.58,19.14±7.05和16.42±5.83,F=16.427,13.205,8.719,均P<0.01]。HPV阳性宫颈癌患者血浆miR-150表达水平与临床分期、淋巴结转移及浸润深度相关(P<0.05)。血浆miR-150诊断HPV阳性宫颈癌和CIN的最佳截值分别为8.25和3.46,敏感度和特异度分别为87.0%和84.3%,81.0%和80.2%。Logistic回归分析显示,血浆miR-150和SCCA水平升高是HPV阳性宫颈癌发生的独立危险因素[OR(95%CI)=2.107(1.915～2.814),OR(95%CI)=1.583(1.327～2.036)]。结论 血浆miR-150在HPV阳性宫颈癌患者中明显上调,可作为HPV阳性宫颈癌早期诊断和CIN鉴别诊断的生物学标志物。     

慢病毒介导人乳头瘤病毒16型E6基因短发夹状干扰RNA对宫颈癌细胞感染效率和侵袭能力的影响

目的 研究以慢病毒为载体靶向人乳头瘤病毒16型(HPV16)E6基因的短发夹状干扰RNA(shRNA)对宫颈癌Caski细胞侵袭能力的影响.方法 将靶向HPV16型E6的shRNA表达序列(干扰组)和无义表达(无义干扰组)克隆到慢病毒工作质粒中包装成病毒颗粒,感染宫颈癌Caski细胞.观察绿色荧光阳性细胞的比例以了解慢病毒对Caski细胞的感染效率;用RT-PCR法检测shRNA作用后3组Caski细胞HPV16型E6 mRNA含量的变化;Western blot法检测E6蛋白表达的变化;Transwell小室法检测肿瘤细胞侵袭能力的变化.结果 慢病毒对Caski细胞的最佳每个细胞中的病毒颗粒数目(MOI)为2.5;与无义干扰组相比,干扰组细胞中HPV16E6的mRNA含量减少70%,蛋白表达水平降低63%,Caski细胞的侵袭能力显著下降.结论 慢病毒携带的shRNA能干扰宫颈癌Caski细胞中HPV16型E6的表达,并抑制癌细胞的侵袭能力.     

荧光定量聚合酶链反应检测人类乳头瘤病毒在尖锐湿疣中的应用

目的：对尖锐湿疣(CA)患者进行人类乳头瘤病毒(HPV)DNA定量测定及HPV分型。方法：用荧光定量聚合酶链反应(FQ-PCR)对146例CA患者疣体组织进行HPV6，11型和HPV 16．18型的分型检测及HPV DNA定量测定。结果：146例CA患者标本中检出HPV 6．11和HPV 16．18型感染阳性143例(97．95％)。其中单独HPV 6．11型阳性104例，占71．24％；单独HPV 16.18型阳性16例，占10．96％；HPV 6．11和HPV 16．18型同时阳性23例，占15．75％；HPV 6.11和HPV 16.18型同时阴性3例，占2．05％。定量测定结果显示：HPV 6.11型患者病毒载量最高1．0×10^8拷贝／mL，最低2．8×10^3拷贝／mL，HPV 16.18型患者病毒载量最高1.0×10^8拷贝／mL，最低2．57×10^4拷贝／mL。结论：CA患者应用FQ-PCR检测HPV阳性率高，并可快速区分高危型及低危型HPV感染，对尖锐湿疣复发及癌变作出可能性预测。     

艾滋病病毒感染者合并丙型肝炎病毒感染的临床研究

目的　探讨人类免疫缺陷病毒 (HIV)和丙型肝炎病毒 (HCV)混合感染的临床特点。方法　收集 2 8份HIV合并HCV感染血清、血浆和 2 4份单纯HIV阳性血清、血浆。进行HIV、HCV载量、T淋巴细胞、血常规、肝功能等检测 ,分析 2组实验室检测指标的差别。结果　HIV合并感染者的HCV载量与各项检测指标无明显相关性。HIV感染组的HIV载量 (4 8± 0 9lg拷贝 /ml)高于HIV合并感染组 [(4 1± 1 0 )lg拷贝 /ml,P <0 0 5 ],而前者红细胞和血红蛋白值低于后者 (P <0 0 5 )。HIV合并感染组的丙氨酸转氨酶异常者 [(87 0± 6 9 6 )U/L ,占 6 4 % ]明显高于HIV感染组 [(2 0 6± 13 6 )U/L ,占 10 % ,P <0 0 1]。结论　HIV合并感染组的HIV载量低于HIV感染组 ,而肝功能损害明显重于HIV感染组 ,HCV载量与各项实验室指标无明显相关性。     

血清HBeAg阴性HBV感染者病毒含量与基因突变的临床关系

目的 :了解HBV感染者血清HBeAg阴转后HBVDNA含量与C基因启动子 (basalcorepromoter,BCP)和C基因 (pre-core/core ,preC/C)变异的关系。方法 :采用PCR微板核酸杂交方法检测 342例血清HBeAg阴性HBV感染者血清HBVDNA含量及BCP与preC/C基因变异。结果 :在HBsAg/HbcAb/HbeAb和HBsAg/HbcAb感染模式中检测到HBVDNA阳性率为 2 2 8% (36 / 15 8)及 31 6 % (31/ 98)。两者无显著差异 (P >0 0 5 ) ;两种模式中HBVDNA阳性血清的BCP变异检出率分别为 ;6 3 9% (2 3/ 36 )、 5 1 6 % (16 / 31) ;preC/C变异检出率 77 8% (2 8/36 )、 5 8 1% (18/ 31) ;HBVDNA血清浓度平均为 4 9 2± 36 7pg/ml、 5 1 7± 31 5pg/ml,均无显著差异 (P >0 0 5 )。两种基因变异间无相关关系 (P >0 0 5 )。但BCP变异组HBVDNA (6 1 8± 34 1pg/ml)显著高于非变异组 (34 4±2 9 7pg/ml) ;preC/C变异组HBVDNA (5 5 7± 31 5pg/ml)显著高于非变异组 (38 7± 2 4 1pg/ml)。结论 :BCP基因变异与preC/C变异是随机存在于HBeAg转换中 ,突变后血清HBVDNA浓度显著升高     

手足口病相关肠道病毒感染血清IgM抗体交叉反应及动态变化

目的分析手足口病相关肠道病毒间IgM抗体交叉反应及动态产生过程,评价抗体捕获法检测肠道病毒71型(EV71)及柯萨奇病毒A组16型(CA16)感染的早期诊断价值。方法收集广州市妇女儿童医疗中心319名手足口病患者及疑似患者咽拭子319份、血标本565份。荧光定量PCR检测咽拭子标本,血清中和试验检测配对血清,结合两法结果为判断标准,将手足口病患者分为EV71、CA16及其他肠道病毒感染组;以捕获ELISA法检测不同发病时间EV71-IgM抗体及CA16-IgM抗体。结果发病第一天均能检测出EV71-IgM抗体及CA16-IgM抗体,阳性率分别为33.0%和25.0%,检出率分别在第5天和第7天达到100%,抗体可持续检出时间达数月。EV71感染血清CA16-IgM捕获ELISA法交叉反应率为25.0%,CA16感染血清EV71-IgM捕获ELISA法交叉反应率为28.0%。EV71-IgM和CA16-IgM抗体存在严重交叉反应,通过两者450 nm处吸光度(A450 nm)比值可以成功诊断97.1%EV71感染和93.0%CA16感染。同一天采集的肛拭子及血清标本检测结果表明,荧光定量PCR与ELISA法诊断EV71及CA16感染的结果差异无统计学意义。结论 EV71及CA16抗体捕获ELISA法是一种简单、有效的诊断方法,联合两种方法检测可以有效提高诊断特异性。     

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.     

Guns and Knives in New Mexico: Patterns of Penetrating Trauma, 1978–1993

Objective: To identify patterns of nonfatal and fatal penetrating trauma among children and adults in New Mexico using ED and medical examiner data.
Methods: The authors retrospectively sampled in 5-year intervals all victims of penetrating trauma who presented to either the state Level-1 trauma center or the state medical examiner from a 16-year period (1978–1993). Rates of nonfatal and fatal firearm and stabbing injury were compared for children and adults.
Results: Rates of nonfatal injury were similar (firearm, 34.3 per 100,000 person-years; stabbing, 35.1). However, rates of fatal injury were significantly different (firearm, 21.9; stabbing, 2.7; relative risk: 8.2; 95% confidence interval: 5.4, 12.5). From 1978 to 1993, nonfatal injury rates increased for children (p = 0.0043) and adults (p < 0.0001), while fatal penetrating injury remained constant. The increase in nonfatal injury in children resulted from increased firearm injury rates. In adults, both stabbing and firearm nonfatal injury rates increased.
Conclusions: Nonfatal injury data suggest that nonfatal violence has increased; fatal injury data suggest that violent death rates have remained constant. Injury patterns vary by age, mechanism of trauma, and data source. These results suggest that ED and medical examiner data differ and that both are needed to guide injury prevention programs.