应用抑制消减杂交技术构建哮喘病人嗜酸细胞差异表达基因消减cDNA文库

目的：构建哮喘病人治疗前后嗜酸细胞差异表达基因的消减cDNA文库。方法：采用新近建立的抑制消减杂交方法，哮喘治疗前后的嗜酸细胞为试验和对比材料，分离哮喘病人嗜酸细胞中差异表达基因的cDNA片段，将其与T载体进行T／A连接构建文库，将连接产物用氯化钙转化法转化大肠杆菌进行文库扩增和蓝白斑筛选。随机挑取100个白色克隆用茵落PCR进行鉴定。结果：扩增消减cDNA文库获得300余个白色阳性克隆，随机挑取100个白色克隆用PCR进行扩增，90％的克隆中均有200～600bp的插入片段，这些片段可能是哮喘嗜酸细胞差异表达基因的cDNA片段。结论：用SSH法及T／A克隆技术成功构建了哮喘病人治疗前后嗜酸细胞差异表达基因消减cDNA文库，该消减cDNA文库的建立为进一步筛选、克隆哮喘病人嗜酸细胞差异表达的新基因奠定了基础。     

化脓性链球菌高毒力株特异基因文库的构建和分析

目的构建化脓性链球菌毒力基因文库,克隆和筛选化脓性链球菌高毒力株特异表达基因。方法采用抑制消减杂交方法,以从患者体内分离的链球菌作为毒力菌株,分离毒力基因,将其与PMD18-T载体进行T/A连接构建文库,将连接产物转化感受态大肠杆菌TOP10进行文库扩增后,将转化菌液涂布于LB固体平板,构建化脓性链球菌毒力株特异基因消减文库,用斑点杂交初步筛选后,将获得的阳性克隆进行测序和同源性分析。结果酶切产物为100~2 000 bp,连接效率大于50%,成功构建了化脓性链球菌毒力基因消减文库,所得阳性克隆经斑点杂交筛选后测序,与Genebank数据库进行同源性比对,5个未知序列可能为新基因,7个与已知基因有高度的同源性。结论用抑制消减杂交技术及T/A克隆技术成功构建了化脓性链球菌毒力基因文库;5个未知的新序列可能与化脓性链球菌的高毒力有关。     

急性白血病多药耐药相关消减cDNA文库的构建与鉴定

研究目的是构建急性白血病多药耐药相关消减cDNA文库，以用于白血病多药耐药(MDR)相关基因的筛选。采用改良的消减杂交方法建立急性白血病耐药细胞差异表达基因cDNA文库，提取HL-60／VCR与HL-60细胞mRNA，将HL-60／VCR细胞mRNA用Cap-Finder法反转录成cDNA，与以常规反转录的HL-60细胞cDNA为模板．PCR合成生物素标记的扣除探针进行杂交。杂交产物用Sephacryl S-400柱和具有链亲和素作用的磁殊纯化后，得到差异表达cDNA。PCR法特异扩增，进行T-A克隆建成消减cDNA文库，并通过反向点杂交鉴定其质量。结果表明：构建急性白血病多药耐药相关消减cDNA文库所需样本量少且时间短，全长或近全长的cDNA分子较多(约达总数的2／3)，质量较高。结论：成功构建急性白血病多药耐药相关消减cDNA文库，为筛选白血病耐药相关基因奠定了基础。     

Lin-CD34-与Lin-CD34+细胞差异表达基因的鉴定

目的 克隆并鉴定Lin^-CD34^-细胞区别于Lin^-CD34^ 细胞的可能与其性状相关的基因。方法 以uLin^-CD34^-细胞作为检测对象，Lin^-CD34 细胞作为驱动细胞，应用抑制消减杂交技术构建Lin^-CD34^-细胞的cDNA消减库。选取部分克隆测序，测序结果与GenBank进行同源性比较和功能分析。结果 共获得593个含有插入片段的克隆，片段的长度为300～500bp。随机选取53条EST进行测序，其中37条EST代表了10个已知基因，其余16条EST。代表了4个未知序列。结论 利用抑制消减杂交技术鉴定出部分Lin^-CD34^-细胞特异表达的基因，可能与Lin^-CD34^-细胞的特性相关。     

应用抑制性消减杂交技术克隆和筛选白血病多药耐药相关基因

目的 克隆和筛选白血病多药耐药（MDR）相关基因。方法 以MDR白血病细胞株K562/DOX为检测样本。以K562细胞株为驱赶样本（对照）。应用抑制性消减杂交（SSH）技术分离和克隆K562/DOX细胞中过高表达的基因。构建cDNA消减库；通过反向Northern斑点杂交法进一步筛选消减库，对阳性克隆进行序列测定和同源性分析；并采用RT-PCR和Northern blot方法对某些阳性克隆进行进一步的验证。结果 发现了11个在K562/DOX细胞中高表达的基因。包括S3核糖体蛋白（S3ribosomal protein,S3rp)基因，尼克酰胺腺嘌呤二核苷酸脱氢酶亚单位2（NADHdehydrogenase subunit2,ND2）基因，My023基因等，其中多数基因与肿瘤MDR的关系尚未见报道。结论 筛选到几个可能与白血病MDR形成有关的基因。提示了白血病MDR发生的新机制。     

日本血吸虫微管蛋白基因的获得和分析

目的从日本血吸虫(schistosoma japonicum，Sj)成虫cDNA文库中获得并分析日本血吸虫新的表达基因，为日本血吸虫病的防治提供药物靶标或侯选疫苗。方法构建日本血吸虫成虫cDNA文库，随机挑取重组阳性克隆进行测序，对部分序列进行步移法测序获取全长cDNA，并进行生物信息学分析和登录。结果获得了1个日本血吸虫新基因，全长1439bp，编码443个氨基酸，与肝片形吸虫微管蛋白基因具有79％的同源性。结论表达序列标签法、步移法测序和生物信息学技术三者相结合，有利于发现日本血吸虫新基因。     

抑制性差减杂交技术分离白血病多药耐药基因

目的分离及鉴定与白血病多药耐药性相关的差异表达基因。方法采用抑制性差减杂交(SSH)技术分离非耐药细胞株K562与耐药细胞株K562/DOX差异表达基因。提取总RNA,逆转录合成cDNA,经限制性内切酶RsaⅠ酶切后,分别与不同的接头(adopter1和adopter2R)连接;连接产物插入pMD19-T载体后转入大肠埃希菌中,构建cDNA差减文库;挑取阳性克隆提取质粒进行测序及同源序列分析,确定差异表达基因。结果筛选获得220个差异表达基因,包括血红蛋白、核糖体和线粒体等相关基因,以及热休克因子结合蛋白(HSPB1)基因等其他基因。结论采用SSH技术及分子克隆技术可构建耐药及非耐药肿瘤细胞株差异表达基因的差减cDNA文库,能够为进一步筛选、克隆肿瘤细胞多药耐药性相关差异表达基因奠定基础。     

白血病原代骨髓基质细胞对柔红霉素作用下Jurkat细胞基因表达的影响

目的研究白血病患者骨髓基质细胞（BMSCs）抑制柔红霉素（DNR）诱导Jurkat细胞凋亡过程中Jurkat细胞相关基因表达的变化,并进一步分析差异表达基因在BMSCs对Jurkat细胞保护过程中的意义.方法采用Percoll体外分离初治急性白血病患者骨髓单个核细胞,贴壁培养BMSCs;采用抑制性消减杂交技术等分子生物学方法建立白血病BMSCs保护的Jurkat细胞差异表达基因的cDNA文库;并对差异表达的基因进行初步鉴定与分析.结果成功地建立了白血病BMSCs保护的Jurkat细胞上调和下调差异表达基因的cDNA文库;初步筛选克隆到30个上调差异表达基因和22个下调差异表达基因cDNA片段;其功能主要与细胞周期调控、细胞凋亡、细胞能量代谢相关.结论白血病患者BMSCs能诱导Jurkat细胞基因发生差异表达,为探讨白血病患者BMSCs保护白血病细胞逃避化疗药物杀伤的分子机制进行了前期研究.     

瘢痕疙瘩抑制消减cDNA文库构建和质量分析对筛选和克隆瘢痕疙瘩相关基因的意义

目的:构建瘢痕疙瘩抑制消减cDNA文库,为进一步筛选、克隆瘢痕疙瘩特异表达的基因奠定基础。方法:实验于2003-09/2004-08在南方医科大学基因工程研究所完成。从同一标本的瘢痕疙瘩和正常皮肤组织分离poly(A)+RNA,经反转录后,利用抑制消减杂交方法,通过两轮杂交和两次抑制聚合酶链反应构建了两种组织间差异表达基因的cDNA消减文库。结果:挑取了84个克隆进行聚合酶链反应扩增检测是否有插入片段,结果显示71个克隆有插入片段,其中45个克隆片段大小范围为200～750bp,符合预期的文库构建要求。结论:应用消减杂交的方法,在去除相同遗传背景的条件下,可以建立较特异的瘢痕疙瘩cDNA消减文库,该文库为进一步批量筛选瘢痕疙瘩发生相关基因群和克隆瘢痕疙瘩特异表达基因奠定了基础,为创伤临床康复治疗提供理论依据。     

人胚胎造血的AGM区基质细胞基因表达谱分析

为了筛选在主动脉-性腺-中肾（AGM）区基质细胞中差异表达的基因，以药物流产的孕30-35天的人胚胎AGM区来源基质细胞为“实验方”，以源自意外而致流产的孕4—5月的水囊引产的胎儿肝组织的基质细胞为“驱动方”，建立了在人AGM区基质细胞中差异表达基因的消减杂交文库。进一步用基因芯片技术对所建立的抑制消减杂交文库进行筛选，并挑选其中明显差异表达的18个克隆进行序列测定和同源性分析。结果表明：在所构建的AGM抑制消减杂交文库中，经过PCR鉴定有特异性扩增带且扩增带分子量在200—700bp的克隆有211个，阳性率高达76.4％。经过基因芯片筛选，挑选出ratio值大于5的克隆共18个进行序列测定显示，在测序的18个明显差异表达的阳性克隆中，4个为未知基因，14个为已知基因，其中这些已知基因分别参与了细胞迁移、分化、生长、细胞周期、信号转导及血管发生等细胞重要生命过程。这些基因大多数在胚胎分化造血发育中的作用尚未见报道。结论 筛选AGM区基质细胞中差异表达的基因为进一步研究胚胎造血发育的分化调控的分子机理提供了重要的理论基础。     

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.     

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.     

手转胎头术失败的原因与分娩结局临床分析

目的 探讨手转胎头术失败的原因与分娩结局.方法 选择2008年1月至2010年12月于我院住院分娩的持续性枕横位、枕后位产妇198例,根据行手转胎头术后结果分为成功组126例、失败组72例.比较两组分娩结局,对比分析失败原因.结果 失败组胎儿体质量≥3500 g的发生率[76.4％(55/72)]明显高于成功组[31.7％(40/126)],差异有统计学意义(x2=30.177,P=0.001)、失败组宫缩乏力发生率[58.3％(42/72)]高于成功组[38.1％ (48/126)],差异有统计学意义(x2=7.569,P=0.006)、失败组骨盆临界或轻度狭窄发生率[38.9％ (28/72)]高于成功组[23.8％(30/126)],差异有统计学意义(x2 =5.030,P=0.002)、失败组手转胎头时机不当(宫口开大＜6 cm、胎头位于坐骨棘上及宫口开大8～10 cm、胎头位于坐骨棘下≥2 cm)发生率[61.1％(44/72)]高于成功组[38.9％(49/126)],差异有统计学意义(x2=9.084,P=0.003).失败组母儿并发症(产后出血、产褥病率、胎儿窘迫、新生儿窒息)发生率高于成功组(x2 =9.586,P=0.002、x2=9.334,P=0.002、x2=5.910,P=0.015、x2=5.240,P=0.022)、失败组剖宫产发生率[72.2％(52/72)]明显高于成功组[34.1 ％(43/126),x2=26.641,P=0.001)].结论 手转胎头术能使难产变顺产,降低剖宫产率,减少母儿并发症,但须积极预防、处理导致手转胎头术失败的原因,对矫正失败后继续矫正及试产应慎重.     

Evidence-Based Pain Management and Palliative Care in Issue Four for 2009 of The Cochrane Library

ABSTRACT

The Cochrane Library of Systematic Reviews is published quarterly. Issue 4 for 2009 contains 4027 complete reviews, 1906 protocols for reviews in production, and 11447 one-page summaries of systematic reviews published in the general medical literature. In addition, there are citations of 600,000 randomized controlled trials, and 12,200 cited papers in the Cochrane methodology register. The health technology assessment database contains over 7500 citations. This edition of the Library contains 90 new reviews, of which 19 have potential relevance for practitioners in pain and palliative medicine.     

Wie zuverlässig sind Angaben zu nichtentzündlichen ausgedehnten Schmerzen?

ZusammenfassungFragestellung Es wurde geprüft, wie sich der Differenziertheitsgrad zweier Schmerzmessmethoden auf Angaben zur Ausgedehntheit klinischer Schmerzen auswirkt. Zugleich wurde der Referenzzeitraum variiert, über den die Patienten berichten sollten.Methode Erfasst wurde der Einfluss zu Lasten der Befragungsdifferenziertheit durch den Vergleich zweier Körperschema-Bildvorlagen. Drei Referenzzeiträume (Schmerz aktuell, letzte Woche, letztes halbes Jahr) wurden vorgegeben.Ergebnisse Patienten mit ausgedehnten Schmerzen gaben bei differenzierter Befragung um so mehr Schmerzen an, je weiter die Schmerzen zurück lagen und je größer der Berichtszeitraum war. Patienten mit gelenknahen Schmerzen gaben bei hoch differenzierter Befragung weniger ausgedehnte Schmerzen in der Vergangenheit an als bei globaler Einschätzung. Patienten mit Rückenschmerzen berichteten bei differenzierter Befragung zum aktuellen Schmerz über weniger ausgedehnte Schmerzen als bei globaler Befragung.Schlussfolgerung Die Angaben zur Schmerzausdehnung variieren vor allem bei Patienten mit ausgedehnten Schmerzen in Abhängigkeit von der Differenziertheit der Befragung. In diesen Fällen ist die Wahrscheinlichkeit erhöht, dass sich die Beschwerdesymptomatik zumindest teilweise erst in der Reaktion auf die situativen Befragungsbedingungen konstituiert und daher nicht auf andere Befragungsbedingungen generalisiert werden kann.