双表达外源性4-1BBL/RANTES的K562细胞活化外周血淋巴细胞的研究

目的探讨K562细胞表达外源性蛋白4-1BBL/RANTES后,对T细胞、NK细胞有无活化作用,为进一步构建人工抗原提呈细胞提供前提。方法采用分子克隆技术,分别将4-1BBL和RANTES基因插入双表达载体pVITRO-2,命名为pV4-1BBL-RANTES。经测序鉴定后,利用脂质体介导的转染及潮霉素筛选,获得稳定双表达4-1BBL、RANTES分子的K562细胞(K562/4-1BBL/RANTES)。经流式细胞仪(FACS)分选后,K562/4-1BBL/RANTES细胞用丝裂霉素C处理,与外周血淋巴细胞孵育24,FACS检测淋巴细胞表面活化性受体CD69的表达;对NK细胞同时检测了活化性受体NKG2D的表达情况。结果受K562/4-1BBL/RANTES细胞刺激后,CD3+T细胞、CD4+T细胞、γδT细胞和NK细胞的活化性受体CD69的表达与未受刺激前相比,均有明显上调;但与单纯K562细胞刺激组相比,无显著差异。另外,CD8+T细胞CD69的表达及NK细胞NKG2D的表达无明显变化。结论将4-1BBL和RANTES共表达于K562细胞,不具备活化淋巴细胞的效应。     

双表达外源性4—1BBL／RANTES的K562细胞活化外周血淋巴细胞的研究

目的探讨K562细胞表达外源性蛋白4-1BBL／RANTES后，对T细胞、NK细胞有无活化作用，为进一步构建人工抗原提呈细胞提供前提。方法采用分子克隆技术，分别将4-IBBL和RANTES基因插入双表达载体pVITRO-2，命名为pV4-1BBL-RANTES。经测序鉴定后，利用脂质体介导的转染及潮霉素筛选，获得稳定双表达4-1BBL、RANTES分子的K562细胞（K562／4—1BBL/RANTES）。经流式细胞仪（FACS）分选后，K562／4—1BBL／RANTES细胞用丝裂霉素C处理，与外周血淋巴细胞孵育24，FACS检测淋巴细胞表面活化性受体CD69的表达；对NK细胞同时检测了活化性受体NKG2D的表达情况。结果受K562／4-1BBL／RANTES细胞刺激后，CD3^＋T细胞、CD4^＋T细胞、γδT细胞和NK细胞的活化性受体CD69的表达与未受刺激前相比，均有明显上调；但与单纯K562细胞刺激组相比，无显著差异。另外，CD8^＋T细胞CD69的表达及NK细胞NKG2D的表达无明显变化。结论将4-1BBL和RANTES共表达于K562细胞，不具备活化淋巴细胞的效应。     

人上皮细胞黏附分子的真核表达及鉴定

目的构建人上皮细胞黏附分子(EpCAM)全基因的真核表达质粒,研究其在HepG2细胞中的表达。方法将EpCAM基因克隆入真核表达载体pcDNA3.1(+),构建真核表达质粒pcDNA3.1(+)-EpCAM,转染人肝癌HepG2细胞。通过免疫荧光、Western Blotting及流式细胞检测对转染后细胞内EpCAM蛋白的表达进行鉴定。结果双酶切鉴定结果表明重组质粒构建成功。免疫荧光、Western Blotting及流式细胞检测结果一致表明,转染后EpCAM基因在HepG2细胞中表达成功。结论成功构建了人EpCAM的真核表达载体,并在HepG2细胞中表达,为研究高表达人EpCAM的肝癌细胞的功能打下基础。     

人NKG2A、CD94的基因克隆和真核共表达载体的构建

目的构建真核共表达载体pNKG2A-IRES-CD94。方法提取人外周血淋巴细胞总RNA,逆转录-聚合酶链反应(RT-PCR)扩增NKG2A、CD94基因,分别克隆pMD-NKG2A、pMD-CD94,对克隆基因进行DNA序列分析。pMD-CD94经XbaⅠ和SalⅠ酶切,插入相应酶切的真核表达载体pIRES(多克隆位点B),NKG2A经XhoⅠ和MluⅠ酶切,插入相应酶切的pIRES-CD94(多克隆位点A)。结果扩增的DNA片段和预期的人NKG2A和CD94大小一致;DNA序列分析显示,克隆的基因序列和GenBank数据库中人NKG2A和CD94基因序列一致。酶谱分析显示,NKG2A和CD94正向插入表达载体pIRES。结论成功构建了含人NKG2A和CD94的真核共表达质粒pNKG2A-IRES-CD94,为研究NKG2A和CD94基因的功能奠定了基础。     

新趋化因子DMC对顺铂致HT-29细胞凋亡的影响

目的:研究新DMC基因高表达对人结肠癌HT-29细胞凋亡及抗肿瘤药物敏感性的影响.方法:将DMC蛋白融合表达载体pcDNA3.1(-)/DMC和对照质粒分别转染细胞,顺铂诱导细胞凋亡,荧光显微镜和电子显微镜观察细胞形态,以流式细胞术检测细胞凋亡率变化.结果:pcDNA3.1(-)/DMC转染细胞株凋亡率均明显低于空白细胞株(P<0.05);电子显微镜结果与流式细胞术检测结果一致.结论:pcDNA3.1(-)/DMC转染HT-29细胞后,能够使肿瘤细胞对化疗药物的敏感性降低.     

人NKG2A、CD94的基因克隆和真核共表达载体的构建

目的构建真核共表达载体pNKG2A-IRES-CD94。方法提取人外周血淋巴细胞总RNA,逆转录-聚合酶链反应（RT-PCR）扩增NKG2A、CD94基因,分别克隆pMD-NKG2A、pMD-CD94,对克隆基因进行DNA序列分析。pMD-CD94经XbaⅠ和saIⅠ酶切,插入相应酶切的真核表达载体pIRES（多克隆位点B）,NKG2A经XhoⅠ和MluⅠ酶切,插入相应酶切的pIRES-CD94（多克隆位点A）。结果扩增的DNA片段和预期的人NKG2A和CD94大小一致;DNA序列分析显示,克隆的基因序列和GenBank数据库中人NKG2A和CD94基因序列一致。酶谱分析显示,NKG2A和CD94正向插入表达载体pIRES。结论成功构建了含人NKG2A和CD94的真核共表达质粒pNKG2A-IRES-CD94,为研究NKG2A和CD94基因的功能奠定了基础。     

大鼠膜联蛋白A_2真核表达载体的构建及表达

目的:克隆大鼠背根神经节膜联蛋白A2(annexin Ⅱ)的基因,构建真核表达载体并检测其表达。方法:以RT-PCR法扩增大鼠背根神经节(DRG)细胞中膜联蛋白A2(annexin Ⅱ)的基因,构建pGMT-Anxa2克隆载体,以此为模板插入flag标签扩增Anxa2-flag序列,将其定向插入真核表达载体pcDNA3.1(+),获得表达载体pcDNA3.1(+)-Anxa2-flag。以PCR、双酶切及序列测定法鉴定该质粒。以脂质体法将表达载体转染到HEK293细胞中,Western印迹和细胞免疫荧光检测annexin Ⅱ蛋白表达及细胞内定位。结果:PCR扩增Anxa2-flag片段大小与预期相同。通过PCR、HindⅢ和NotⅠ双酶切及测序鉴定,证明构建出了含大鼠annexin Ⅱ基因的真核表达载体。Western印迹和细胞免疫荧光的检测表明大鼠annexin Ⅱ基因稳定表达,并可在胞膜和胞质中广泛表达。结论:成功构建pcDNA3.1(+)-Anxa2-flag真核表达载体,并稳定表达蛋白,为研究annexin Ⅱ调控DRG伤害性感受传导的分子机制奠定基础。     

重组VEGF165在哺乳动物细胞中表达及其生物学活性

目的构建真核表达载体pcDNA3.1(+)-血管内皮生长因子(VEGF)165并在哺乳动物细胞中实现目的基因的表达,对目的蛋白进行鉴定和生物学活性分析。方法分子生物学方法将扩增的VEGF165基因克隆入真核表达载体pcDNA3.1(+),在脂质体介导下将重组质粒转染至哺乳动物细胞NIH 3T3并进行抗生素压力筛选,采用双抗夹心ELISA、SDS-PAGE和免疫印迹方法对目的蛋白在转染细胞中的特异表达进行鉴定,在血管内皮细胞ECV304上对表达产物的细胞结合活性和促增殖活性进行分析。结果构建的真核表达载体pcDNA3.1(+)-VEGF165成功转染NIH3T3,获得加压筛选的重组细胞,目的基因得到稳定表达,目的蛋白能与血管内皮细胞ECV304特异结合,并具有促内皮细胞增殖活性。结论应用pcDNA3.1(+)-VEGF165载体转染的NIH 3T3细胞可稳定表达具生物学活性的VEGF165重组蛋白。     

白细胞相关免疫球蛋白样受体2(CD306)真核表达载体构建及蛋白的纯化与鉴定

背景:目前国内外对白细胞相关免疫球蛋白样受体(leukocyte-associated immunoglobulin like-receptor,LAIR)家族中LAIR1 的生物学功能研究已较清晰,而关于LAIR-2分子在机体内的生物学功能尚缺乏相关报道.目的:为进一步研究LAlR-2的体内生物学功能,构建其真核表达载体,纯化并鉴定蛋白.设计、时间及地点:单一样本观察实验,于2007-06/2008-06在解放军第四军医大学完成.材料:载体pig/3c由英国牛津大学徐小宁博士惠赠.载体pcDNA3.1由荷兰Meyaard博士惠赠.中国仓鼠卵巢细胞系CHO由解放军第四军医大学免疫学教研室冻存.方法:构建plg/3c-LAIR-2及pcDNA3.1-LAIR-2两个真核表达载体,电转染法转染CHO细胞建立稳定表达LAIR-2-Fc融合蛋白及LAIR-2全长蛋白分子的细胞株,通过Western blot、细胞免疫化学、流式细胞术分析实验鉴定真核表达LAIR-2蛋白分子与抗原核表达LAIR-2 mAbs的结合活性.主要观察指标:稳定转染细胞系的建立及真核表达蛋白的纯化和鉴定.LAIR-2蛋白与相应单克隆抗体的结合活性.结果:构建真核表达载体并成功转染CHO细胞,建立稳定分泌LAIR-2-Fc融合蛋白的转染细胞系及稳定分泌LAIR-2蛋白的转染细胞系,分别命名为CHO/LAIR-2-Fc以及CHO/LAIR-2.Western blot实验表明真核表达的LAIR-2全长蛋白分子均可与三株抗LAIR-2 mAb 1A7、3H12及4A9发生特异性结合反应.免疫细胞化学、流式细胞术分析实验结果表明抗原核细胞表达蛋白的三株LAIR-2单克隆抗体中,1A7不能结合pcDNA3.1-LAIR-2转染的CHO细胞内的LAIR-2,面3H12和4A9均可以很好结合细胞内的LAIR-2.结论:实验成功构建了plg/3c-LAIR-2及pcDNA3.1-LAIR-2两个真核表达载体,成功转染CHO细胞并建立稳定表达LAIR-2-Fc融合蛋白及LAIR-2全长蛋白分子的细胞株.真核表达的LAIR-2蛋白分子与抗原核表达的LAIR-2 mAbs有良好的结合活性.     

肌腱愈合及粘连形成机制的研究:胰岛素样生长因子1基因真核表达载体的构建及表达

目的:探讨真核表达载体介导外源性胰岛素样生长因子-1(IGF-1)基因转染肌腱细胞的可能性。方法:组织块法培养原代肌腱细胞,体外重组真核表达载体pcDNA3.1(+)-IGF-1,并以脂多胺DOGS为介导转染培养的肌腱细胞,反转录多聚酶链反应(RT-PCR)检测IGF-1mRNA的表达,ELISA法检测IGF-1活性蛋白表达。结果:成功构建真核表达载体pcDNA3.1(+)-IGF-1;RT-PCR检测显示转染后的肌腱细胞成功表达IGF-1mRNA;ELISA结果显示转染组IGF-1蛋白表达较对照组明显增高(t=-2.873,P<0.05)。结论:重组的真核表达载体pcDNA3.1(+)-IGF-1能够将IGF-1cDNA成功导入肌腱细胞并表达。     

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.