抗人甘露聚糖结合凝集素单抗的制备、鉴定及应用

目的　制备抗人甘露聚糖结合凝集素单克隆抗体及建立应用方法。方法　用亲和层析法自人血清中提取甘露聚糖结合凝集素 (MBL) ,免疫Balb/c小鼠 ,通过细胞融合的方法 ,得到 3株稳定分泌抗人MBL单克隆抗体 (mAb)的杂交瘤细胞株。建立检测MBL的ELISA。结果　本组mAb识别相对分子质量 (Mr)为 310 0 0的MBL分子 ,与MBL作用后可明显抑制MBL 甘露聚糖复合物的抗补体效应。用ELISA检测 186例健康人血清中MBL水平为 2 6 9± 1 5 8mg/L。结论　成功地获得了抗MBL的mAb并用于建立ELISA。     

用合成多肽制备抗胰蛋白酶原激活肽单克隆抗体的研究

目的建立以合成的小分子肽胰蛋白酶原激活肽制备单克隆抗体(mAb)的技术路线.方法以合成多肽TAP与载体KLH交联的偶联物为免疫原,免疫BALB/c小鼠,应用细胞融合技术建立能稳定分泌抗TAP mAb的杂交瘤细胞株.结果共获得10株能稳定分泌TAP mAb的杂交瘤细胞株,经稳定性试验,对其分泌的mAb进行效价测定、中和抑制试验、特异性鉴定,均表明所制备的杂交瘤细胞株稳定性好,其所分泌的mAb效价高、特异性好.结论利用合成多肽TAP成功制备了抗TAP单克隆抗体.     

抗HCCR单克隆抗体的制备及其鉴定

目的制备抗人宫颈癌癌基因（HCCR）单克隆抗体（mAb）,并进行鉴定。方法以HCCR重组蛋白免疫BALB/c小鼠,取免疫小鼠的脾细胞和同系小鼠的骨髓瘤细胞SP2/0进行细胞融合,常规筛选杂交瘤细胞。腹水诱导法制备抗人HCCR单克隆抗体,并经蛋白A亲合层析进行纯化。用Western blot和免疫荧光等方法检测HCCR mAb的特异性。用纯化HCCR蛋白进行包被,建立间接ELISA方法,检测系列稀释的HCCR mAb,确定其灵敏度。结果筛选出2株分泌抗人HCCR mAb的杂交瘤细胞株。Western blot结果表明抗人HCCR抗体能与HCCR蛋白特异性结合,免疫荧光结果显示肝癌HepG2细胞中胞浆和胞膜均呈阳性染色。间接ELISA方法检测HCCR mAb的灵敏度可达到1μg/L。结论成功制备并鉴定了特异性抗人HCCR mAb的杂交瘤细胞株,为进一步深入研究HCCR的生物学特征和临床应用打下基础。     

用合成多肽制备抗胰蛋白酶原激活肽单克隆抗体的研究

目的 建立以合成的小分子肽胰蛋白酶原激活肽制备单克隆抗体(mAb)的技术路线。方法 以合成多肽TAP与载体KLH交联的偶联物为免疫原，免疫BALB／c小鼠，应用细胞融合技术建立能稳定分泌抗TAP mAb的杂交瘤细胞株。结果 共获得10株能稳定分泌TAP mAb的杂交瘤细胞株．经稳定性试验．对其分泌的mAb进行效价测定、中和抑制试验、特异性鉴定，均表明所制备的杂交瘤细胞株稳定性好．其所分泌的mAb效价高、特异性好。结论 利用合成多肽TAP成功制备了抗TAP单克隆抗体。     

人Survivin蛋白的纯化、单克隆抗体的制备及在肿瘤细胞的表达

目的表达人Survivin重组蛋白、制备单克隆抗体及检测Survivin在多种肿瘤细胞中的表达。方法诱导含有pMS-Survivin重组质粒的E.coli.表达人MS2-Survivin重组蛋白,并作为抗原免疫BALB/c小鼠,取其脾细胞与Sp2/0骨髓瘤细胞融合,经ELISA双筛,获得稳定分泌抗人Survivin mAb的杂交瘤细胞株。ELISA检测Survivin mAb的特异性。免疫细胞化学检测多种肿瘤细胞中天然Survivin的表达情况。结果诱导菌在相对分子质量为30KD左右表达出一新的蛋白条带。筛选出2株稳定分泌特异性抗人Survivin mAb的杂交瘤细胞株,亚类鉴定均为IgG1类。两株单克隆抗体的效价均达到6.4×105。ELISA结果显示出抗人Survivin mAb的特异性。在胃癌细胞、肺癌细胞、宫颈癌细胞中均强表达,且主要在细胞浆中。结论成功制备出特异性抗人Survivin mAb,为以后应用于临床检测奠定了基础,也为其进一步开发成诊断性试剂盒奠定了基础。     

抗人心肌肌钙蛋白T杂交瘤细胞系的建立及初步鉴定

[目的]建立稳定分泌抗人心肌肌钙蛋白T(cardic troponin,cTnT)单克隆抗体的杂交瘤细胞株.[方法]以纯化的cTnT(分子质量为3.7×107)加福氏佐剂免疫8周龄雌性Balb/c小鼠,取免疫脾细胞与骨髓瘤细胞sp2/0融合,以酶联免疫吸附法(ELISA)筛选.阳性克隆以有限稀释法进行克隆化、Western blot法鉴定所制备的单克隆抗体.[结果]共筛出4株稳定分泌抗cTnT单克隆抗体的杂交瘤细胞株,所分泌的抗体效价高达10-5,经免疫学鉴定其分泌的免疫球蛋白类型均为IgG1、κ型,且Western b1ot鉴定4株细胞株所分泌的抗体均与人cTnT均有很好的反应性.[结论]获得4株稳定分泌抗人cTnT单克隆抗体的杂交瘤细胞株,为cTnT的临床研究提供了可能性.     

人H-FABP单克隆抗体制备和纯化及其特性鉴定

目的制备人心肌型脂肪酸结合蛋白(Heart type fatty acid-binding protein;H-FABP)单克隆抗体。方法(1)应用淋巴细胞杂交瘤技术,将重组人H-FABP腹腔注射免疫BALB/C小鼠。(2)将鼠脾细胞与鼠骨髓瘤细胞(SP2/0)融合、培养。通过HAT和间接ELISA法筛选、鉴定制备稳定分泌抗人H-FABP单克隆抗体的杂交瘤细胞株。(3)利用Protein G Sepharose 4 Fast Flow亲合纯化体内诱生法制备的腹水抗体,并对抗体特性进行鉴定。结果得到7株特异性识别人H-FABP单克隆抗体,并纯化两株亲合力高、空间位点远的单克隆抗体。结论成功制备了稳定分泌抗人H-FABP单克隆抗体的杂交瘤细胞株。     

抗胎儿血红蛋白单克隆抗体的制备及特性鉴定

目的 制备和鉴定抗胎儿血红蛋白(fetal hemoglobin,HbF)单克隆抗体(mAb),为相关疾病的研究提供支持与工具.方法 用脐带血红细胞裂解物免疫Balb/c小鼠,取其脾细胞与小鼠骨髓瘤细胞(NS-1)融合制备杂交瘤,经筛选和三次克隆化,从制备的腹水中纯化单克隆抗体.用ELISA以及western blot等方法鉴定单克隆抗体的特性(效价、Ig亚类、特异性以及亲和力).结果 筛选到3株可稳定分泌抗HbF单克隆抗体的杂交瘤细胞6A5、5C8、2C8,腹水效价分别为1×10-6、 2×10-6和4×10-6.单抗亚类均为IgG1,抗体亲和力分别为7.8×109、2.2×108和8.6×109.ELISA和western blot结果显示,6A5、5C8、2C8 mAb只与HbF的二聚体特异性结合,与其他类型血红蛋白不发生交叉反应.结论 获得3株能特异性识别HbF的高亲和力mAb,可结合二聚体与单体HbF,为临床检测HbF水平及相关研究提供了有效的工具.     

人心肌肌钙蛋白I的重组表达及其单克隆抗体的制备

目的构建人心肌肌钙蛋白I(cTnI)的表达载体,表达cTnI重组蛋白,并制备其单克隆抗体(mAb)。方法以化学方法合成cTnI基因并插入融合表达载体pBV220的多克隆位点,构建重组表达质粒pBV220/cTnI。以重组质粒转化大肠杆菌DH5α,筛选阳性重组子,经热激诱导目的蛋白的表达,表达产物的免疫学活性用Westernblot进行鉴定。以基因重组的cTnI蛋白为抗原,常规方法免疫BALB/c小鼠,取其脾细胞与NS1细胞融合,获得稳定分泌cTnImAb的杂交瘤细胞株,ELISA检测mAb的相对亲和力;Westernblot检测mAb的特异性。结果酶切鉴定和DNA测序显示cTnI重组表达载体中含有人cTnI全长编码序列。将该重组载体转化入大肠杆菌DH5α中表达所得蛋白经WesternBlot验证为目的蛋白。筛选出2株能稳定分泌特异性抗cTnI的mAb杂交瘤细胞株,免疫球蛋白亚类均为IgG类;Westernblot结果显示,两株单抗识别相对分子量为24000的cTnI单一条带;中和抑制试验表明培养上清中的抗体能明显被cTnI中和;cTnI单克隆抗体的亲和常数为Kaff=1.62×109(mol/L)-1。结论成功地构建了cTnI表达载体、表达出cTnI重组蛋白,制备出抗cTnImAb,为进一步用于cTnI的体外免疫学检测奠定了基础。     

人5型腺病毒六邻体蛋白单克隆抗体的制备及鉴定

目的制备抗人5型腺病毒(HAdV5)六邻体(Hexon)蛋白的单克隆抗体并对其进行鉴定。方法用纯化的Hexon蛋白免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞融合,经ELISA间接法筛选和克隆化培养,获得4株分泌抗HAdV5的Hexon蛋白单克隆抗体的杂交瘤细胞。纯化获得单克隆抗体,使用ELISA和Western blot鉴定单克隆抗体的敏感性和特异性。结果 ELISA和Western blot结果表明这4株单克隆抗体与HAdV5的Hexon蛋白可以特异性结合。单克隆抗体株4A9-HRP标记和8D6建立的ELISA夹心法具备较高灵敏度和特异性。结论获得4株抗HAdV5的Hexon蛋白的单克隆抗体,为HAdV5相关的疫苗研制和HAdV5感染性疾病的早期快速诊断奠定了基础。     

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.     

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.     

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.     

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.