甲状腺功能低下对大鼠中枢神经细胞增殖及其迁移的影响

妊娠大鼠用丙基硫氧嘧啶造成甲状腺功能低下(甲低)后,其新生仔鼠~3H-胸腺嘧啶核苷示踪,在注射后不同时间取脑制成切片并进行自显影。结果表明:甲低时,①嗅球嗅室壁室管膜及其下层细胞增殖延迟,细胞向内颗粒层迁移的速度减慢;②齿状回多形层细胞向颗粒层迁移的速度减慢;③小脑皮质外颗粒层细胞增殖延迟,外颗粒层细胞向内颗粒层迁移的速度减慢;④大脑皮质视、听区侧脑室壁室管膜及其下层细胞向浅层迁移的速度减慢,致使浅层的形成延迟。作者认为细胞增殖及迁移的延缓,影响神经回路的建成,可能是克汀病患者聋哑、智力低下、步态不稳的原因之一。     

介绍一种简便的组织蜡块修边法

在病理组织制片过程中,传统的组织蜡块修边法常常是技术人员用刀具或者其它工具对蜡块逐一刮削的方法,修掉蜡块周围余蜡(组织周围要留有余蜡)[1]。随着病理标本的增多,蜡块数量也不断增加。手工逐一刮削蜡块包埋盒的修边方法已满足不了日益增加的工作需要,严重影响制片效率,且人工成本较大,安全性低,影响切片的质量。     

微柱凝胶法交叉配血试验及其影响因素的探讨

目的:探讨微柱凝胶法(MGT)用于交叉配血试验及其影响因素。方法:应用MGT法和试管生理盐水法,对1200例受血者和供血者的血标本进行交叉配血试验,对MGT法交叉配血试验阳性的标本,再用试管法间接抗人球蛋白试验做血型不规则抗体筛选和交叉配血试验,以便进行对照比较。结果:在1200例血标本中,MGT法交叉配血试验阴性者1173例(97.8%),阳性27例(2.3%)。在27例阳性标本中,由血型不规则抗体引起凝集者2例(7.4%),由非血型因素引起的非特异性凝集者25例(92.6%)。结论:应用MGT法进行交叉配血试验,能及时检出血型抗原抗体引起的特异性凝集反应和非血型因素引起的非特异性假凝集反应;而盐水法则不能检出上述真假凝集反应,因此不能有效地防止免疫性溶血性和非溶血性输血反应的发生。     

凝胶-浇注法制备多孔羟基磷灰石的抗压强度正交试验分析

多孔羟基磷灰石 (简称 HA)具有良好的引导骨形成的能力 ,是目前骨缺损修复术中替代自体骨的常用材料之一。利用凝胶 -浇注成型工艺制备多孔 HA,工艺简单 ,并能制备形状复杂的陶瓷坯体。本研究利用正交试验法 ,对抗压强度实验结果进行分析 ,找出有机单体 AM、交联剂 MBAM、引发剂 APS及水等工艺参数的主次关系和较优水平 ,并研究了干燥、烧结工艺等因素对多孔羟基磷灰石特性的影响。结果表明 ,凝胶 -浇注成型工艺过程中各因素的主次关系依次为 :有机单体 AM、引发剂 APS、交联剂 MBAM、水。根据各因子的较优水平 ,选用合适的料浆组成和干燥、烧结工艺 ,制备出了抗压强度为 6～ 7MPa的多孔羟基磷灰石。     

介绍一种准确简便的CIC测定法——PEG-AC法

<正> 本文以手(山岛)秀毅的PEG-CC法为基础,对其CIC的沉淀步骤、测定终点的显示及结果的表示法进行了改进,定名为聚乙二醇-抗补体法(PEG-AC法),该法其原理为     

介绍一种简便的少量游离细胞电镜样品制备法

游离细胞的电镜样品制备通常采用离心固定法、琼脂包埋法、血浆凝块法等[1,2],但当细胞量很少时(<1×104),就不易收集到细胞团块,给样品制备造成困难。有人采用“琼脂模具收集法”收集细胞,效果虽好,但操作烦琐。我们经过实践摸索出一种更简便的制备少量游离细胞电镜样品的方法,效果满意,现介绍如下:1　材料与方法　收集少量体外分离的小鼠脾脏树突状细胞,将细胞连同1640培养基放在尖头离心管中离心5分钟(800转/分)后弃上清,加入4%多聚甲醛(用pH7.30,0.1mol/L磷酸缓冲液配制)并轻轻吹打使细胞混悬于固定液中于4℃固定1小时。然后用0.1mol/…     

一种简便的皮肤、肌肉石蜡制片方法

皮肤层次丰富，结构较复杂。由于皮肤各层臻密度不同，其制片过程中常需特殊处理，才能获得较理想的切片。皮肤制片改进的方法有操作繁琐，有的仍难以避免真皮（网状层）出现松散、分离或浅层出现较多裂隙等现象。     

介绍一种改进的骨内血管透明法

骨内血管的透明标本往往显示不佳。我们在进行手舟骨血供研究时,为了较好地显示舟骨内血管构筑,经过摸索,改进了透明法,获得了显示比较完美的骨内血管透明标本。现介绍如下:透明主要是利用一些化学药品或物理方法处理机体组织,使其折光率与透明剂近似。以便与所需要显示并已进行染色的结构区别。     

介绍一种显示肥大细胞快捷简便的染色方法

目前关于肥大细胞的染色方法很多,如经典的甲苯胺蓝染色、阿尔辛蓝-番红花红染色、硫堇染色、地衣红染色等,因种种原因目前尚无法广泛应用,本文现介绍一种显示肥大细胞的染色方法,该方法安全、简便、快捷,并且可用于新鲜或固定的组织标本。     

介绍一种简便的组织切片脱汞方法

介绍一种简便的组织切片脱汞方法梁英杰，凌启波在进行某些特殊染色和免疫组化染色时，为了更好地保存组织成分或组织抗原，往往需要选择最佳的固定液。常用的固定液如Ｂ５固定液、Ｚｅｎｋｅｒ固定液，因含有汞盐，在染色前需经脱汞处理。本文介绍的方法是将脱汞的操作过...     

两种化学发光电泳迁移率变动分析技术的比较

采用DIG及Biotin标记寡核苷酸,研究了两种非放射性标记电泳迁移率变动分析的实验方法,即化学发光电泳迁移率变动分析实验(chemi-EMSA)的可靠性,并对两种标记体系的化学发光电泳迁移率变动分析实验进行了比较,以确立一种最佳的化学发光电泳迁移率变动分析实验技术。结果表明:通过对转录因子蛋白NF-κB的实验研究,两种标记体系均可以获得良好的电泳迁移率变动分析检测效果,其中“Biotin-streptavidin-HRP-ECL化学发光电泳迁移率变动分析”实验体系更好。     

Determination of Optimal Conditions for the Immobilization of Cells in a Cell Capture Enzyme Immunoassay (CC-EIA) by a Simple Giemsa Assay

To determine the optimal conditions for the immobilization of cells in a cell capture enzyme immunoassay (CC-EIA), the most suitable diluent, and the optimal pH, temperature and period of incubation were examined using WI-38, a human embryonic lung fibroblast cell line. For the evaluation, we devised a simple Giemsa assay method, in which immobilized cells on a microplate were stained with Giemsa solution, the stained dye was eluted with ethanol after washing the plate, and the optimal density (O.D.) was measured at wavelength 620 nm. the optimal conditions for the immobilization were determined to be treatment with 5% formalin in phosphate-buffered saline (PBS) (pH 7.2) for 15 minutes at room temperature, which were confirmed to be suitable for the measurement of cell associated collagen by CC-EIA. Additionally, we found that the simple Giemsa staining method was also useful for evaluating the number of immobilized cells on the microplate after CC-EIA.     

A Simple Stained Protein Assay (SPA) for Rapid Total Protein Determination Applied to Spinal Fluids

A rapid, simple technique measures small amounts of total protein applied on an agarose gel slab. Alternating current through the gel may reduce diffusion of the protein, which is chemically fixed. The intensity of the Coomassie-Blue-stained spots is a function of the amount of protein determined by comparison with simultaneously run standards. Densitometric and visual estimates of protein in spinal fluids by the stained protein assay correlated with Lowry values highly significantly (n = 28; P less than 0.001, Spearman test of rank correlation). As little as 0.2 mug protein was measured; this figure should be compared with 10 mug for the Lowry method. The reproducibility of the method was +/- 5%.     

The Assessment of Histocompatibility by Mixed Lymphocyte Reaction as Measured by the Macrophage Electrophoretic Mobility (MEM) Test

The mixed lymphocyte reaction (MLR) between donor and recipient lymphocytes has been measured by the macrophage electrophoretic mobility (MEM) test and the modified (MOD-MEM) test. Its value as a measure of compatibility has been assessed by comparison with conventional HL-A serotyping and with the outcome of renal transplantation. Thirty-six living donor/recipient pairs and 59 cadaver donor/recipient pairs for transplantation have been studied. Whilst uniovular twins gave lymphocyte interactions, measured as macrophage slowings of about 1%, the slowing produced by paired allogeneic lymphocytes ranged from 2% to 26% depending on the number of HL-A matches. The test measurement of lymphocytic interaction was significantly correlated with histocompatibility measured by HL-A serotyping, in both living and cadaver donors. One way MEM-MLR showed the dominant role of the second HL-A sublocus in mixed lymphocyte reactivity. The long term success of the renal graft correlated with the pre-transplant initial reaction between donor and recipient lymphocytes. The test has advantages in the field of human histocompatibility assessment since no particular reference to individual antigens is made and it may be performed in a matter of hours.     

稳定表达鼻咽癌来源潜伏膜蛋白1基因的鼻咽癌细胞系的建立

目的:建立稳定表达鼻咽癌(NPC)来源潜伏膜蛋白1(LMP1)的鼻咽癌细胞系。方法:利用基因重组技术构建NPC来源LMP1的一般性真核表达载体及上皮特异性表达载体,并将其转染鼻咽癌细胞系CNE-2,用PCR、RT-PCR及蛋白印迹等检测N-LMP1在CNE-2中的整合和表达。结果:①成功构建了N-LMP1的一般性和上皮特异性表达载体。②N-LMP1基因在CNE-2细胞中获得了正确表达。结论:成功建立了稳定表达NPC来源LMP1的鼻咽癌细胞系,为进一步研究LMP1在鼻咽癌细胞中的作用机制奠定基础。     

A Simple Assay for Evaluating Inhibitors of Proteoglycan-Ligand Binding

Proteoglycans, once thought to primarily serve as structural components of extracellular matrix, are now being focused on for their role in tissue and cell regulation, particularly angiogenesis. Many growth factors, notably the fibroblast growth family (FGF) which now numbers 19 members, bind to heparin and heparan sulfate proteoglycans and this binding has been shown to have a significant impact on the availability and activity of these growth factors. Proteoglycans can serve as both temporal and spatial regulators and effective inhibitor design may depend on disruption of these interactions. We have developed a simple assay for evaluating small inhibitors of proteoglycan-ligand binding. The assay is based on cell-free incubation of the reactants and filtration across a cationic membrane. Conditions were established that allow one to semiquantitatively determine binding constants for both direct proteoglycan as well as soluble inhibitor affinity. The assay has been demonstrated using a model heparan sulfate proteoglycan preparation (perlecan from cultured bovine endothelial cells) and FGF-2. Protamine sulfate, sucrose octasulfate, and heparin were analyzed as model inhibitor molecules. This type of assay may have wide application as a fast and easy screening tool for small potential agonists and antagonists of proteoglycan-protein interactions. © 2000 Biomedical Engineering Society. PAC00: 8714Ee, 8715Kg, 8715Rn, 8780-y     

检测人博卡病毒抗体间接酶联免疫吸附法的建立及临床应用

目的 建立间接酶联免疫吸附法(ELISA)检测人博卡病毒(HBoV)抗体,探讨其临床应用的可行性.方法 以重组表达的HBoV融合蛋白VP2作为包被抗原,建立检测HBoV抗体的间接ELISA,优化该检测方法的最佳条件,观察其精密度、敏感度和特异性,并与荧光定量PCR方法对比检测40份临床样本,同时采用免疫印迹法(Western blot)确认其符合率.结果 所建立的间接ELISA最佳抗原包被浓度为2 mg/L,酶标二抗工作浓度为1∶5000,血清标本最佳稀释度为1∶200.该方法平均批内变异系数为6.87%,平均批间变异系数为4.67%.40份临床样本间接ELISA检测结果与荧光定量PCR及免疫印迹结果一致,符合率100%.结论 利用VP2融合蛋白作为抗原,建立的检测血清HBoV抗体间接ELISA方法特异性强、重复性好,可用于HBoV抗体的定量和定性检测.     

Detection of Lassa Virus Antinucleoprotein Immunoglobulin G (IgG) and IgM Antibodies by a Simple Recombinant Immunoblot Assay for Field Use

The nucleoprotein of Lassa virus, strain Josiah, was expressed in Escherichia coli as an N-terminally truncated, histidine-tagged recombinant protein. Following affinity purification the protein was completely denatured and spotted onto nitrocellulose membrane. A total of 1 μg of protein was applied for detection of Lassa virus antibodies (LVA) in a simple immunoblot assay. Specific anti-Lassa immunoglobulin M (IgM) antibodies could be detected by increasing the amount of protein to 5 μg. A panel of 913 serum specimens from regions in which Lassa virus was endemic and from regions in which Lassa virus was not endemic was used for evaluating the sensitivity and specificity of the LVA immunoblot in comparison to those of an indirect immunofluorescence (IIF) assay. The sera originated from field studies conducted in the Republic of Guinea (570 serum samples) and Liberia (99 serum samples), from inpatients of the clinical department of the Bernhard-Nocht-Institute, Hamburg, Germany (94 serum samples), and from healthy German blood donors (150 serum samples). In comparison to the IIF assay the LVA immunoblot assay had a specificity of 90.0 to 99.3%, depending on the origin of the specimens. The sensitivity was found to be highest for the Guinean samples (90.7%) and was lower for the Liberian samples (75%). Acute Lassa fever was diagnosed by PCR in 12 of 59 (20.3%) patients with fever of unknown origin (FUO) from the Republic of Guinea. On admission to the hospital, nine Lassa fever patients (75%) were reactive by the IgM immunoblot assay. One of the patients was infected with a new Lassa variant, which showed 10.4% variation on the amino acid level in comparison to the prototype strain of Lassa virus, Josiah. Seven PCR-negative patients were reactive by immunoblotting. The positive and negative predictive values of a single IgM immunoblot result for acute, PCR-confirmed Lassa fever were therefore 53.6 and 93.0%, respectively. Because of its high negative predictive value, a single IgM immunoblot result will be valuable for excluding acute Lassa fever for cases of FUO in areas where Lassa fever is endemic.     

Evaluation of a Microplate Latex Agglutination Method (Verotox-F Assay) for Detecting and Characterizing Verotoxins (Shiga Toxins) in Escherichia coli

The performance of a commercial microplate latex agglutination assay, the Verotox-F assay, was compared with that of the Vero cell assay for the detection and characterization of Escherichia coli verocytotoxins (VTs). Culture filtrates of 68 VT-positive E. coli strains (65 human isolates [33 of serotype O157:H7/H−, 32 of non-O157 serotypes] and 3 reference strains) and 104 VT-negative strains (100 human isolates and 4 reference strains) were investigated. The toxin phenotypes and genotypes of the 68 VT-positive isolates were VT1 only (18 strains), VT2 and/or VT2c (33 strains), and VT1 plus VT2 (17 strains). The Verotox-F assay involved incubation of serial dilutions of culture filtrates with equal volumes of latex particles sensitized with anti-VT1 antibody or anti-VT2 antibody in 96-well microtiter plates with appropriate controls and examination for latex agglutination after 20 to 24 h. Compared to the results of the Vero cell assay, the Verotox-F assay was 100% sensitive and 100% specific for the detection of VTs in culture filtrates and correctly identified the toxin types of all 68 VT producers. By checkerboard titration with purified toxins, the sensitivity of the Verotox-F assay was found to be 14 pg (0.7 ng/ml) for VT1, 12 pg (0.6 ng/ml) for VT2, and 350 pg (17.5 ng/ml) for VT2c; this sensitivity is comparable to that of the bioassay. The anti-VT2 latex reagent detected both VT2 and VT2c and did not cross-react with VT1. The anti-VT1 reagent showed a low-level cross-reaction with VT2c only at levels (≥4.5 μg/ml) that were about 1,000-fold higher than those found in culture filtrates. We conclude that the Verotox-F assay is highly sensitive and specific for the detection and characterization of VTs in culture filtrates of human E. coli isolates. The test is rapid, reliable, and easy to perform; its results are easy to interpret; and it should allow testing for VT to become more widely performed.