类风湿关节炎滑膜成纤维细胞MICA及其受体NKG2D表达的初步研究

目的:探讨共育培养体系中RA患者滑膜成纤维细胞MICA mRNA及T淋巴细胞表面活化受体NKG2D mRNA表达的改变及其在RA免疫机制中的作用.方法:RA滑膜成纤维细胞与Jurkat T细胞株以相同浓度共育培养.MTT法测定共育培养后T淋巴细胞的扩增能力,实时荧光定量PCR方法测定T淋巴细胞NKG2D mRNA的表达水平及滑膜成纤维细胞MICA mRNA、TNF-α mRNA的表达水平,并分别与单纯培养组比较.结果:共育培养组与单纯培养组比较,第4天T淋巴细胞增殖率明显增加(P＜0.001),其NKG2D mRNA的表达水平明显升高;同时滑膜成纤维细胞MICA mRNA及TNF-α mRNA表达水平也升高.结论:共育培养的RA滑膜成纤维细胞和T淋巴细胞可能通过MICA-NKG2D结合激活T淋巴细胞,RA滑膜成纤维细胞分泌的TNF-α很可能在诱导T淋巴细胞NKG2D表达升高中发挥着重要作用.     

外周血淋巴细胞穿孔素和颗粒酶B mRNA检测对肾移植急性排斥反应的早期诊断价值

探讨外周血淋巴细胞(peripheral blood lymphocyte,PBL)穿孔素和颗粒酶B mRNA检测对肾移植急性排斥反应(AR)的早期诊断价值。采用RT-PCR方法动态检测67例肾移植AR患者外周血淋巴细胞穿孔素和颗粒酶B mRNA的表达水平,同时检测所有患者肌酐(Cr)表达水平情况,比较各时间点穿孔素、颗粒酶B mRNA及Cr表达水平,并探讨穿孔素、颗粒酶B与AR的关系。移植后1d与移植前1d比较,PBL穿孔素、颗粒酶B mRNA表达水平比较无明显差异性(P>0.05),Cr表达水平明显下降(P<0.05);AR前3d较之前各时间点比较,PBL穿孔素、颗粒酶B mRNA表达水平明显上升(P<0.05),而Cr表达水平明显下降(P<0.05);AR后1d时PBL穿孔素、颗粒酶B mRNA表达水平达到最高峰,随后逐渐下降(P<0.05),而AR后5d时Cr表达水平达到最高峰,随后逐渐下降(P<0.05);PBL穿孔素、颗粒酶B mRNA表达水平升高时间比Cr早4d,AR患者经MP/OKT3冲击治疗后,PBL穿孔素和颗粒酶B mRNA表达水平逐渐降低至原有基础水平。PBL穿孔素和颗粒酶B mRNA在AR早期诊断和抗排斥反应疗效评估等方面具有一定的临床价值。     

人外周血NK细胞在细胞因子刺激下扩增后穿孔素、颗粒酶B基因表达的变化

目的 探讨经过纯化及在多种细胞因子的作用下扩增后的NK细胞,其穿孔素(per-furin)、颗粒酶B(granzyme B)表达水平的改变与细胞杀伤率的关系.方法 应用竞争性定量RT-PCR方法 检测了8例供者纯化、扩增后NK细胞的穿孔素、颗粒酶B基因表达水平,同时检测其对K562细胞的杀伤率.结果 经纯化、扩增后的NK细胞,在多种细胞因子作用下穿孔素和颗粒酶B的基因表达水平明显提高,且.IL-2+IL-15组、IL-2+IL-12+IL-15组基因的表达量均显著高于其他组,NK细胞对K562的杀伤率结果 与基因表达水平一致.结论 应用细胞因子后可使NK细胞的穿孔素、颗粒酶B基因表达水平明显提高,同时提高了NK细胞的杀伤功能.     

人NKG2D的基因克隆及其在CHO细胞中的表达

目的:构建重组人NKG2D真核表达载体并在CHO细胞表达重组人NKG2D。方法:用RT-PCR方法从NK-92细胞中调取NKG2D基因片段,克隆到pGEM-TEasy载体并对克隆的DNA片段进行序列分析。用限制内切酶EcoRI和BamHI消化pGEM-TEasy/NKG2D重组质粒,分离NKG2D片段,并插入真核表达质粒pEGFP-N1的相应限制酶位点,酶谱分析鉴定重组表达载体pEGFP-N1/NKG2D。然后经脂质体介导转染CHO细胞。应用荧光显微镜观测、Westernblot方法和免疫组化染色对转染细胞内pEGFP-N1/NKG2D的表达进行鉴定。结果:RT-PCR扩增获得650bp基因片段,经DNA序列分析证明所获得的DNA序列与文献报道的NKG2D序列一致。转染的CHO细胞在荧光显微镜下发出强绿色荧光,Westernblot分析显示重组蛋白能特异地与抗人NKG2D单克隆抗体结合;免疫组化检测显示,转染的CHO中有棕色颗粒,证明所构建的NKG2D真核表达载体可以在细胞中表达。结论:构建了人NKG2D的哺乳动物细胞表达载体,并成功地在CHO细胞中获得重组人NKG2D的表达。     

石蜡包埋乳腺癌组织中用荧光差别多聚酸链反应检测erbB2基因扩增

作者基于差别多聚酶键反应（D－PCR）和荧光DNA技术已发展了一种迅速的非放射性方法，为了在人类肿瘤标本库中定量检测ERBβ2基因扩增。来自ERBβ2基因和来自单拷贝参照基因序列同时用PCR扩增，其中每对引物中有一个用荧光标记。PCR产物在自动荧光DNA测序仪上，在激光激活发光后用适当的软件扫描直接定量。这种荧光差别多聚酶链反应（FD－PCR）法被用来定量检测195列甲醛固定、石腊包理乳腺癌组织中的ERBβ2基因扩增，其中52例（26％）发现有ERBβ2基因扩增，并与肿瘤大小、缺乏雌激素受体（ER）和PS2表达有显著相关，但与孕…     

结核杆菌H37Ra免疫小鼠后脾淋巴细胞的杀伤活性及免疫机制的研究

目的探讨结核杆菌H37Ra免疫小鼠后,产生特异性的脾淋巴细胞杀伤活性及其免疫机制。方法(1)分别以结核杆菌H37Ra、BCG和PBS免疫小鼠后第30天及第60天,分离各组小鼠脾淋巴细胞作为效应细胞,以表达Ag85B分泌蛋白的Sp2/0细胞为靶细胞,用MTT比色法检测免疫小鼠脾淋巴细胞的杀伤率;(2)在免疫后第60天,小鼠脾淋巴细胞经PPD刺激后,以RT-PCR法检测穿孔素mRNA及颗粒酶BmRNA的表达。结果(1)结核杆菌H37Ra组脾淋巴细胞的杀伤率明显高于PBS对照组(P<0.05),略高于BCG组;(2)H37Ra组和BCG组穿孔素、颗粒酶mRNA的表达量均显著高于PBS组(P<0.05),H37Ra组穿孔素mRNA的表达量显著高于BCG组(P<0.05),但颗粒酶BmRNA的表达量两组无差异。结论结核杆菌H37Ra免疫小鼠后,能增强脾淋巴细胞的杀伤活性,其机制可能与增加穿孔素、颗粒酶B的表达有关。     

NKG2D真核表达载体的构建及其对YT细胞生物学功能的影响

目的：建立NK细胞受体NKG2D真核表达载体，通过转染NK细胞系YT，初步探讨NKG2D分子对YT细胞系杀伤功能的增强作用。方法：用RT-PCR方法从NK-92细胞中调取NKG2D基因片段，克隆到pGEM-TEasy载体并对克隆的DNA片段进行序列分析。用限制内切酶EcoRⅠ和BamHⅠ消化pGEM-T Easy／NKG2D重组质粒，分离NKG2D片段，并插入真核表达质粒pEGFP-N1的相应限制酶位点，酶谱分析鉴定重组表达载体pEGFP-N1／NKG2D。然后经脂质体介导转染CHO细胞和YT细胞。应用荧光显微镜观测、Western blot方法和免疫组化染色对转染细胞内pEGFP-NilNKG2D的表达进行鉴定，MTF方法观察YT细胞对肿瘤细胞的杀伤功能。结果：RT-PCR扩增获得650bp基因片段，经DNA序列分析证明所获得的DNA序列与文献报道的NKG2D序列一致。转染的CHO细胞在荧光显微镜下发出强绿色荧光，Western blot分析显示重组蛋白能特异地与抗人NKG2D单克隆抗体结合；免疫组化检测显示，转染的CHO中有棕色颗粒，证明所构建的NKG2D真核表达载体可以在细胞中表达；转染NKG2D真核表达载体的YT细胞对乳腺癌细胞具有更强的杀伤效果。结论：所获得的表达NKG2D分子的真核表达载体，通过转染YT细胞，初步鉴定所表达NKG2D分子具有生物学功能，可以提高NK细胞对肿瘤细胞的杀伤活性。     

实时荧光定量PCR方法检测非霍奇金淋巴瘤患者B淋巴细胞刺激因子表达水平

目的:建立实时荧光定量PCR方法(RFQ-PCR)检测非霍奇金淋巴瘤(NHL)患者B淋巴细胞刺激因子(BAFF)表达水平。方法:47例NHL患者及20例健康对照,采用实时荧光定量PCR方法检测其外周血单个核细胞中的B淋巴细胞刺激因子表达水平。结果:RFQ-PCR检测BAFF含量的示灵敏度为10 pg/ml,低浓度样本批内和批间变异系数(CV)分别为8.56%和11.32%,高浓度样本批内和批间CV分别为0.76%和4.58%。NHL患者和健康对照者BAFF mRNA表达量分别为(0.48±0.023,0.25±0.023),两者具有显著性差异(P=0.0001)。结论:RFQ-PCR检测BAFF mRNA含量的方法,具有较好的检测灵敏度和重复性。NHL患者BAFF mRNA高表达,提示BAFF可能在NHL发生发展中发挥重要作用。     

RT-qPCR检测人IL-6 mRNA的方法建立与运用

目的：建立实时定量检测人IL-6mRNA的方法并加以应用。方法：抽取人外周静脉血，EDTA抗凝，用淋巴细胞分离液分离外周血单个核细胞，用Trizol裂解液提取总RNA，将总RNA逆转录为cDNA，然后用IL-6引物进行实时荧光定量PCR扩增，扩增体系中加入SYBR GreenⅠ。结果：该法检测IL-6 mRNA线性范围广。灵敏度高，特异性好，重复性好，方法简单。结论：该法可较为精确地定量IL-6 mRNA。     

水飞蓟宾增强人CIK细胞杀伤胃癌细胞株BCG-823的体外研究

为探讨水飞蓟宾对人CIK细胞杀伤胃癌细胞株BCG-823的影响及作用机制,分离健康志愿者外周血单个核细胞,体外诱导培养成人CIK细胞;收集培养扩增7d的CIK细胞,将其用不同浓度的水飞蓟宾诱导24h、48h、72h,CCK-8法检测其增殖情况;流式细胞术检测CIK细胞穿孔素、颗粒酶B、CD107a和IFN-γ的表达变化及水飞蓟宾对其凋亡的影响;LDH法检测CIK细胞对胃癌细胞株BCG-823的杀伤活性。结果显示,与对照组相比,浓度为(1.6~50)μg/mL的水飞蓟宾作用CIK细胞72h后,增殖率较对照组显著增加(P<0.05);穿孔素、颗粒酶B、CD107a和IFN-γ的表达以及活细胞率均不同程度增加(P<0.05);且对胃癌细胞BGC-823的杀伤活性显著增强(P<0.05)。以上实验结果提示一定浓度的水飞蓟宾对CIK细胞有促增殖作用,且能增强其对胃癌细胞株BCG-823的杀伤活性。     

Skeleton-based cerebrovascular quantitative analysis

This study was performed to evaluate the ability of contrast-enhanced ultrasonography (CEUS) with time-intensity curve analysis to demonstrate an increased enhancement of the liver parenchyma adjacent to the inflamed gallbladder, as seen on contrast-enhanced computed tomography. The Ethics Committee of our institution approved the study protocol (Kawasaki Medical School, registration number 1277). From April to November 2013, 11 consecutive patients with acute cholecystitis and 16 patients without cholecystitis consented to CEUS (Sonazoid™) and were enrolled in this study. The gallbladder and liver were scanned by one gastroenterologist using harmonic imaging with a low mechanical index. The raw imaging data were stored. Another physician, blinded to all clinical information, constructed the time-intensity curve. The major axis of the region of interest (ROI) was set in segment 5 (pericholecystic area), and the control ROI in segment 8 at the same depth. The intensity ratio (IR) was defined as the peak intensity of segment 5 divided by the simultaneous value of segment 8. The characteristics of the patient with and without acute cholecystitis were compared. The correlation between the IR and the presence of acute cholecystitis was analyzed using binomial logistic regression analysis. A receiver operating characteristic (ROC) curve analysis was performed as well. The IR was significantly higher in the group with than without acute cholecystitis (p = 0.006). The IR correlated significantly with the presence of acute gallbladder inflammation (p = 0.043). The area under the ROC curve was estimated as 0.852 (95 % confidence interval, 0.709–0.995). A cut-off value of 2.72 had a sensitivity of 81.8 % and a specificity of 81.3 %. The IR obtained by CEUS with time-intensity curve analysis generally demonstrated increased enhancement of the liver parenchyma adjacent to the inflamed gallbladder.     

Progress towards in vitro quantitative imaging of human femur using compound quantitative ultrasonic tomography

The objective of this study is to make cross-sectional ultrasonic quantitative tomography of the diaphysis of long bones. Ultrasonic propagation in bones is affected by the severe mismatch between the acoustic properties of this biological solid and those of the surrounding soft medium, namely, the soft tissues in vivo or water in vitro. Bone imaging is then a nonlinear inverse-scattering problem. In this paper, we showed that in vitro quantitative images of sound velocities in a human femur cross section could be reconstructed by combining ultrasonic reflection tomography (URT), which provides images of the macroscopic structure of the bone, and ultrasonic transmission tomography (UTT), which provides quantitative images of the sound velocity. For the shape, we developed an image-processing tool to extract the external and internal boundaries and cortical thickness measurements. For velocity mapping, we used a wavelet analysis tool adapted to ultrasound, which allowed us to detect precisely the time of flight from the transmitted signals. A brief review of the ultrasonic tomography that we developed using correction algorithms of the wavepaths and compensation procedures are presented. Also shown are the first results of our analyses on models and specimens of long bone using our new iterative quantitative protocol.     

Problems of quantitative urine analysis

Quantitative determinations of various analytes in urine represent only a small part of all analyses performed in the routine clinical chemistry laboratory. Consequently, analytical as well as pre-analytical problems are less often considered in urine analysis. In the first part of this paper, pre-analytical factors affecting quantitative determinations are discussed, e.g., physical activity, posture, circadian rhythms, different solubilities of the various substances, errors in urine collecting, or drug effects. Subsequently, aspects of linearity and specificity of the routine determinations will be illustrated with special consideration given to protein, calcium, and enzyme determinations as well as some specialized measurements, e.g., porphyrins, catecholamines and their metabolites, or 5-hydroxyindole-acetic acid. Effects of pre-analytical and analytical factors on intra-assay and between-assay variations found in urine analysis are discussed. Using the results of surveys, it is shown that the standardization of methods as well as the use of suitable standards are mandatory for an effective amelioration of the present quality of quantitative urine analysis.     

Antibody arrays for quantitative immunophenotyping

Detection of multiple surface antigens expressed on living cell is an important step for cell processing and clinical diagnosis. Here we describe the preparation of antibody arrays that allow parallel detection of multiple surface antigens through affinity binding of living cells. An antibody array was fabricated by photo-assisted patterning of an alkanethiol monolayer formed on a gold-coated glass plate and subsequent immobilization of antibodies specific for cell surface antigens in an array format. We demonstrate here that rapid phenotyping can be performed on the array for both adhesion-dependent and non-dependent cells by direct cell binding assays. The density of bound cells on each antibody spot was in accordance with their contents in an original suspension. This result suggests the feasibility of the array-based method for quantitative assessment of multiple antigen expression. These findings will serve to extend the range of fundamental and clinical applications of antibody arrays.     

High resolution quantitative EEG analysis

Summary High resolution spectral methods are explored as an alternative to broad band spectral parameters (BBSP) in quantitative EEG analysis. In a previous paper (Valdes et al. 1990b) regression equations (Developmental surfaces) were introduced to characterize the age-frequency distribution of the mean and standard deviation of the log spectral EEG power in a normative sample. These normative surfaces allow the calculation of z transformed spectra for all derivations of the 10/20 system and z maps for each frequency. Clinical material is presented that illustrates how these procedures may pinpoint frequencies of abnormal brain activity and their topographic distribution, avoiding the frequency and spatial smearing that may occur using BBSP. The increased diagnostic accuracy of high resolution spectral methods is demonstrated by means of receiver operator characteristic (ROC) curve analysis. Procedures are introduced to avoid type I error inflation due to the use of more variables in this type of procedure.     

Segregation analysis of quantitative traits

A review of advanced methods of segregation analysis of quantitative traits on human pedigree data is presented. Special attention was paid to formulation of genetic models tested in the analysis, to the possibility of statistical distinguishing between these models, to the power of the used transmission probability tests, and to the possibility of unambiguous interpretation of the analysis results.     

A quantitative approach to cytoarchitectonics

Summary The cerebral cortex of the guinea pig has been examined by means of a quantitative cytoarchitectonic method (Schleicher et al. 1978; Zilles et al. 1978a). In this method, a computer-controlled automatic image analyzer determines the grey level index of microscopic fields measuring 50x50 m in Nissl-stained sections by a systematic scanning procedure. Computer plots of serially sectioned histological slides from three hemispheres were produced by printing selected ranges of grey level indices. The delineation of cortical areas was worked out in these plots based on quantitative criteria. Cortical maps of the areal pattern were reconstructed graphically.The resulting cortical map of the guinea pig differes from that of Rose (1912), but it corresponds to the results of Friede (1960) and is in agreement with neurophysiological studies. In general, the areal pattern of the guinea pig is similar to that of the rat (Zilles et al. 1980), but there are also some differences. These differences are discussed with respect to functional considerations.Supported by Deutsche Forschungsgemeinschaft grants Zi 192/4-3     

Aging and quantitative sensory thresholds

Cutaneous thresholds for vibration and thermal sensitivity were quantitated at the index finger and great toe in 54 subjects without obvious neurological disease. Twelve patients over sixty years of age also consented to standard nerve conduction studies which proved normal. Vibration thresholds progressively increased with age to a significant degree at the finger (p less than 0.05), and especially at the toe (p less than 0.001). Thermal sensitivity thresholds showed no significant relationship with age. These findings confirm the clinical notion that vibration discrimination decreases with normal aging, especially in the toes.     

A quantitative approach to cytoarchitectonics

Summary Fourteen allocortical areas of Tupaia belangeri are described after measuring the grey level index (Zilles et al., 1978b) with a new, automatic, high resolution measuring procedure. The laminar pattern of the cortical areas is quantitatively described after vertical changes in this index occurred from the pial surface to the white matter. The registered profiles show characteristic features for the different allocortical brain regions.The results are compared with lamination systems gained by comparative cytoarchitectonic studies using purely qualitative criteria.(Direktor: Professor Dr. H. Leonhardt)(Abteilungsvorsteher: Professor Dr. H.-J. Kretschmann)Supported by the Deutsche Forschungsgemeinschaft grants Kr 289/8, 289/9 and Zi 192/1Dedicated to Professor Dr. R. Ortmann on the occasion of his 65th birthday