免疫荧光定量分析仪设计

设计了免疫荧光定量分析仪,用以对人体血液和尿液中的各种分析物(CRP、PCT、NTpro BNP、c Tn I等)含量进行快速准确的定量分析。光源采用大功率LED灯珠,采用窄带干涉滤光片对激发光和荧光进行滤光,采用内置运放的光电转换芯片OPT101进行荧光强度的检测。采用步进电机驱动,皮带传动方式带动双排滚珠宽体滑块在单根精密直线导轨上滑动,实现对检测样品的扫描式检测。通过与标准仪器进行对比试验,结果表明样机在小型化、快速性以及低成本的基础上,测量结果准确,测量精度高,稳定性好,能满足临床应用要求。     

Visual learning and classification of human epithelial type 2 cell images through spontaneous activity patterns

Identifying the presence of anti-nuclear antibody (ANA) in human epithelial type 2 (HEp-2) cells via the indirect immunofluorescence (IIF) protocol is commonly used to diagnose various connective tissue diseases in clinical pathology tests. As it is a labour and time intensive diagnostic process, several computer aided diagnostic (CAD) systems have been proposed. However, the existing CAD systems suffer from numerous shortcomings due to the selection of features, which is commonly based on expert experience. Such a choice of features may not work well when the CAD systems are retasked to another dataset. To address this, in our previous work, we proposed a novel approach that learns a set of filters from HEp-2 cell images. It is inspired by the receptive fields in the mammalian's vision system, since the receptive fields can be thought as a set of filters for similar shapes. We obtain robust filters for HEp-2 cell classification by employing the independent component analysis (ICA) framework. Although, this approach may be held back due to one particular problem; ICA learning requires a sufficiently large volume of training data which is not always available. In this paper, we demonstrate a biologically inspired solution to address this issue via the use of spontaneous activity patterns (SAP). The spontaneous activity patterns, which are related to the spontaneous neural activities initialised by the chemical release in the brain, are found as the typical stimuli for the visual cell development of newborn animals. In the classification system for HEp-2 cells, we propose to model SAP as a set of small image patches containing randomly positioned Gaussian spots. The SAP image patches are generated and mixed with the training images in order to learn filters via the ICA framework. The obtained filters are adopted to extract the set of responses from a HEp-2 cell image. We then employ regions from this set of responses and stack them into “cubic regions”, and apply a classification based on the correlation information of the features. We show that applying the additional SAP leads to a better classification performance on HEp-2 cell images compared to using only the existing patterns for training ICA filters. The improvement on classification is particularly significant when there are not enough specimen images available in the training set, as SAP adds more variations to the existing data that makes the learned ICA model more robust. We show that the proposed approach consistently outperforms three recently proposed CAD systems on two publicly available datasets: ICPR HEp-2 contest and SNPHEp-2.     

Germline stem cell lineage tracing by free-floating immunofluorescent assay of mouse seminiferous tubule

Whole-mount immunohistochemistry (whole-mount IH) of the seminiferous tubule is widely used to investigate the self-renewal and differentiation of spermatogonial stem cells (SSCs). Examination of the length of spermatogonial cysts is critical for tracing SSCs lineage by using Whole-mount IH. However, it is difficult for antibody molecules to penetrate into the depth of seminiferous epithelium because its thickness and the tight peritubular myoid and basement membrane outside. Here, we developed a free-floating immunofluorescent procedure of mouse seminiferous tubules using regular incubation time and normal antibody concentration. Microscopic results showed that undifferentiated spermatogonia were positively labeled by promyelocytic leukemia zinc finger protein, E-cadherin, and glial cell line-derived neurotrophic factor family receptor alpha 1, respectively. Spermatogonial cysts in varied length were revealed clearly and spermatogonia subpopulations including A(single) (A(s)), A(paired) (A(pr)), and A(aligned) (A(al)) were distinguished in lower background images. This method provides us an alternate simple way to trace the lineage of individual SSCs and show their three-dimensional locations and distributions within their niches anatomically in next step.     

Steric hindrance in immunolabelling

In this paper we describe immunocytochemical detection of PhoE pore protein in the outer membrane of E. coli K-12 cells in dependence of a variety of labelling approaches. Immuno-gold labelling on ultrathin cryosections showed a uniform, dense labelling of the outer membrane of all cells. However, using immunofluorescence, whole-mount or freeze-etch labelling methods, labelling was limited to less than 5% of the cell population. In order to understand this phenomenon, immunoincubated cells exhibiting less than 5% labelling were processed for cryo-ultramicrotomy. Reincubation of the sections with antibody and probe resulted in labelling of all of the cells. If an E. coli Gal-U mutant strain, defective in the lipopolysaccharide (LPS) carbohydrate chain length, was used, each approach rendered 100% labelling. From these results it is concluded that the antigenic sites of the PhoE pore protein are not accessible in intact ‘wild-type’ cells due to steric hindrance caused by the LPS carbohydrate chains. In cryosections this steric hindrance is partly precluded since antigenic determinants are exposed at the section surface in transversely cut membranes. Our results emphasize the necessity to compare results obtained by means of several, basically different approaches.     

自水解预处理对杨木聚木糖分布的影响

采用免疫荧光标记法结合共聚焦显微镜(CLSM)研究了自水解强度对杨木聚木糖分布的影响。结果表明,聚木糖荧光信号较均匀地分布于杨木纤维细胞壁中,随强度因子的增大细胞壁中心部位荧光信号下降逐渐增多,细胞壁边缘荧光信号下降较少,有些部位甚至会稍许增加。采用高效液相色谱(HPLC)检测法测得强度因子增强到3.72时,聚木糖含量从原料的14.64%下降到7.33%;对聚木糖荧光图谱进行区域强度统计,可得出与HPLC检测法结果相同的变化趋势。     

几种常见抗体介导的肾小球肾炎免疫荧光和超微结构的形态对比观察

本文通过比较观察几种常见抗体介导的肾小球肾炎的免疫荧光和超微结构的特征,以进一步提高对其识别能力和诊断水平。结果显示,一些常见抗体介导的肾小球病,如IgA肾病（包括紫癜性肾炎）、膜性肾病、急性弥漫增生性肾小球肾炎、膜性增生性肾小球肾炎（Ⅰ型）、狼疮性肾炎（Ⅳ型）的免疫荧光和电镜检查所显示的抗体或免疫反应产物有较好的可比性和一致性。提高对抗体介导的肾小球肾炎典型肾小球超微结构形态特征的认识和鉴别能力将十分有助于对肾小球疾病作出正确的病理学诊断。     

Functional display of amylase on yeast surface from Rhizopus oryzae as a novel enzyme delivery method

Fungal amylase has great importance in fermentation industry such as brewing, food fermentation, starch hydrolysis and for improving microbial populations in chicken intestine through feed applications. In the present investigation, alpha amylase cDNA from Rhizopus oryzae was cloned, sequenced, and successfully surface anchored in functional form using Saccharomyces cerevisiae EBY100 as host, yielding enzyme activity of 4.35 (±0.5) U/ml. The surface displayed yeast expressed amylase activity using plate assay, produced glucose and maltose as hydrolysis products using starch as substrate. The targeted and armed yeast with displayed enzyme was evaluated for its characterization at various pH and temperatures. The engineered yeast showed optimal activity at neutral pH and at 50°C incubation temperature. Reducing sugars produced by displayed enzyme were visualized by paper chromatography. The data suggested successful heterologous expression and display of amylase enzyme on yeast cell surface. The displayed system could further be utilized in fermentation industry for improving cost-effectiveness of the process.     

Intranucleolar localization of DNA topoisomerase IIalpha is a distinctive feature of necrotic, but not of apoptotic, Jurkat T-cells

Two distinct types of cell death have been described: apoptosis and necrosis. However, it is becoming increasingly clear that the differences between these two types are far less numerous than initially thought. Morphological analyses might provide important information to distinguish apoptotic from necrotic samples. We recently reported that in necrotic, but not apoptotic, HL-60 human myeloid leukaemia cells, the nuclear protein topoisomerase IIalpha concentrated in nucleoli. In order to ascertain whether or not this phenomenon was restricted to a peculiar cell type or could be detected also in cells of lymphoid lineage, we performed an investigation aimed at defining the localization of topoisomerase IIalpha in apoptotic and necrotic Jurkat human T lymphoblastoid cells. Immunofluorescence staining demonstrated that topoisomerase IIalpha was excluded from the condensed chromatin of apoptotic cells, whereas in necrotic cells it was localized in discrete nuclear dots. Immuno-electron microscopy analysis showed that topoisomerase IIalpha was undetectable in nucleoli of normal and apoptotic cells, whereas it was present in the nucleolus of necrotic cells irrespectively of the type of inducer used (ethanol, H(2)O(2), HgCl(2)). Taken together, our findings identify topoisomerase IIalpha as a potential morphological marker useful to discriminate between apoptotic and necrotic cells.     

细胞凋亡过程中细胞骨架改变的激光共聚焦显微镜观察

目的：了解细胞凋亡过程中细胞骨架的形态学改变。方法：应用高浓度全反式维甲酸诱导卵巢癌细胞凋亡,激光共聚焦显微镜观察经荧光染色的细胞微管蛋白的形态学变化。结果：凋亡早期,微管蛋白在荧光相由正常按一定方向分布的丝网状结构变成杂乱分布的网状结构,晚期呈细颗粒状分布,荧光强度随凋亡发展而逐渐变弱。讨论：在凋亡过程中,细胞骨架随凋亡发展出现相应的形态学改变,凋亡早期,细胞骨架在分布上出现变化,伴随细胞形态出现变化,凋亡晚期,细胞骨架蛋白发生降解,此时的细胞形态改变是不可逆的。     

Determination of surface-induced platelet activation by applying time-dependency dissipation factor versus frequency using quartz crystal microbalance with dissipation

Platelet adhesion and activation rates are frequently used to assess the thrombogenicity of biomaterials, which is a crucial step for the development of blood-contacting devices. Until now, electron and confocal microscopes have been used to investigate platelet activation but they failed to characterize this activation quantitatively and in real time. In order to overcome these limitations, quartz crystal microbalance with dissipation (QCM-D) was employed and an explicit time scale introduced in the dissipation versus frequency plots (Df–t) provided us with quantitative data at different stages of platelet activation. The QCM-D chips were coated with thrombogenic and non-thrombogenic model proteins to develop the methodology, further extended to investigate polymer thrombogenicity. Electron microscopy and immunofluorescence labelling were used to validate the QCM-D data and confirmed the relevance of Df–t plots to discriminate the activation rate among protein-modified surfaces. The responses showed the predominant role of surface hydrophobicity and roughness towards platelet activation and thereby towards polymer thrombogenicity. Modelling experimental data obtained with QCM-D with a Matlab code allowed us to define the rate at which mass change occurs (A/B), to obtain an A/B value for each polymer and correlate this value with polymer thrombogenicity.