一种碳/碳复合材料表面制备高结合强度羟基磷灰石涂层的新方法

背景：目前在碳/碳表面制备羟基磷灰石涂层的方法有很多种,但所制涂层与基体的结合强度不高。 目的：提出一种在碳/碳复合材料表面制备高结合强度羟基磷灰石涂层的新方法。 方法：首先利用感应加热法在基体表面制备出无水磷酸氢钙涂层,而后对其进行水热处理,转变为羟基磷灰石相。扫描电镜观察涂层的形貌,划痕法测试涂层的临界载荷,顶出法测试剪切强度。 结果与结论：感应热沉积法可以在碳/碳复合材料表面制备出致密的块状晶粒结构的无水磷酸氢钙涂层;通过水热处理可以将其转变成结晶完好致密的羟基磷灰石涂层。涂层的临界载荷为13.31 N,剪切强度约为47 MPa。     

A novel epitope of N-CAM defines precursors of human adherent NK cells

Activated, adherent natural killer (A-NK) cells represent a distinct subpopulation of interleukin (IL)-2-stimulated NK cells, which are selectively endowed with the increased expression of integrins and ability to adhere to solid surfaces, migrate into, infiltrate, and destroy cancerous tissues. The present study defines the phenotype and functions of precursors of A-NK (pre-A-NK) cells in humans. Peripheral blood pre-A-NK cells, in contrast to the rest of NK cells, express a novel epitope of CD56 neuronal cell adhesion molecule, termed ANK-1, and increased cell-surface levels of integrins. Pre-A-NK cells also express low levels of CD56 and CD161, and some express CD162 receptor, do not express CD25 or activation markers, and are effective mediators of NK cytotoxicity. Thus, pre-A-NK cells are generally similar to CD56(dim) NK cells. However, pre-A-NK cells differ from the main NK cell subpopulation by having a lower expression level of CD16 and a lower ability to mediate redirected antibody-dependent, cell-mediated cytotoxicity. More importantly, pre-A-NK cells are preferentially endowed with the ability to rapidly respond to IL-2 by integrin-mediated adherence to endothelial cells, extracellular matrix, and plastic. This early, specific response of pre-A-NK cells to IL-2 is followed by their activation, vigorous proliferation, and differentiation into phenotypically and functionally similar A-NK cells. Pre-A-NK cells represent only approximately 26% of peripheral blood NK cells but encompass the majority of NK cells in normal and cancerous, solid tissues. We conclude that pre-A-NK cells represent a distinct subset of resting, mature NK cells with the characteristics indicative of their ability to migrate and reside in solid tissues.     

一种新的培养嗅鞘细胞方法(英文)

为了建立一种新的培养嗅鞘细胞的方法,从而为神经诱导修复材料的研究提供种子细胞。本研究取新生鼠的嗅球最外两层经胰蛋白酶消化成单细胞悬液,经差速贴壁法纯化,观察并记录其形态特征;经HE染色以及神经生长因子蛋白受体p75(NGFR-p75)和S-100免疫组化染色鉴定并计算其纯度。结果显示,获得的嗅鞘细胞突起呈双极或三级,p75和S-100阳性细胞纯度达到91%。上述结果提示该方法经济易行,所获得的嗅鞘细胞纯度高、活性好。     

A simple method for counting adherent cells: application to cultured human monocytes, macrophages and multinucleated giant cells

A simple method was devised for counting small numbers (10(4)-10(6)) of adherent mononuclear phagocytes, including populations containing multinucleated giant cells, which often arise during cultivation of human blood monocytes. Coverslips with adherent cells were transferred into small volumes (50-200 microliters) of 0.1 M citric acid, pH 2.2, containing 0.05% naphthol blue black and 1.0% of either Triton X-100 or Cetavlon. Triton X-100 was adequate for use with monocytes and macrophages from early cultures. However, Cetavlon was preferable for use with older cultures of adherent human mononuclear cells in order to prevent aggregation of the nuclei from giant cells. When multinucleated cells were present, separately stained coverslips were inspected to determine the mean number of nuclei per cell. This value, together with the number of nuclei per coverslip, permitted calculation of the number of cells per coverslip. The latter value is not readily derived from measurements of protein or DNA content in populations containing multinucleated giant cells. This counting method was simpler and more sensitive than several previously reported methods for enumerating adherent macrophages.     

A novel fluorescent sensitive assay for detection of differential T cell mediated lysis of multiple adherent target cells

There are few studies that have investigated T cell mediated lysis of adherent cells. We have developed a novel, rapid and sensitive fluorescent dye-swap assay that allows efficient detection of adherent target cell lysis. The assay allows simultaneous use of multiple differentially sensitised targets and facilitates concomitant surface or intracellular effector cell phenotypic analysis.     

一种高选择性、高效的牛视网膜微血管内皮细胞培养方法

目的建立一种高效、可行、特异性高的牛视网膜微血管内皮细胞（BREC）培养方法。方法取新鲜牛眼,分离视网膜并用DMEM进行冲洗、匀浆剪碎,过75μm筛,将滤网上滤渣转移至50ml离心管,用Ⅰ型胶原酶、DNaseⅠ及蛋白酶等多种酶混合液消化20min,人血清中和后,过46μm网筛并冲洗,离心5min,将组织扣在培养皿中,转入15ml离心管,用10%人血清含生长因子ECGS的DMEM培养液培养于25cm2,选择性培养视网膜血管内皮细胞,接种于明胶包被培养瓶中,采用ECGS配合肝素培养液促进内皮细胞生长,观察细胞形状生长特性,并用免疫化学荧光方法进行鉴定。结果选择性培养视网膜微血管内皮细胞成单层、镶嵌铺路石状生长,Ⅷ因子相关抗原免疫荧光检测为阳性纯度均大于95%以上,将细胞种在凝固的基质胶表面,12～18h形成官腔结构。结论本方法过程简单、可靠,培养的内皮细胞纯度高,生长状态良好,稳定传代,为视网膜血管生成疾病研究建立了模型。     

A novel bulk-culture method for generating mature dendritic cells from mouse bone marrow cells

We established a novel culture method for generating dendritic cells (DC) from mouse bone marrow (BM) cells. Unfractionated bulk BM cells were cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 5-7 days and a DC population was isolated by gradient centrifugation with 14.5% (w/v) metrizamide. Through this method, 30-40 x 10(6)/mouse DC with 85-95% purity was obtained on day 7; this yield was higher than those of conventional DC generated by Inaba's method either with GM-CSF alone (conventional-GM DC) or GM-CSF and IL-4 (conventional-GM/4 DC). Bulk-cultured DC have a more matured phenotype than both conventional-GM and -GM/4 DC as shown by higher expression of CD86, MHC class II and CD40. Functional analyses reveal that (1) bulk-DC show less ability in endocytosis than conventional-GM DC and are comparable in IL-12 p70 production with conventional-GM and -GM/4 DC. (2) Bulk-DC exhibit stronger stimulatory capacity in allogeneic T-cell proliferation than conventional DC. (3) By using ovalbumin (OVA) and OVA-specific T-cell receptor (TCR) transgenic mice (DO11.10) system, OVA protein-loaded bulk-DC stimulated CD4 T cells of DO11.10 mice more than conventional-GM DC and comparable with conventional-GM/4 DC. (4) Furthermore, OVA peptide-pulsed bulk-DC stimulated CD4 T cells more than conventional-GM and -GM/4 DC. These data indicate that bulk-DC are functionally more mature than conventional DC. Taken together, bulk-culture method is a simple technique for generating functionally mature BM-DC in large quantities and high purity.     

A novel five-colour flow cytometric assay to determine NK cell cytotoxicity against neuroblastoma and other adherent tumour cells

For the evaluation of novel therapies, and for initial in vitro testing of potential in vivo graft-versus-tumour-effects (GvT), cytotoxicity of effector cells against target tumour cells needs to be determined in a reliable fashion. Recently Zimmermann et al. [Zimmermann, S.Y., Esser, R., Rohrbach, E., Klingebiel, T., Koehl, U., 2005. A novel four-colour flow cytometric assay to determine natural killer cell or T-cell-mediated cellular cytotoxicity against leukaemia cells in peripheral or bone marrow specimens containing greater than 20% of normal cells. J. Immunol. Methods. 296(1-2), 63-76] introduced a single platform, no-wash flow cytometric assay to quantify natural killer (NK) cell cytotoxicity against leukaemia cells. Here we have optimised this method introducing a novel five-colour flow cytometric assay for the evaluation of NK cell activity against adherent tumour cells, in particular neuroblastoma cells (NB cells). Beside an enhanced cytotoxic activity corresponding to increasing effector/target (E:T) ratios, we could demonstrate an increasing cytotoxicity in a time-dependent manner over a time period of 8 h. The usefulness of this novel method was also confirmed with human tumour cells lines of various other origin including breast and ovarian carcinoma and Wilms tumour cells freshly isolated from a patient after surgery. In addition to flow cytometric analysis, we monitored NK-cell-mediated induction of target cell apoptosis via the caspase cascade in attacked NB cells by fluorescence microscopy after immunofluorescence staining of activated Caspase-3 (Casp-3) in combination with detection of CD45(+) and CD9(+) for discrimination between NK and NB cells. In summary, this novel flow cytometric cytotoxicity assay enables efficient quantification of the phenotype of both, effector and adherent target tumour cells, and therefore represents a useful tool for research on immunotherapies that rely on cytotoxic effector cells.     

A novel method for assessing adherent single-cell stiffness in tension: design and testing of a substrate-based live cell functional imaging device

Various micro-devices have been used to assess single cell mechanical properties. Here, we designed and implemented a novel, mechanically actuated, two dimensional cell culture system that enables a measure of cell stiffness based on quantitative functional imaging of cell-substrate interaction. Based on parametric finite element design analysis, we fabricated a soft (5 kPa) polydimethylsiloxane (PDMS) cell substrate coated with collagen-I and fluorescent micro-beads, thus providing a favorable terrain for cell adhesion and for substrate deformation quantification, respectively. We employed a real-time tracking system that analyzes high magnification images of living cells under stretch, and compensates for gross substrate motions by dynamic adjustment of the microscope stage. Digital image correlation (DIC) was used to quantify substrate deformation beneath and surrounding the cell, leading to an estimate of cell stiffness based upon the ability of the cell to resist the applied substrate deformation. Sensitivity of the system was tested using chemical treatments to both “soften” and “stiffen” the cell cytoskeleton with either 0.5 μg/ml Cytochalasin-D or 3% Glutaraldehyde, respectively. Results indicate that untreated osteosarcoma cells (SAOS-2) exhibit a 1.5 ± 0.7% difference in strain from an applied target substrate strain of 8%. Compared to untreated cells, those treated with Cyochalasin-D passively followed the substrate (0.5 ± 0.5%, p < 0.001), whereas Glutaraldehyde enhanced cellular stiffness and the ability to resist the substrate deformation (2.9 ± 1.6%, p < 0.001). Nano-indentation testing showed differences in cell stiffness based on culture treatment, consistent with DIC findings. Our results indicate that mechanics and image analysis approaches do hold promise as a method to quantitatively assess tensile cell constitutive properties.     

ELISpot for measuring human immune responses to vaccines

The enzyme-linked immunosorbent spot (ELISpot) assay is one of the most commonly used methods to measure antigen-specific T cells in both mice and humans. Some of the primary reasons for the popularity of the method are that ELISpot is highly quantitative, can measure a broad range of magnitudes of response and is capable of assessing critical cellular immune-related activities such as IFN-γ secretion and granzyme B release. Furthermore, ELISpot is adaptable not only to the evaluation of a variety of T-cell functions, but also to B cells and innate immune cells. It is no wonder that ELISpot has evolved from a research tool to a clinical assay. Recent Phase I and II studies of cancer vaccines, tested in a variety of malignancies, have suggested that ELISpot may be a useful biomarker assay to predict clinical benefit after therapeutic immune modulation. This article will discuss the most common applications of ELISpot, overview the efforts that have been undertaken to standardize the assay and apply the method in the analysis of human clinical trials, and describe some important steps in the process of developing a clinical-grade ELISpot.     

A novel method for patch-clamp automation

An increasing demand of the pharmaceutical industry for automated electrophysiological stations for ion channel drug discovery has recently resulted in the development of several commercial platforms for secondary and safety screening of ion channel modulators. These commercial systems have demonstrated an enhanced throughput, however, often at the expense of some quality-sensitive aspects of traditional patch-clamp recordings. To improve data quality and content, we have developed a patch-clamp robot that fully automates manual patch-clamp recordings, including patch pipette handling, gigaseal formation, obtaining whole-cell or perforated-cell configuration, drug application, and data acquisition. Utilization of glass micropipettes results in high-quality electrophysiological recordings with an overall success rate of about 30% in perforated-cell mode. A fast drug application system with low volume requirements (1–1.5 ml) allows the study of ligand-gated ion channels on a millisecond scale. As proof-of-concept, we present two assays developed for voltage-gated human ether-a-go-go-related and ligand-gated α₇ nicotinic receptor ion channels. The system throughput was a single concentration–response curve every 30–40 min or 12–17 6-point concentration–response curves daily, representing a significant improvement of typical manual patch-clamp throughput. This system represents an efficient method for patch-clamp automation without the need for a complex and expensive electrophysiological set-up for cell visualization.     

Optically micropatterned culture of adherent cells

We used a liquid-crystal spatial light modulator to project 473 nm light patterns surrounding a region of adherent cells and achieved an arbitrarily micropatterned cell culture. For a group of ～60 cells, the cell boundaries fit the pattern of light within 15% deviation of the side length. We also demonstrated a wound-healing experiment with a definite starting temporal point by using this technique. While observing mitochondrial structures in the illuminated cells, we found that the 473 nm light damaged the integrity of mitochondria and thus prohibited cell proliferation in the illuminated region.     

A novel method for generating xeno-free human feeder cells for human embryonic stem cell culture

Long-term cultures of human embryonic stem (hES) cells require a feeder layer for maintaining cells in an undifferentiated state and increasing karyotype stability. In routine hES cell culture, mouse embryonic fibroblast (MEF) feeders and animal component-containing media (FBS or serum replacement) are commonly used. However, the use of animal materials increases the risk of transmitting pathogens to hES cells and therefore is not optimal for use in cultures intended for human transplantation. There are other limitations with conventional feeder cells, such as MEFs, which have a short lifespan and can only be propagated five to six passages before senescing. Several groups have investigated maintaining existing hES cell lines and deriving new hES cell lines on human feeder layers. However, almost all of these human source feeder cells employed in previous studies were derived and cultured in animal component conditions. Even though one group previously reported the derivation and culture of human foreskin fibroblasts (HFFs) in human serum-containing medium, this medium is not optimal because HFFs routinely undergo senescence after 10 passages when cultured in human serum. In this study we have developed a completely animal-free method to derive HFFs from primary tissues. We demonstrate that animal-free (AF) HFFs do not enter senescence within 55 passages when cultured in animal-free conditions. This methodology offers alternative and completely animal-free conditions for hES cell culture, thus maintaining hES cell morphology, pluripotency, karyotype stability, and expression of pluripotency markers. Moreover, no difference in hES cell maintenance was observed when they were cultured on AF-HFFs of different passage number or independent derivations.     

A novel method for the serum-free plating of stage-24 chick limb bud cells

Summary A modification of the standard plating of Stage 24 chick limb bud cells in serum-containing media (SCM) is described in which the cells are incubated in SCM for 3 hours before direct plating in a serum-free chemically defined media (SFM). This results in equal plating efficiencies to those obtained with plating in SCM and may allow for more defined culture conditions when testing mitogenic growth factors found in serum.     

The automated counting of spots for the ELISpot assay

An automated method for counting spot-forming units in the ELISpot assay is described that uses a statistical model fit to training data that is based on counts from one or more experts. The method adapts to variable background intensities and provides considerable flexibility with respect to what image features can be used to model expert counts. Point estimates of spot counts are produced together with intervals that reflect the degree of uncertainty in the count. Finally, the approach is completely transparent and "open source" in contrast to methods embedded in current commercial software. An illustrative application to data from a study of the reactivity of T-cells from healthy human subjects to a pool of immunodominant peptides from CMV, EBV and flu is presented.     

A simple and efficient method for the monitoring of antigen-specific T cell responses using peptide pool arrays in a modified ELISpot assay

In this study, we describe a simple and efficient method for both the monitoring of antigen-specific CD4 and CD8 T cell responses as well as the identification of novel CD4 and CD8 T cell epitopes using a modified ELISpot assay and pools of 20mer peptides. We have demonstrated that pools containing as many as 64 20mer peptides may be used to screen for CD4 and CD8 T cell responses to HPV16 L1, E1, and E7 in mice. Using arrays of pools of overlapping 20mer peptides, we have identified novel CD4 and CD8 epitopes in both HPV16L1 and HPV16E1 which are presented in Balb/c mice. We have further shown that the use of 20mer peptides is equivalent to using minimal 9mer epitopes for the stimulation of CD8 T cell responses in our assay. While our experiments are conducted in mice, the use of peptide pool arrays allows for the identification of epitope-specific responses using far fewer cells than is required for testing a panel of overlapping peptides individually, making this strategy particularly useful in clinical settings where immune cells may be limiting.     

A novel real-time PCR method for KIR genotyping

Genes encoding killer cell immunoglobulin-like receptors (KIRs) are variable among individuals. Sequence-specific primer-directed polymerase chain reaction (PCR) amplification (PCR-SSP) and sequence-specific oligonucleotide hybridization of the PCR-amplified products (PCR-SSO) are the methods currently used to characterize the diversity of KIR gene content. Both these methods include time-consuming post-PCR analyses. Here, we developed a real-time PCR method that identifies the presence or absence of 16 KIR genes during PCR and avoids post-PCR analyses. This method is specific, sensitive, shortens the turnaround time compared with the conventional PCR-SSP and PCR-SSO methods, and it can be easily adapted for automation.