药物筛选模型研究进展

药物筛选模型 (ｄｒｕｇｓｃｒｅｅｎｉｎｇｍｏｄｅｌ,ｄｒｕｇｓｃｒｅｅｎ ｉｎｇａｓｓａｙｍｅｔｈｏｄ)是用于证明某种物质具有药理活性(生物活性、治疗作用 )的实验方法 ,这些实验方法是寻找和发现药物的重要条件之一。人们在长期寻找药物的实践过程中 ,建立了大量用于新药筛选的各类模型 ,在新药发现和研究中发挥了积极作用。随着生命科学的发展 ,新的药物筛选模型不断出现 ,这些新的筛选模型不仅促进了药物的发现 ,而且对药物筛选的方法、理论、技术都产生了巨大影响[1] 。本文对近年来药物筛选模型的研究进展作简单综述。1　常用…     

An antiviral drug screening system for enterovirus 71 based on an improved plaque assay: A potential high-throughput method

Plaque assay plays an irreplaceable role in a variety of virological studies, including determining titers of viruses. Our previous study showed that a simple and highly repeatable plaque assay could be used for enterovirus 71 (EV-A71). Now, we show that using a subclone of a clinical EV-A71 isolate and a rhabdomyosarcoma cell line (RD), a plaque assay based on an EV-A71/RD model could exhibit the most rapid formation of plaques (<2 days), with much higher repeatability and consistency. Inspired by a plaque inhibitory test for testing ribavirin and interferon, as well as a plaque reduction neutralization test, this modified method has been used to establish a convenient system by using 96-well plates for screening anti-EV-A71 drugs from a 130-compound library containing multiple types of inhibitors. Nine candidate effective compounds for EV-A71 have been screened out, and among them, nobiletin (flavonoid) was found to be a novel effective compound at the concentration of 10 μM. Our findings imply that this improved method based on an EV-A71/RD model proved to be a potential high-throughput method in screening novel antiviral drugs for EV-A71. Undoubtedly, this method can also be applied to other viruses that can produce an obvious cytopathic effect.     

A Cell-Based Pharmacokinetics Assay for Evaluating Tubulin-Binding Drugs

Increasing evidence reveals that traditional pharmacokinetics parameters based on plasma drug concentrations are insufficient to reliably demonstrate accurate pharmacological effects of drugs in target organs or cells in vivo. This underscores the increasing need to improve the types and qualities of cellular pharmacokinetic information for drug preclinical screening and clinical efficacy assessments. Here we report a whole cell-based method to assess drugs that disturb microtubule dynamics to better understand different formulation-mediated intracellular drug release profiles. As proof of concept for this approach, we compared the well-known taxane class of anti-microtubule drugs based on paclitaxel (PTX), including clinically familiar albumin nanoparticle-based Abraxane™, and a polymer nanoparticle-based degradable paclitaxel carrier, poly(L-glutamic acid)-paclitaxel conjugate (PGA-PTX, also known as CT-2103) versus control PTX. This in vitro cell-based evaluation of PTX efficacy includes determining the cellular kinetics of tubulin polymerization, relative populations of cells under G2 mitotic arrest, cell proliferation and total cell viability. For these taxane tubulin-binding compounds, the kinetics of cell microtubule stabilization directly correlate with G2 arrest and cell proliferation, reflecting the kinetics and amounts of intracellular PTX release. Each individual cell-based dose-response experiment correlates with published, key therapeutic parameters and taken together, provide a comprehensive understanding of drug intracellular pharmacokinetics at both cellular and molecular levels. This whole cell-based evaluating method is convenient, quantitative and cost-effective for evaluating new formulations designed to optimize cellular pharmacokinetics for drugs perturbing tubulin polymerization as well as assisting in explaining drug mechanisms of action at cellular levels.     

A flexible polymer tube lab-chip integrated with microsensors for smart microcatheter

A flexible polymer tube lab-chip integrated with physical and biochemical sensor modules mounted on a flexible spiral structure for measuring physiological (temperature/flow rate) and metabolic data (glucose concentration) in a catheter application was designed, fabricated and characterized in this work. This new approach not only provides a unique way to assemble multiple sensors on both the inside and outside the flexible polymer tube using standard microfabrication methods while avoiding wiring and assembling problems associated with previous methods, but also maintains catheter inherent lumen potency for in situ drug delivery or insertion of medical tools. Three well-known sensors: temperature sensor (RTD), flow rate sensor (hot film anemometry) and glucose biosensor (amperometric sensor) have been successfully fabricated and fully integrated outside the spirally rolled polymer tube (ID = 500 μm, OD = 650 μm) of this demonstration device. The fabricated sensors showed good performances not only in a planar configuration but also in a spirally rolled configuration. This flexible micro tube lab-chip system provides a generic platform for developing patient-specific “smart” microcatheters that incorporate microsensors, microactuators, microfluidic devices and wireless signal communication modules that are tailored for the patients’ unique condition.     

A rapid, automated colorimetric assay for measuring antibody binding to cell surface antigens

An automated, colorimetric procedure is described for detecting antibodies specific for cell surface antigens. The procedure entails (a) coating the wells of 96-well microplates with either protein A or anti-immunoglobulin antibodies and (b) preincubating either the microplate or target cells with the test antibody. Target cells which react with the test antibody bind to the wells of the microplate and bound cells are quantitated by staining with the dye Rose Bengal. A microplate spectrophotometer is used to measure absorbance in each well of the plate, providing a rapid, automated measure of antibody titre. The assay is simple to perform, uses readily available reagents and gives comparable sensitivity to rosetting assays. With these features, and the capacity for handling large numbers of trays quickly, this method has obvious advantages in screening for antibody activity in culture supernatants of hybridoma clones.     

基于细胞3D打印技术的肿瘤药物筛选细胞芯片研究

现有的药物筛选评价技术中,动物筛选模型存在种属差异和周期长等缺点,高通量筛选和细胞筛选模型则与体内环境差异大,药物筛选准确率低。细胞3D打印技术为在体外构建仿真的组织器官模型提供了可能,当其与细胞芯片技术结合则为体外构建高效准确的药物筛选模型提供了新的技术空间。本研究构建了含有多个叉指电极（IDEs）阵列的细胞芯片,用细胞3D打印技术在芯片上组装了卵巢癌细胞HO-8910和人肝间充质干细胞HMSC-H组织模型,并通过对组织模型内细胞阻抗变化的检测,反映细胞生长、贴附、增殖、凋亡的过程及药物对细胞活性的影响等,最终基于该模型检测了抗癌药物顺铂和环磷酰胺对肿瘤细胞的杀伤和肝毒性。结果显示：支架微丝直径和孔径约为200～300 μm,肿瘤细胞和肝细胞在三维结构里生长良好;DMEM作为电解液,芯片在10.4 Hz可准确检测到三维结构中细胞增殖引起的阻抗变化,20 h后阻抗升高69.6%;基于该筛选模型,能同步检测到药物的抗肿瘤作用和肝毒性,并筛选出需要肝的二次代谢产物才能产生抗肿瘤性的药物环磷酰胺。     

Automated live cell screening system based on a 24-well-microplate with integrated micro fluidics

In research, pharmacologic drug-screening and medical diagnostics, the trend towards the utilization of functional assays using living cells is persisting. Research groups working with living cells are confronted with the problem, that common endpoint measurement methods are not able to map dynamic changes. With consideration of time as a further dimension, the dynamic and networked molecular processes of cells in culture can be monitored. These processes can be investigated by measuring several extracellular parameters. This paper describes a high-content system that provides real-time monitoring data of cell parameters (metabolic and morphological alterations), e.g., upon treatment with drug compounds. Accessible are acidification rates, the oxygen consumption and changes in adhesion forces within 24 cell cultures in parallel. Addressing the rising interest in biomedical and pharmacological high-content screening assays, a concept has been developed, which integrates multi-parametric sensor readout, automated imaging and probe handling into a single embedded platform. A life-maintenance system keeps important environmental parameters (gas, humidity, sterility, temperature) constant.     

A microELISA Raji cell assay to detect immune complexes

The Raji cell assay to detect immune complexes has been modified to a microtiter ELISA system. Raji cells were fixed to microplate wells, then reacted with serum samples or aggregated human IgG. Horseradish peroxidase-conjugated anti-human IgG was used to detect bound complexes. There was a linear relationship between aggregated IgG added and optical density reading, with less than 2 micrograms/ml of aggregated IgG readily detected. When applied to human serum this technique gave results comparable to those obtained with the standard Raji cell assay. The Raji micro-ELISA is simpler to perform than the standard assay, is equally reliable, and avoids the hazards of radioactivity.     

Short-chain acyl-coenzyme A dehydrogenase deficiency

Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is a disorder of mitochondrial fatty acid oxidation that leads to the accumulation of butyrylcarnitine and ethylmalonic acid in blood and urine. Originally described with a relatively severe phenotype, most patients are now diagnosed through newborn screening by tandem mass spectrometry and remain asymptomatic. Molecular analysis of affected individuals has identified a preponderance of private inactivating point mutations and one common one present in high frequency in individuals of Ashkenazi Jewish ancestry. In addition, two polymorphic variants have been identified that have little affect on enzyme kinetics but impair folding and stability. Individuals homozygous for one of these variants or compound heterozygous for one of each often show an increased level of ethylmalonic acid excretion that appears not to be clinically significant. The combination of asymptomatic affected newborns and the frequent variants can cause much confusion in evaluating and treating individuals with SCADD. The long-term consequences and the need for chronic therapy remain current topics of contention and investigation.     

A comprehensive approach to identification of pathogenic FANCA variants in Fanconi anemia patients and their families

Fanconi anemia (FA) is a rare recessive DNA repair deficiency resulting from mutations in one of at least 22 genes. Two‐thirds of FA families harbor mutations in FANCA. To genotype patients in the International Fanconi Anemia Registry (IFAR) we employed multiple methodologies, screening 216 families for FANCA mutations. We describe identification of 57 large deletions and 261 sequence variants, in 159 families. All but seven families harbored distinct combinations of two mutations demonstrating high heterogeneity. Pathogenicity of the 18 novel missense variants was analyzed functionally by determining the ability of the mutant cDNA to improve the survival of a FANCA‐null cell line when treated with MMC. Overexpressed pathogenic missense variants were found to reside in the cytoplasm, and nonpathogenic in the nucleus. RNA analysis demonstrated that two variants (c.522G > C and c.1565A > G), predicted to encode missense variants, which were determined to be nonpathogenic by a functional assay, caused skipping of exons 5 and 16, respectively, and are most likely pathogenic. We report 48 novel FANCA sequence variants. Defining both variants in a large patient cohort is a major step toward cataloging all FANCA variants, and permitting studies of genotype–phenotype correlations.     

MUTYH mutations associated with familial adenomatous polyposis: functional characterization by a mammalian cell‐based assay

MUTYH‐associated polyposis (MAP) is a colorectal cancer syndrome, due to biallelic mutations of MUTYH. This Base Excision Repair gene encodes for a DNA glycosylase that specifically mitigates the high mutagenic potential of the 8‐hydroxyguanine (8‐oxodG) along the DNA. Aim of this study was to characterize the biological effects, in a mammalian cell background, of human MUTYH mutations identified in MAP patients (137insIW [c.411_416dupATGGAT; p.137insIleTrp]; R171W [c.511C>T; p.Arg171Trp]; E466del [c.1395_1397delGGA; p.Glu466del]; Y165C [c.494A>G; p.Tyr165Cys]; and G382D [c.1145G>A; p.Gly382Asp]). We set up a novel assay in which the human proteins were expressed in Mutyh^?/? mouse defective cells. Several parameters, including accumulation of 8‐oxodG in the genome and hypersensitivity to oxidative stress, were then used to evaluate the consequences of MUTYH expression. Human proteins were also obtained from Escherichia coli and their glycosylase activity was tested in vitro. The cell‐based analysis demonstrated that all MUTYH variants we investigated were dysfunctional in Base Excision Repair. In vitro data complemented the in vivo observations, with the exception of the G382D mutant, which showed a glycosylase activity very similar to the wild‐type protein. Our cell‐based assay can provide useful information on the significance of MUTYH variants, improving molecular diagnosis and genetic counseling in families with mutations of uncertain pathogenicity. Hum Mutat 30:1–8, 2009. © 2009 Wiley‐Liss, Inc.     

膜联蛋白A5低表达对人宫颈癌HeLa细胞迁移和侵袭的影响

目的探讨膜联蛋白A5(ANXA5)低表达对人宫颈癌HeLa细胞迁移和侵袭的影响。方法将细胞分为干扰组、阴性对照组、转染试剂对照组和空白对照组,采用脂质体转染法将siRNA转染干扰组HeLa细胞,在转染72h后采用RT-PCR和Western blotting检测各组ANXA5的表达以鉴定抑制效率;细胞划痕实验检测各组HeLa细胞的迁移能力;Transwell实验检测各组HeLa细胞的侵袭能力;RT-PCR和Western blotting分别检测ANXA5低表达对E-钙黏蛋白(E-cadherin)和基质金属蛋白酶(MMP)-9 mRNA及蛋白表达的影响。结果干扰组ANXA5蛋白及mRNA的表达均明显低于阴性对照组(P0.05);其表达均被显著抑制,干扰组细胞的迁移及侵袭能力比阴性对照组明显增强(P0.05),E-cadherin mRNA及蛋白的表达显著低于阴性对照组(P0.05);干扰组MMP-9mRNA及蛋白的表达均显著高于阴性对照组(P0.05)。结论靶向ANXA5的siRNA可有效抑制ANXA5的表达,且ANXA5低表达可能通过影响E-cadherin和MMP-9的表达来促进HeLa细胞的迁移和侵袭能力。     

AMPK: A metabolic switch for CD8+ T‐cell memory

Adenosine monophosphate‐activated protein kinase (AMPK) is a serine/threonine kinase and is crucial for cellular energy homeostasis. The exact role of AMPK during memory CD8⁺ T‐cell differentiation, a process that changes from the metabolically active state of effector T cells to one of quiescence in memory cells is not well understood; however, a report by Cantrell and colleagues [Eur. J. Immunol. 2013. 43: 889‐896] in this issue of the European Journal of Immunology shows that AMPK, by sensing glucose stress, is an important upstream molecule of mammalian target of rapamycin (mTOR) complex 1 for memory CD8⁺ T‐cell differentiation. This study provides new insights into how AMPK monitors energy stress to control effector and memory CD8⁺ T‐cell formation as discussed in this Commentary.     

A New Workflow for Whole‐Genome Sequencing of Single Human Cells

Unbiased amplification of the whole‐genome amplification (WGA) of single cells is crucial to study cancer evolution and genetic heterogeneity, but is challenging due to the high complexity of the human genome. Here, we present a new workflow combining an efficient adapter‐linker PCR‐based WGA method with second‐generation sequencing. This approach allows comparison of single cells at base pair resolution. Amplification recovered up to 74% of the human genome. Copy‐number variants and loss of heterozygosity detected in single cell genomes showed concordance of up to 99% to pooled genomic DNA. Allele frequencies of mutations could be determined accurately due to an allele dropout rate of only 2%, clearly demonstrating the low bias of our PCR‐based WGA approach. Sequencing with paired‐end reads allowed genome‐wide analysis of structural variants. By direct comparison to other WGA methods, we further endorse its suitability to analyze genetic heterogeneity.     

Detection of active human cytomegalovirus by the promyelocytic leukemia body assay in cultures of PBMCs from patients undergoing hematopoietic stem cell transplantation

A novel detection system was established previously for cells infected with the human cytomegalovirus (HCMV) in vitro that utilizes the unique IE1-dependent nuclear dispersion of promyelocytic leukemia (PML) bodies early in the HCMV replication cycle. This assay system, designated "the PML assay," makes use of the GFP-PML-expressing cell line SE/15, and allows real-time monitoring of infected cells by fluorescence microscopy without any staining procedures. A rapid and quantitative drug susceptibility testing was developed for low-titer clinical isolates propagated in fibroblasts in vitro. The present study sought to exploit the PML assay for evaluating in vivo status of HCMV without virus isolation. Progeny viruses were detected directly from peripheral blood mononuclear cells (PBMCs) infected in vivo obtained from hematopoietic stem cell transplantation recipients. The overall positivity of the PML assay tended to correlate with the levels of genomic DNA. Direct phenotypic susceptibility testing detected one ganciclovir (GCV)-resistant case among 19 samples, which was confirmed further by genomic and plaque reduction assays. However, in another patient with the sequence-proven mutant confirmed by sequencing, the progeny viruses exhibiting GCV-resistance were not detected. Studies on the isolated virus from the latter patient suggested the possibility that replication efficiency may differ between PBMCs and lesions infected in vivo, which may hamper the detection of GCV-resistant viruses by the PML assay, at least in this case. Taken together, the PML assay is sufficiently sensitive to monitor replication-competent HCMV directly from PBMCs infected in vivo, and provides a novel tool for comparing the characteristics of HCMV strains infected in vivo.     

Culture of HepG2 liver cells on three dimensional polystyrene scaffolds enhances cell structure and function during toxicological challenge

Cultured cells are dramatically affected by the micro-environment in which they are grown. In this study, we have investigated whether HepG2 liver cells grown in three dimensional (3-D) cultures cope more effectively with the known cytotoxic agent, methotrexate, than their counterparts grown on traditional two dimensional (2-D) flat plastic surfaces. To enable 3-D growth of HepG2 cells in vitro, we cultured cells on 3-D porous polystyrene scaffolds previously developed in our laboratories. HepG2 cells grown in 3-D displayed excellent morphological characteristics and formed numerous bile canaliculi that were seldom seen in cultures grown on 2-D surfaces. The function of liver cells grown on 3-D supports was significantly enhanced compared to activity of cells grown on 2-D standard plasticware. Unlike their 2-D counterparts, 3-D cultures were less susceptible to lower concentrations of methotrexate. Cells grown in 3-D maintained their structural integrity, possessed greater viability, were less susceptible to cell death at higher levels of the cytotoxin compared to 2-D cultures, and appeared to respond to the drug in a manner more comparable to its known activity in vivo. Our results suggest that hepatotoxicity testing using 3-D cultures might be more likely to reflect true physiological responses to cytotoxic compounds than existing models that rely on 2-D culture systems. This technology has potential applications for toxicity testing and drug screening.     

A phenotypically severe,biochemically “silent” case of HIBCH deficiency in a newborn diagnosed by rapid whole exome sequencing and enzymatic testing

3‐Hydroxyisobutyryl‐CoA dehydrogenase (HIBCH) deficiency is a rare error in valine catabolism associated with a Leigh syndrome‐like phenotype, mitochondrial dysfunction, and increased C4‐OH. We report the most severe case to date in a full‐term female who presented with poor feeding and nystagmus on day of life (DOL) 1. Although initial neuroimaging findings were concerning for metabolic disease, further metabolic testing was nondiagnostic and she was discharged on DOL 18. She was readmitted on DOL 22 after severe apneic episodes requiring intubation, with EEG demonstrating multifocal seizures and MRI/MRS demonstrating worsening findings. Care was withdrawn DOL 27 and she expired. Rapid whole exome sequencing (WES) demonstrated compound heterozygous variants in HIBCH with a paternal pathogenic variant (c.852delA, p.L284FfsX10 ) and a maternal likely pathogenic variant (c.488G>T, p.C163F). Fibroblast enzymatic testing demonstrated marked reduction in HIBCH levels. This case demonstrates the importance of rapid WES and follow‐up functional testing in establishing a diagnosis when metabolic disease is suspected but lacks an expected biochemical signature.     

SULT1A1 copy number variation: ethnic distribution analysis in an Indian population

Cytosolic sulfotransferases (SULTs) are phase II detoxification enzymes involved in metabolism of numerous xenobiotics, drugs and endogenous compounds. Interindividual variation in sulfonation capacity is important for determining an individual’s response to xenobiotics. SNPs in SULTs, mainly SULT1A1 have been associated with cancer risk and also with response to therapeutic agents. Copy number variation (CNVs) in SULT1A1 is found to be correlated with altered enzyme activity. This short report primarily focuses on CNV in SULT1A1 and its distribution among different ethnic populations around the globe. Frequency distribution of SULT1A1 copy number (CN) in 157 healthy Indian individuals was assessed using florescent-based quantitative PCR assay. A range of 1 to >4 copies, with a frequency of SULT1A1 CN =2 (64.9%) the highest, was observed in our (Indian) population. Upon comparative analysis of frequency distribution of SULT1A1 CN among diverse population groups, a statistically significant difference was observed between Indians (our data) and African-American (AA) (p =?0.0001) and South African (Tswana) (p SULT1A1 varies significantly among world populations and may be one of the determinants of health and diseases.     

Inferring the effect of genomic variation in the new era of genomics

Accurate and detailed understanding of the effects of variants in the coding and noncoding regions of the genome is the next big challenge in the new genomic era of personalized medicine, especially to tackle newer findings of genetic and phenotypic heterogeneity of diseases. This is necessary to resolve the gene‐variant–disease relationship, the pathogenic variant spectrum of genes, pathogenic variants with variable clinical consequences, and multiloci diseases. In turn, this will facilitate patient recruitment for relevant clinical trials. In this review, we describe the trends in research at the intersection of basic and clinical genomics aiming to (a) overcome molecular diagnostic challenges and increase the clinical utility of next‐generation sequencing (NGS) platforms, (b) elucidate variants associated with disease, (c) determine overall genomic complexity including epistasis, complex inheritance patterns such as “synergistic heterozygosity,” digenic/multigenic inheritance, modifier effect, and rare variant load. We describe the newly emerging field of integrated functional genomics, in vivo or in vitro large‐scale functional approaches, statistical bioinformatics algorithms that support NGS genomics data to interpret variants for timely clinical diagnostics and disease management. Thus, facilitating the discovery of new therapeutic or biomarker options, and their roles in the future of personalized medicine.     

A simple preparation method for mouse eosinophils and their responses to anti-allergic drugs

Objective and design: A simple method for preparing mouse eosinophils was established, and the characteristics of the eosinophils were assessed including their responses to anti-allergic drugs. Materials or subjects: Mouse eosinophils were prepared from peritoneal exudate cells of BALB/c mice primed and boosted with antigen ovalbumin (OVA). Methods: Surface phenotypes, migration activities and leukotriene C₄ (LTC₄) production abilities of these eosinophils were examined. In addition, the effects of anti-allergic drugs, oxatomide and tranilast, on generation of LTC₄ from mouse eosinophils were examined. Results: Eosinophils of mice boosted with OVA were phenotypically and functionally identical with human eosinophils. Around 1 × 10⁷ eosinophils were obtained from mouse peritoneal exudate. It was found that these mouse eosinophils enabled to migrate in response to eotaxin as well as platelet-activating factor (PAF), and generated LTC₄ by IL-5 stimulation. Moreover, it was revealed that clinically used anti-allergic drugs inhibited LTC₄-production dose-dependently. Conclusions: The present study provides a convenient method to obtain fully functional mouse eosinophils that are useful for drug screening and for evaluating implications of eosinophils in allergic responses. Received 20 April 2006; returned for revision 6 July 2006; returned for final revision 29 August 2006; accepted by M. Katori 29 September 2006