共查询到20条相似文献,搜索用时 15 毫秒
1.
In vivo bioluminescence imaging 总被引:3,自引:0,他引:3
In vivo bioluminescent imaging (BLI) is a versatile and sensitive tool that is based on detection of light emission from cells or tissues. Bioluminescence, the biochemical generation of light by a living organism, is a naturally occurring phenomenon. Luciferase enzymes, such as that from the North American firefly (Photinus pyralis), catalyze the oxidation of a substrate (luciferin), and photons of light are a product of the reaction. Optical imaging by bioluminescence allows a low-cost, noninvasive, and real-time analysis of disease processes at the molecular level in living organisms. Bioluminescence has been used to track tumor cells, bacterial and viral infections, gene expression, and treatment response. Bioluminescence in vivo imaging allows longitudinal monitoring of a disease course in the same animal, a desirable alternative to analyzing a number of animals at many time points during the course of the disease. We provide a brief introduction to BLI technology, specific examples of in vivo BLI studies investigating bacterial/viral pathogenesis and tumor growth in animal models, and highlight some future perspectives of BLI as a molecular imaging tool. 相似文献
2.
Many different cells' signalling pathways are universally regulated by Ca(2+) concentration [Ca(2+)] rises that have highly variable amplitudes and kinetic properties. Optical imaging can provide the means to characterise both the temporal and spatial aspects of Ca(2+) signals involved in neurophysiological functions. New methods for in vivo imaging of Ca(2+) signalling in the brain of Drosophila are required for probing the different dynamic aspects of this system. In studies here, whole brain Ca(2+) imaging was performed on transgenic flies with targeted expression of the bioluminescent Ca(2+) reporter GFP-aequorin (GA) in different neural structures. A photon counting based technique was used to undertake continuous recordings of cytosolic [Ca(2+)] over hours. Time integrals for reconstructing images and analysis of the data were selected offline according to the signal intensity. This approach allowed a unique Ca(2+) response associated with cholinergic transmission to be identified by whole brain imaging of specific neural structures. Notably, [Ca(2+)] transients in the Mushroom Bodies (MBs) following nicotine stimulation were accompanied by a delayed secondary [Ca(2+)] rise (up to 15 min. later) in the MB lobes. The delayed response was sensitive to thapsigargin, suggesting a role for intra-cellular Ca(2+) stores. Moreover, it was reduced in dunce mutant flies, which are impaired in learning and memory. Bioluminescence imaging is therefore useful for studying Ca(2+) signalling pathways and for functional mapping of neurophysiological processes in the fly brain. 相似文献
3.
Understanding biological processes in the context of intact organ systems with fine temporal resolution has required the development of imaging strategies that reveal cellular and molecular changes in the living body. Reporter genes that confer optical signatures on a given biological process have been used widely in cell biology and have been used more recently to interrogate biological processes in living animal models of human biology and disease. The use of internal biological sources of light, luciferases, to tag cells, pathogens, and genes has proved to be a versatile tool to provide in vivo indicators that can be detected externally. The application of this technology to the study of animal models of infectious disease has not only provided insights into disease processes, but has also revealed new mechanisms by which pathogens may avoid host defences during infection. 相似文献
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In vivo bioluminescence imaging to evaluate estrogenic activities of endocrine disrupters 总被引:2,自引:0,他引:2
Reporter gene technology is widely used to measure activity of hormone analogs, and bioluminescent in vitro assays have allowed rapid screening of numerous chemicals either to identify new agonists or antagonists of hormones or to detect the presence of endocrine disrupters in the environment. Stable bioluminescent cell lines have been established and they provide reproducible dose–response curves and accurate determination of in vitro efficiencies of various chemicals. In vivo, however, these molecules can be metabolized, bound by proteins, or stored in fats and thus could display efficiencies different from those observed in vitro. In vivo assays, such as the uterotrophic bioassay, require numerous sacrificed animals, and responses not only are dependent on an estrogenic action but also imply other factors. For a faster assay and to avoid the use of numerous animals, we developed an in vivo biosensor constituted of stable bioluminescent cells implanted in nude mice. MCF-7 bioluminescent cell lines were chosen since their proliferation is low in the absence of estrogen and the xenograft size can thus be stable for several weeks. Luciferase gene expression was monitored noninvasively with a cooled charge-coupled device camera. Quantitative analysis allowed us to compare in vitro and in vivo actions of different estrogenic compounds (estradiol, estrone) and endocrine disruptors (ethynylestradiol, genistein, octylphenol, and 2,4′-dichlorodiphenyldichloroethylene) in the same cell lines and to follow hormone action on a living animal as a function of time. Different administration protocols have been used and good correlation was observed for most products. However, we found that ethynylestradiol was the most efficient chemical when orally administered. 相似文献
5.
In vivo magnetic resonance imaging of acute brain inflammation using microparticles of iron oxide 总被引:5,自引:0,他引:5
McAteer MA Sibson NR von Zur Muhlen C Schneider JE Lowe AS Warrick N Channon KM Anthony DC Choudhury RP 《Nature medicine》2007,13(10):1253-1258
Multiple sclerosis is a disease of the central nervous system that is associated with leukocyte recruitment and subsequent inflammation, demyelination and axonal loss. Endothelial vascular cell adhesion molecule-1 (VCAM-1) and its ligand, alpha4beta1 integrin, are key mediators of leukocyte recruitment, and selective inhibitors that bind to the alpha4 subunit of alpha4beta1 substantially reduce clinical relapse in multiple sclerosis. Urgently needed is a molecular imaging technique to accelerate diagnosis, to quantify disease activity and to guide specific therapy. Here we report in vivo detection of VCAM-1 in acute brain inflammation, by magnetic resonance imaging in a mouse model, at a time when pathology is otherwise undetectable. Antibody-conjugated microparticles carrying a large amount of iron oxide provide potent, quantifiable contrast effects that delineate the architecture of activated cerebral blood vessels. Their rapid clearance from blood results in minimal background contrast. This technology is adaptable to monitor the expression of endovascular molecules in vivo in various pathologies. 相似文献
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The discovery of endogenous neural stem cells (eNSCs) in the adult mammalian brain with their ability to self-renew and differentiate into functional neurons, astrocytes and oligodendrocytes has raised the hope for novel therapies of neurological diseases. Experimentally, those eNSCs can be mobilized in vivo, enhancing regeneration and accelerating functional recovery after, e.g., focal cerebral ischemia, thus constituting a most promising approach in stem cell research. In order to translate those current experimental approaches into a clinical setting in the future, non-invasive imaging methods are required to monitor eNSC activation in a longitudinal and intra-individual manner. As yet, imaging protocols to assess eNSC mobilization non-invasively in the live brain remain scarce, but considerable progress has been made in this field in recent years. This review summarizes and discusses the current imaging modalities suitable to monitor eNSCs in individual experimental animals over time, including optical imaging, magnetic resonance tomography and-spectroscopy, as well as positron emission tomography (PET). Special emphasis is put on the potential of each imaging method for a possible clinical translation, and on the specificity of the signal obtained. PET-imaging with the radiotracer 3’-deoxy-3’-[18F]fluoro-L-thymidine in particular constitutes a modality with excellent potential for clinical translation but low specificity; however, concomitant imaging of neuroinflammation is feasible and increases its specificity. The non-invasive imaging strategies presented here allow for the exploitation of novel treatment strategies based upon the regenerative potential of eNSCs, and will help to facilitate a translation into the clinical setting. 相似文献
7.
Immunological reactions have a key role in health and disease and are complex events characterized by coordinated cell trafficking to specific locations throughout the body. Clarification of these cell-trafficking events is crucial for improving our understanding of how immune reactions are initiated, controlled and recalled. As we discuss here, an emerging modality for revealing cell trafficking is bioluminescence imaging, which harnesses the light-emitting properties of enzymes such as luciferase for quantification of cells and uses low-light imaging systems. This strategy could be useful for the study of a wide range of biological processes, such as the pathophysiology of graft-versus-host and graft-versus-leukaemia reactions. 相似文献
8.
Background
Plaque vulnerability to rupture has emerged as a critical correlate to risk of adverse coronary events but there is as yet no clinical method to assess plaque stability in vivo. In the search to identify biomarkers of vulnerable plaques an association has been found between macrophages and plaque stability—the density and pattern of macrophage localization in lesions is indicative of probability to rupture. In very unstable plaques, macrophages are found in high densities and concentrated in the plaque shoulders. Therefore, the ability to map macrophages in plaques could allow noninvasive assessment of plaque stability. We use a multimodality imaging approach to noninvasively map the distribution of macrophages in vivo. The use of multiple modalities allows us to combine the complementary strengths of each modality to better visualize features of interest. Our combined use of Positron Emission Tomography and Magnetic Resonance Imaging (PET/MRI) allows high sensitivity PET screening to identify putative lesions in a whole body view, and high resolution MRI for detailed mapping of biomarker expression in the lesions.Methodology/Principal Findings
Macromolecular and nanoparticle contrast agents targeted to macrophages were developed and tested in three different mouse and rat models of atherosclerosis in which inflamed vascular plaques form spontaneously and/or are induced by injury. For multimodal detection, the probes were designed to contain gadolinium (T1 MRI) or iron oxide (T2 MRI), and Cu-64 (PET). PET imaging was utilized to identify regions of macrophage accumulation; these regions were further probed by MRI to visualize macrophage distribution at high resolution. In both PET and MR images the probes enhanced contrast at sites of vascular inflammation, but not in normal vessel walls. MRI was able to identify discrete sites of inflammation that were blurred together at the low resolution of PET. Macrophage content in the lesions was confirmed by histology.Conclusions/Significance
The multimodal imaging approach allowed high-sensitivity and high-resolution mapping of biomarker distribution and may lead to a clinical method to predict plaque probability to rupture. 相似文献9.
Bioluminescent imaging (BLI) is a widely used in vivo method to determine the location and relative intensity of luciferase expression in mice. Luciferase expression is observed following an i.p. dose of d-luciferin, resulting in bioluminescence that is detected in anesthetized mice by a charge-coupled device camera. To establish whether BLI could be used as a quantitative measurement of non-viral-mediated luciferase expression, precise quantities of plasmid DNA encoding the luciferase gene were hydrodynamically dosed in mice. The results established a linear correlation between the DNA dose and the BLI response measured in liver which spanned five orders of magnitude. The level of luciferase expression was found to be a direct function of d-luciferin dose. The time course of luciferase expression and the influence of multidosing of substrate were measured by BLI. The recovery of luciferase from the liver of hydrodynamically dosed mice allowed calibration of the BLI measurements. The results establish BLI's limit-of-detection at 20 pg of luciferase per liver following a hydrodynamic dose of 100 pg of plasmid DNA. These results demonstrate that BLI is both sensitive and linear and should allow for the direct comparison of the efficiency of gene transfer vectors that target the liver. 相似文献
10.
The CB1 cannabinoid receptor is expressed in the brain at levels sufficient to serve as potential target for in vivo imaging using positron emission tomography (PET) or single photon emission computed tomography methodology. To date, the most promising radioligands for the in vivo imaging of this receptor have structures based on that of the cannabinoid antagonist, SR141716A. Rodent data obtained using these in vivo radiotracers has demonstrated that both the behavioral and neurochemical effects of cannabinoids occur at very low levels of receptor occupancy. More recently, an agonist radiotracer based on the structure of aminoalkylindole cannabinoids has also been examined for in vivo labeling of CB1 receptors. Although rodent studies have indicated that in vivo imaging of CB1 receptors is feasible, at the present time this receptor has still to be successful imaged in a human PET study. 相似文献
11.
In vivo imaging of transplanted hematopoietic stem and progenitor cells (HSPCs) was developed to investigate the relationship between HSPCs and components of their microenvironment in the bone marrow. In particular, it allows a direct observation of the behavior of hematopoietic cells during the first few days after transplantation, when the critical events in homing and early engraftment are occurring. By directly imaging these events in living animals, this method permits a detailed assessment of functions previously evaluated by crude assessments of cell counts (homing) or after prolonged periods (engraftment). This protocol offers a new means of investigating the role of cell-intrinsic and cell-extrinsic molecular regulators of hematopoiesis during the early stages of transplantation, and it is the first to allow the study of cell-cell interactions within the bone marrow in three dimensions and in real time. In this paper, we describe how to isolate, label and inject HSPCs, as well as how to perform calvarium intravital microscopy and analyze the resulting images. A typical experiment can be performed and analyzed in ~1 week. 相似文献
12.
ABSTRACT: BACKGROUND: Tumor cell lines that can be tracked in vivo during tumorigenesis and metastasis provide vital tools for studying the specific cellular mechanisms that mediate these processes as well as investigating therapeutic targets to inhibit them. The goal of this study was to engineer imageable mouse mammary tumor cell lines with discrete propensities to metastasize to bone in vivo. Two novel luciferase expressing cell lines were developed and characterized for use in the study of breast cancer metastasis to bone in a syngeneic mouse model. RESULTS: The 4T1.2 luc3 and 66c14 luc2 cell lines were shown to have high levels of bioluminescence intensity in vitro and in vivo after orthotopic injection into mouse mammary fat pads. The 4T1.2 luc3 cell line was found to closely model the sites of metastases seen in human patients including lung, liver, and bone. Specifically, 4T1.2 luc3 cells demonstrated a high incidence of metastasis to spine, with an ex-vivo BLI intensity three orders of magnitude above the commercially available 4T1 luc2 cells. 66c14 luc2 cells also demonstrated metastasis to spine, which was lower than that of 4T1.2 luc3 cells but higher than 4T1 luc2 cells, in addition to previously unreported metastases in the liver. High osteolytic activity of the 4T1.2 luc3 cells in vivo in the bone microenvironment was also detected. CONCLUSIONS: The engineered 4T1.2 luc3 and 66c14 luc2 cell lines described in this study are valuable tools for studying the cellular events moderating the metastasis of breast tumor cells to bone. 相似文献
13.
In vivo bioluminescence imaging (BLI) has the advantages of high sensitivity and low background. By counting the number of photons emitted from a specimen, BLI can quantify biological events such as tumour growth, gene expression and drug response. The intensities and kinetics of the BL signal are affected by many factors and may confound the quantitative results acquired from consecutive imaging sessions or different specimens. We used three different mouse models of tumours to examine whether anaesthetics, positioning and tumour growth may affect the consistency of the BL signal. The results showed that BLI signal could be affected by different anaesthetics and repetitive positioning. Using the same anaesthetics produced consistent peak times, while other factors were held constant. However, as the tumours grew the peak times shifted and the time course of BL signals had different shapes, depending on the positioning of the mice. The data indicate that a carefully designed BLI experiment is required to generate optimal and consistent results. Copyright © 2008 John Wiley & Sons, Ltd. 相似文献
14.
In vivo bioluminescence: new potentials for microbiology 总被引:8,自引:0,他引:8
G.S.A.B. STEWART 《Letters in applied microbiology》1990,10(1):1-8
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Feng Liu Zhi-da Liu Nan Wu Jiang-Hua Wang Heng-Hui Zhang Ran Fei Xu Cong Hong-song Chen Lai Wei 《In vitro cellular & developmental biology. Animal》2013,49(7):537-547
Transplantation of bone marrow (BM)-derived endothelial progenitor cells (EPCs) has been reported to improve liver fibrosis, but there is no direct evidence for the mechanism of improvement. We investigated the mechanism in vitro by coculturing BM-derived EPCs with activated hepatic stellate cells (HSCs) to mimic the hepatic environment. EPCs and HSCs were cultured alone and indirectly cocultured at a 1:1 ratio in a Transwell system. The characteristics of HSCs and EPCs were examined at different time points. An invasion assay showed the time-dependent effect on degradation of the extracellular matrix (ECM) layer in EPCs cultured alone. Real-time PCR and enzyme-linked immunosorbent assay analysis revealed that EPCs served as a source of matrix metalloproteinase-9 (MMP-9), and MMP-9 expression levels significantly increased during the 2 d of coculture. CFSE labeling showed that EPCs inhibited proliferation of HSCs. Annexin-V/PI staining, erminal deoxynucleotidyl transferase X-dUTP nick end labeling analysis, and (cleaved) caspase-3 activity revealed that EPCs promoted HSC apoptosis. However, the proliferation and apoptosis of EPCs were unaffected by cocultured HSCs. Coculturing increased the expression of inducible nitric oxide synthase, vascular endothelial growth factor, and hepatocyte growth factor (HGF) in EPCs, promoted differentiation of EPCs, and reduced the expression of types I and III collagens and transforming growth factor beta 1. Knockdown of HGF expression attenuated EPC-induced activation of HSC apoptosis and profibrotic ability. These findings demonstrated that BM-derived EPCs could degrade ECM, promoting activated HSC apoptosis, suppressing proliferation and profibrotic ability of activated HSCs. HGF secretion by EPCs plays a key role in inducing activated HSC apoptosis and HSC profibrotic ability. 相似文献
18.
骨髓间充质干细胞无血清培养 总被引:1,自引:0,他引:1
为建立一种化学成分明确的、能用于体外扩增骨髓间充质干细胞的无血清培养基, 且骨髓间充质干细胞经无血清培养扩增后仍能保持其多向分化的潜能。采用密度梯度离心结合贴壁法从1月龄新西兰大白兔股骨中分离骨髓间充质干细胞, 比较在含10%胎牛血清的培养基(SCM)和自制的化学成分明确的无血清培养基(CDSFM)中骨髓间充质干细胞的形态、增殖能力, 以及扩增后的骨髓间充质干细胞的细胞周期、集落形成能力和成骨、成脂肪分化能力。经过10 d的培养, 骨髓间充质干细胞在自制的无血清培养基中扩增了50倍, 在含10%胎牛血清的培养基中扩增了40倍。在无血清和有血清培养基中扩增后的细胞中G0/G1期比例分别为(80.31%±0.6%)和(75.24%±4.0%), 两者无显著差异(P>0.05)。无血清培养扩增后的骨髓间充质干细胞集落形成率(12.7%±4.0%)低于有血清培养组(28.7%±4.2%), 两者比较差异显著(P<0.01)。经过无血清培养扩增的骨髓间充质干细胞在成骨、成脂肪诱导分化培养基中能够分化成成骨和脂肪细胞。自制的化学成分明确的无血清培养基能够在体外培养扩增骨髓间充质干细胞, 并且维持其干细胞特性, 可以用于细胞治疗以及生物医学研究。 相似文献
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Oumaïma Ibrahim-Granet Grégory Jouvion Tobias M Hohl Sabrina Droin-Bergère François Philippart Oh Yoen Kim Minou Adib-Conquy Reto Schwendener Jean-Marc Cavaillon Matthias Brock 《BMC microbiology》2010,10(1):105
Background
Invasive aspergillosis (IA) is a major cause of infectious morbidity and mortality in immune compromised patients. Studies on the pathogenesis of IA have been limited by the difficulty to monitor disease progression in real-time. For real-time monitoring of the infection, we recently engineered a bioluminescent A. fumigatus strain. 相似文献20.
Cellular therapeutics show great promise for the treatment of disease, but few noninvasive techniques exist for monitoring the cells after administration. Here we present a magnetic resonance imaging (MRI) technology that uses perfluoropolyether (PFPE) agents to track cells in vivo. Fluorine MRI selectively images only the labeled cells, and a 'conventional' (1)H image places the cells in their anatomical context. We labeled phenotypically defined dendritic cells (DCs) with PFPE ex vivo and observed efficient intracellular uptake of the PFPE with little effect on DC function. We injected labeled DCs into tissue or intravenously in mice and then tracked the cells in vivo using (19)F MRI. Although we focused on DCs, which are being developed as immunotherapeutics for cancer and autoimmune diseases, this technology should be useful for monitoring a wide range of cell types in vivo. 相似文献