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1.
In vivo imaging of molecular events in small animals has great potential to impact basic science and drug development. For this reason, several imaging technologies have been adapted to small animal research, including X-ray, magnetic resonance, and radioisotope imaging. Despite this plethora of visualization techniques, fluorescence imaging is emerging as an important alternative because of its operational simplicity, safety, and cost-effectiveness. Fluorescence imaging has recently become particularly interesting because of advances in fluorescent probe technology, including targeted fluorochromes as well as fluorescent "switches" sensitive to specific biochemical events. While past biological investigations using fluorescence have focused on microscopic examination of ex vivo, in vitro, or intravital specimens, techniques for macroscopic fluorescence imaging are now emerging for in vivo molecular imaging applications. This review illuminates fluorescence imaging technologies that hold promise for small animal imaging. In particular we focus on planar illumination techniques, also known as Fluorescence Reflectance Imaging (FRI), and discuss its performance and current use. We then discuss fluorescence molecular tomography (FMT), an evolving technique for quantitative three-dimensional imaging of fluorescence in vivo. This technique offers the promise of non-invasively quantifying and visualizing specific molecular activity in living subjects in three dimensions.  相似文献   

2.
Molecular imaging enables non-invasive monitoring of tumor growth, progression, and drug treatment response, and it has become an important tool to promote biological studies in recent years. In this study, we comprehensively evaluated the in vivo anti-angiogenic and anti-neoplastic effects of Endostar on liver cancer based on the optical molecular imaging systems including micro-computer tomography (Micro-CT), bioluminescence molecular imaging (BLI) and fluorescence molecular tomography (FMT). Firefly luciferase (fLuc) and green fluorescent protein (GFP) dual labeled human hepatocellular carcinoma cells (HCC-LM3-fLuc-GFP cells) were used to establish the subcutaneous and orthotopic liver tumor model. After the tumor cells were implanted 14∼18 days, Endostar (5 mg/kg/day) was administered through an intravenous tail vein injection for continuous 14 days. The computer tomography angiography (CTA) and BLI were carried out for the subcutaneous tumor model. FMT was executed for the orthotopic tumor model. The CTA data showed that tumor vessel formation and the peritumoral vasculature of subcutaneous tumor in the Endostar treatment group was significantly inhibited compared to the control group. The BLI data exhibited the obvious tumor inhibition day 8 post-treatment. The FMT detected the tumor suppression effects of Endostar as early as day 4 post-treatment and measured the tumor location. The above data confirmed the effects of Endostar on anti-angiogenesis and tumor suppression on liver cancer. Our system combined CTA, BLI, and FMT to offer more comprehensive information about the effects of Endostar on the suppression of vessel and tumor formation. Optical molecular imaging system enabled the non-invasive and reliable assessment of anti-tumor drug efficacy on liver cancer.  相似文献   

3.
Fluorescence imaging is increasingly used to probe protein function and gene expression in live animals. This technology could enhance the study of pathogenesis, drug development, and therapeutic intervention. In this article, we focus on three-dimensional fluorescence observations using fluorescence-mediated molecular tomography (FMT), a novel imaging technique that can resolve molecular function in deep tissues by reconstructing fluorescent probe distributions in vivo. We have compared FMT findings with conventional fluorescence reflectance imaging (FRI) to study protease function in nude mice with subsurface implanted tumors. This validation of FMT with FRI demonstrated the spatial congruence of fluorochrome activation as determined by the two techniques.  相似文献   

4.
The ability to visualize molecular processes and cellular regulators of complex pulmonary diseases such as asthma, chronic obstructive pulmonary disease (COPD), or adult respiratory distress syndrome (ARDS), would aid in the diagnosis, differentiation, therapy assessment and in small animal-based drug-discovery processes. Herein we report the application of normalized transillumination and fluorescence molecular tomography (FMT) for the noninvasive quantitative imaging of the mouse lung in vivo. We demonstrate the ability to visualize and quantitate pulmonary response in a murine model of LPS-induced airway inflammation. Twenty-four hours prior to imaging, BALB/c female mice were injected via tail vein with 2 nmol of a cathepsin-sensitive activatable fluorescent probe (excitation: 750 nm; emission: 780 nm) and 2 nmol of accompanying intravascular agent (excitation: 674 nm; emission: 694 nm). Six hours later, the mice were anesthetized with isoflurane and administered intranasal LPS in sterile 0.9% saline in 25 microl aliquots (one per nostril). Fluorescence molecular imaging revealed the in vivo profile of cysteine protease activation and vascular distribution within the lung typifying the inflammatory response to LPS insult. Results were correlated with standard in vitro laboratory tests (Western blot, bronchoalveolar lavage or BAL analysis, immunohistochemistry) and revealed good correlation with the underlying activity. We demonstrated the capacity of fluorescence tomography to noninvasively and longitudinally characterize physiological, cellular, and subcellular processes associated with inflammatory disease burden in the lung. The data presented herein serve to further evince fluorescence molecular imaging as a technology highly appropriate for the biomedical laboratory.  相似文献   

5.
The complexity of the tumor microenvironment necessitates that cell behavior is studied in a broad, multi-scale context. Although tomographic and microscopy-based far and near infrared fluorescence (NIRF, >650 nm) imaging methods offer high resolution, sensitivity, and depth penetration, there has been a lack of optimized NIRF agents to label and track cells in their native environments at different scales. In this study we labeled mammalian leukocytes with VivoTag 680 (VT680), an amine reactive N-hydroxysuccinimide (NHS) ester of a (benz) indolium-derived far red fluorescent probe. We show that VT680 diffuses into leukocytes within minutes, covalently binds to cellular components, remains internalized for days in vitro and in vivo, and does not transfer fluorescence to adjacent cells. It is biocompatible, keeps cells fully functional, and fluoresces at high intensities. In a tumor model of cytotoxic T lymphocyte (CTL) immunotherapy, we track and quantify VT680-labeled cells longitudinally at the whole-body level with fluorescence-mediated molecular tomography (FMT), within tissues at single cell resolutions by multiphoton and confocal intravital microscopy, and ex vivo by flow cytometry. Thus, this approach is suitable to monitor cells at multiple resolutions in real time in their native environments by NIR-based fluorescence imaging.  相似文献   

6.
X-ray computed tomography (XCT) has been shown to be an effective imaging technique for a variety of materials. Due to the relatively low differential attenuation of X-rays in biological tissue, a high density contrast agent is often required to obtain optimal contrast. The contrast agent, iodine potassium iodide (), has been used in several biological studies to augment the use of XCT scanning. Recently was used in XCT scans of animal hearts to study cardiac structure and to generate 3D anatomical computer models. However, to date there has been no thorough study into the optimal use of as a contrast agent in cardiac muscle with respect to the staining times required, which has been shown to impact significantly upon the quality of results. In this study we address this issue by systematically scanning samples at various stages of the staining process. To achieve this, mouse hearts were stained for up to 58 hours and scanned at regular intervals of 6–7 hours throughout this process. Optimal staining was found to depend upon the thickness of the tissue; a simple empirical exponential relationship was derived to allow calculation of the required staining time for cardiac samples of an arbitrary size.  相似文献   

7.
Imaging of cell trafficking and metastases of paediatric rhabdomyosarcoma   总被引:2,自引:0,他引:2  
Abstract.   Objective : The aim of this study was to establish a preclinical mouse model to study metastases of paediatric rhabdomyosarcoma at the macroscopic and cellular levels, with different imaging methods. Experimental Design : The alveolar rhabdomyosarcoma cell line Rh30 was stably transfected with the red fluorescent protein (DsRed2) then was xenotransplanted (intravenous injection [ n  = 8], and footpad injection [ n  = 8]) into nude mice (NMRI nu/nu). Macroscopic imaging of metastases was performed using DsRed2-fluorescence and flat-panel volumetric computed tomography scan. In a further series of animals ( n  = 8), in vivo cell trafficking of rhabdomyosarcoma cells using cellular imaging with an Olympus OV100 variable-magnification small-animal imaging system was used. Results : Metastases in the pelvis, thoracic wall and skin were visualized by fluorescence imaging. Pelvic metastases were found after tail vein injection and at other metastatic sites after footpad injection. Flat-panel volumetric computed tomography scan data allowed highly specific analysis of contrast between tumour and surrounding tissue. Correlation between fluorescence and flat-panel volumetric computed tomography scan imaging data was observed. Single-cell imaging visualized tumour cells in the vessels and demonstrated the arrest of tumour cells at vessel junctions followed by extravasation of the tumour cells. Conclusion : We established a model for visualization of experimental metastatic invasion and describe relevant tools for imaging childhood rhabdomyosarcoma metastases at the macroscopic and cellular levels. Imaging of cell trafficking visualized the behaviour of tumour cells and development of metastases by accumulation and extravasation of rhabdomyosarcoma cells.  相似文献   

8.
A series of human carbonic anhydrase (hCA) IX inhibitors conjugated to various near-infrared fluorescent dyes was synthesized with the aim of imaging hypoxia-induced hCA IX expression in tumor cells in vitro, ex vivo and in vivo. The resulting compounds were profiled for inhibition of transmembrane hCA IX showing a range of potencies from 7.5 to 116 nM and up to 50-fold selectivity over the cytosolic form hCA II. Some of the compounds also showed inhibition selectivity for other transmembrane forms hCA XII and XIV as well. Compounds incubated in vitro with HeLa cells cultured under normoxic and hypoxic conditions detected upregulation of hCA IX under hypoxia by fluorescence microscopy. A pilot in vivo study in HT-29 tumor bearing mice showed significant accumulation of a fluorescent acetazolamide derivative in tumor tissue with little accumulation in other tissues. Approximately 10% of injected dose was non-invasively quantified in tumors by fluorescence molecular tomography (FMT), demonstrating the promise of these new compounds for quantitative imaging of hCA IX upregulation in live animals.  相似文献   

9.
When small molecules or proteins are injected into live animals, their physical and chemical properties will significantly affect pharmacokinetics, tissue penetration, and the ultimate routes of metabolism and clearance. Fluorescence molecular tomography (FMT) offers the ability to non-invasively image and quantify temporal changes in fluorescence throughout the major organ systems of living animals, in a manner analogous to traditional approaches with radiolabeled agents. This approach is best used with biotherapeutics (therapeutic antibodies, or other large proteins) or large-scaffold drug-delivery vectors, that are minimally affected by low-level fluorophore conjugation. Application to small molecule drugs should take into account the significant impact of fluorophore labeling on size and physicochemical properties, however, the presents studies show that this technique is readily applied to small molecule agents developed for far-red (FR) or near infrared (NIR) imaging. Quantification by non-invasive FMT correlated well with both fluorescence from tissue homogenates as well as with planar (2D) fluorescence reflectance imaging of excised intact organs (r2 = 0.996 and 0.969, respectively). Dynamic FMT imaging (multiple times from 0 to 24 h) performed in live mice after the injection of four different FR/NIR-labeled agents, including immunoglobulin, 20-50 nm nanoparticles, a large vascular imaging agent, and a small molecule integrin antagonist, showed clear differences in the percentage of injected dose per gram of tissue (%ID/g) in liver, kidney, and bladder signal. Nanoparticles and IgG1 favored liver over kidney signal, the small molecule integrin-binding agent favored rapid kidney and bladder clearance, and the vascular agent, showed both liver and kidney clearance. Further assessment of the volume of distribution of these agents by fluorescent volume added information regarding their biodistribution and highlighted the relatively poor extravasation into tissue by IgG1. These studies demonstrate the ability of quantitative FMT imaging of FR/NIR agents to non-invasively visualize and quantify the biodistribution of different agents over time.  相似文献   

10.
G Wang  J Zhang  H Gao  V Weir  H Yu  W Cong  X Xu  H Shen  J Bennett  M Furth  Y Wang  M Vannier 《PloS one》2012,7(6):e39700
We recently elevated interior tomography from its origin in computed tomography (CT) to a general tomographic principle, and proved its validity for other tomographic modalities including SPECT, MRI, and others. Here we propose "omni-tomography", a novel concept for the grand fusion of multiple tomographic modalities for simultaneous data acquisition in a region of interest (ROI). Omni-tomography can be instrumental when physiological processes under investigation are multi-dimensional, multi-scale, multi-temporal and multi-parametric. Both preclinical and clinical studies now depend on in vivo tomography, often requiring separate evaluations by different imaging modalities. Over the past decade, two approaches have been used for multimodality fusion: Software based image registration and hybrid scanners such as PET-CT, PET-MRI, and SPECT-CT among others. While there are intrinsic limitations with both approaches, the main obstacle to the seamless fusion of multiple imaging modalities has been the bulkiness of each individual imager and the conflict of their physical (especially spatial) requirements. To address this challenge, omni-tomography is now unveiled as an emerging direction for biomedical imaging and systems biomedicine.  相似文献   

11.
Cre/loxP-dependent expression of fluorescent proteins represents a powerful biological tool for cell lineage, fate-mapping, and genetic analysis. Live tissue imaging has significantly improved with the development of far-red fluorescent proteins, with optimized spectral characteristics for in vivo applications. Here, we report the generation of the first transgenic mouse line expressing the far-red fluorescent protein Katushka, driven by the hybrid CAG promoter upon Cre-mediated recombination. After germ line or tissue-specific Cre-driven reporter activation, Katushka expression is strong and ubiquitous, without toxic effects, allowing fluorescence detection in fresh and fixed samples from all tissues examined. Moreover, fluorescence can be detected by in vivo noninvasive whole-body imaging when Katuhska is expressed exclusively in a specific cell population deep within the animal body such as pancreatic beta cells. Thus, this reporter model enables early, widespread, and sensitive in vivo detection of Cre activity and should provide a versatile tool for a wide spectrum of fluorescence and live-imaging applications.  相似文献   

12.
Multiphoton imaging has evolved as an indispensable tool in cell biology and holds prospects for clinical applications. When addressing endogenous signals such as coherent anti-Stokes Raman scattering (CARS) or second harmonic generation, it requires intense laser irradiation that may cause photodamage. We report that increasing endogenous fluorescence signal upon multiphoton imaging constitutes a marker of photodamage. The effect was studied on mouse brain in vivo and ex vivo, on ex vivo human brain tissue samples, as well as on glioblastoma cells in vitro, demonstrating that this phenomenon is common to a variety of different systems, both ex vivo and in vivo. CARS microscopy and vibrational spectroscopy were used to analyze the photodamage. The development of a standard easy-to-use model that employs rehydrated cryosections allowed the characterization of the irradiation-induced fluorescence and related it to nonlinear photodamage. In conclusion, the monitoring of endogenous two-photon excited fluorescence during label-free multiphoton microscopy enables to estimate damage thresholds ex vivo as well as detect photodamage during in vivo experiments.  相似文献   

13.
Optical projection tomography (OPT) is a 3D mesoscopic imaging modality that can utilize absorption or fluorescence contrast. 3D images can be rapidly reconstructed from tomographic data sets sampled with sufficient numbers of projection angles using the Radon transform, as is typically implemented with optically cleared samples of the mm‐to‐cm scale. For in vivo imaging, considerations of phototoxicity and the need to maintain animals under anesthesia typically preclude the acquisition of OPT data at a sufficient number of angles to avoid artifacts in the reconstructed images. For sparse samples, this can be addressed with iterative algorithms to reconstruct 3D images from undersampled OPT data, but the data processing times present a significant challenge for studies imaging multiple animals. We show here that convolutional neural networks (CNN) can be used in place of iterative algorithms to remove artifacts—reducing processing time for an undersampled in vivo zebrafish dataset from 77 to 15 minutes. We also show that using CNN produces reconstructions of equivalent quality to compressed sensing with 40% fewer projections. We further show that diverse training data classes, for example, ex vivo mouse tissue data, can be used for CNN‐based reconstructions of OPT data of other species including live zebrafish.   相似文献   

14.
Human hydatid disease (cystic echinococcosis, CE) is a chronic parasitic infection caused by the larval stage of the cestode Echinococcus granulosus. As the disease mainly affects the liver, approximately 70% of all identified CE cases are detected in this organ. Optical molecular imaging (OMI), a noninvasive imaging technique, has never been used in vivo with the specific molecular markers of CE. Thus, we aimed to construct an in vivo fluorescent imaging mouse model of CE to locate and quantify the presence of the parasites within the liver noninvasively. Drug-treated protoscolices were monitored after marking by JC-1 dye in in vitro and in vivo studies. This work describes for the first time the successful construction of an in vivo model of E. granulosus in a small living experimental animal to achieve dynamic monitoring and observation of multiple time points of the infection course. Using this model, we quantified and analyzed labeled protoscolices based on the intensities of their red and green fluorescence. Interestingly, the ratio of red to green fluorescence intensity not only revealed the location of protoscolices but also determined the viability of the parasites in vivo and in vivo tests. The noninvasive imaging model proposed in this work will be further studied for long-term detection and observation and may potentially be widely utilized in susceptibility testing and therapeutic effect evaluation.  相似文献   

15.
Fluorescence‐mediated tomography (FMT) enables noninvasive assessment of the three‐dimensional distribution of near‐infrared fluorescence in mice. The combination with micro‐computed tomography (µCT) provides anatomical data, enabling improved fluorescence reconstruction and image analysis. The aim of our study was to assess sensitivity and accuracy of µCT‐FMT under realistic in vivo conditions in deeply‐seated regions. Accordingly, we acquired fluorescence reflectance images (FRI) and µCT‐FMT scans of mice which were prepared with rectal insertions with different amounts of fluorescent dye. Default and high‐sensitivity scans were acquired and background signal was analyzed for three FMT channels (670 nm, 745 nm, and 790 nm). Analysis was performed for the original and an improved FMT reconstruction using the µCT data. While FRI and the original FMT reconstruction could detect 100 pmol, the improved FMT reconstruction could detect 10 pmol and significantly improved signal localization. By using a finer sampling grid and increasing the exposure time, the sensitivity could be further improved to detect 0.5 pmol. Background signal was highest in the 670 nm channel and most prominent in the gastro‐intestinal tract and in organs with high relative amounts of blood. In conclusion, we show that µCT‐FMT allows sensitive and accurate assessment of fluorescence in deep tissue regions.

  相似文献   


16.
Given that optical tomography is capable of quantitatively imaging the distribution of several important chromophores and fluorophores in vivo, there has been a great deal of interest in developing optical imaging systems with increased numbers of measurements under optimal experimental conditions. In this article, we present a novel system that enables three-dimensional imaging of fluorescent probes in whole animals using a noncontact setup, in parallel with a three-dimensional surface reconstruction algorithm. This approach is directed toward the in vivo imaging of fluorophore or fluorescent protein concentration in small animals. The system consists of a rotating sample holder and a lens-coupled charge-coupled device camera in combination with a fiber-coupled laser scanning device. By measuring multiple projections, large data sets can be obtained, thus improving the accuracy of the inversion models used for quantitative three-dimensional reconstruction of fluorochrome distribution, as well as facilitating a higher spatial resolution. In this study, the system was applied to determining the distribution of green fluorescent protein (GFP)-expressing T lymphocytes in a transgenic mouse model, thus demonstrating the potential of the system for studying immune system function. The technique was used to image and reconstruct fluorescence originating from 32 x 10(6) T cells in the thymus and 3 x 10(5) T cells in the spleen.  相似文献   

17.
Carbonic anhydrase IX (CA IX) is a transmembrane protein that has been shown to be greatly upregulated under conditions of hypoxia in many tumor cell lines. Tumor hypoxia is associated with impaired efficacy of cancer therapies making CA IX a valuable target for preclinical and diagnostic imaging. We have developed a quantitative in vivo optical imaging method for detection of CA IX as a marker of tumor hypoxia based on a near-infrared (NIR) fluorescent derivative of the CA IX inhibitor acetazolamide (AZ). The agent (HS680) showed single digit nanomolar inhibition of CA IX as well as selectivity over other CA isoforms and demonstrated up to 25-fold upregulation of fluorescent CA IX signal in hypoxic versus normoxic cells, which could be blocked by 60%–70% with unlabeled AZ. CA IX negative cell lines (HCT-116 and MDA-MB-231), as well as a non-binding control agent on CA IX positive cells, showed low fluorescent signal under both conditions. In vivo FMT imaging showed tumor accumulation and excellent tumor definition from 6–24 hours. In vivo selectivity was confirmed by pretreatment of the mice with unlabeled AZ resulting in >65% signal inhibition. HS680 tumor signal was further upregulated >2X in tumors by maintaining tumor-bearing mice in a low oxygen (8%) atmosphere. Importantly, intravenously injected HS680 signal was co-localized specifically with both CA IX antibody and pimonidazole (Pimo), and was located away from non-hypoxic regions indicated by a Hoechst stain. Thus, we have established a spatial correlation of fluorescence signal obtained by non-invasive, tomographic imaging of HS680 with regions of hypoxia and CA IX expression. These results illustrate the potential of HS680 and combined with FMT imaging to non-invasively quantify CA IX expression as a hypoxia biomarker, crucial to the study of the underlying biology of hypoxic tumors and the development and monitoring of novel anti-cancer therapies.  相似文献   

18.
In this article, a fluorescence lifetime imaging system for small animals is presented. Data were collected by scanning a region of interest with a measurement head, a linear fiber array with fixed separations between a single source fiber and several detection fibers. The goal was to localize tumors and monitor their progression using specific fluorescent markers. We chose a near-infrared contrast agent, Alexa Fluor 750 (Invitrogen Corp., Carlsbad, CA). Preliminary results show that the fluorescence lifetime for this dye was sensitive to the immediate environment of the fluorophore (in particular, pH), making it a promising candidate for reporting physiologic changes around a fluorophore. To quantify the intrinsic lifetime of deeply embedded fluorophores, we performed phantom experiments to investigate the contribution of photon migration effects on observed lifetime by calculating the fluorescence intensity decay time. A previously proposed theoretical model of migration, based on random walk theory, is also substantiated by new experimental data. The developed experimental system has been used for in vivo mouse imaging with Alexa Fluor 750 contrast agent conjugated to tumor-specific antibodies (trastuzumab [Herceptin]). Three-dimensional mapping of the fluorescence lifetime indicates lower lifetime values in superficial breast cancer tumors in mice.  相似文献   

19.
Considerable progress has been made in adapting existing and developing new technologies to enable increasingly detailed phenotypic information to be obtained in embryonic and newborn mice. Sophisticated methods for imaging mouse embryos and newborns are available and include ultrasound and magnetic resonance imaging (MRI) for in vivo imaging, and MRI, vascular corrosion casts, micro-computed tomography, and optical projection tomography (OPT) for postmortem imaging. In addition, Doppler and M-mode ultrasound are useful noninvasive tools to monitor cardiac and vascular hemodynamics in vivo in embryos and newborns. The developmental stage of the animals being phenotyped is an important consideration when selecting the appropriate technique for anesthesia or euthanasia and for labeling animals in longitudinal studies. Study design also needs to control for possible differences between inter- and intralitter variability, and for possible long-term developmental effects caused by anesthesia and/or procedures. Noninvasive or minimally invasive intravenous or intracardiac injections or blood sampling, and arterial pressure and electrocardiography (ECG) measurements are feasible in newborns. Whereas microinjection techniques are available for embryos as young as 6.5 days of gestation, further advances are required to enable minimally invasive fluid or tissue samples, or blood pressure or ECG measurements, to be obtained from mouse embryos in utero. The growing repertoire of techniques available for phenotyping mouse embryos and newborns promises to accelerate knowledge gained from studies using genetically engineered mice to understand molecular regulation of morphogenesis and the etiology of congenital diseases.  相似文献   

20.
4-[18F]Fluoro-L-m-tyrosine (FMT), a biochemical probe of striatal dopaminergic function, has been synthesized as an L-3,4-dihydroxyphenylalanine analog for positron emission tomography. Biochemical characterization of this compound in the rat 30 min after intrajugular administration indicated that in the brain, selective decarboxylation occurred in the striatum, with the formation of 4-fluoro-3-hydroxyphenylethylamine and its metabolites. Positron emission tomography analysis of brain tissue in monkeys (Macaca nemestrina) after intravenous injection of FMT revealed a true time-dependent, specific accumulation of radioactivity in striatum, with a striatum/cerebellum (nonspecific) ratio of 4 at 180 min. Peripheral metabolism accounted for less than 40% of the total radioactivity in arterial blood samples after 120 min. The amino acid remained as the major component throughout the period of investigation (n = 3; 5 min, 95%; 10 min, 85%; 30 min, 67%; 60 min, 62%; 120 min, 60%), with a plasma clearance t 1/2 of 112 min. 3-O-Methylated metabolites were not observed. The substrate specificity of FMT, coupled with its limited in vivo peripheral metabolism, makes it a useful, new biochemical probe for in vivo, noninvasive evaluation of central dopaminergic mechanisms.  相似文献   

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