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1.
The region in tendons that surrounds bone extremities adapts to compression forces, developing a fibrocartilaginous structure. During maturation, changes occur in the amount and organization of macromolecules of the extracellular matrix of tendons, changing the tissue morphology. To study the effect of maturation on tendons, Pedrês chickens were sacrificed at 1, 5, and 8 months old and had the calcaneal tendon (CT) divided into proximal region, submitted to tension/compression forces ( p ), and distal region submitted to tension force ( d ). Morphological analysis of the p region showed the presence of fibrocartilage in all ages. In the central part of the fibrocartilage, near a diminishment of the metachromasy, there was also a development of a probable fat pad that increased with the maturation. The activity of MMP‐2 and MMP‐9 was higher at 5 and 8 months old, in both regions, compared with 1‐month‐old animals. In SDS‐PAGE analysis, components with electrophoretic migration similar to decorin and fibromodulin increased with maturation, particularly in the d region. The Western blotting confirmed the increased amount of fibromodulin with maturation. In conclusion, our results show that process of maturation leads to the appearance of a probable fat pad in the center of the fibrocartilage of CT, with a reduced amount of glycosaminoglycans and an increased activity of MMPs. Microsc. Res. Tech. 78:949–957, 2015. © 2015 Wiley Periodicals, Inc.  相似文献   

2.
The plantaris longus tendon (PLT) in bullfrog develops a fibrocartilage‐like tissue in the area that is functionally subject to compressive forces. The aim of this study was to analyze the modifications of the pressure‐bearing region in bullfrog PLT with different ages (7, 180, and 1,080 days after the end of metamorphosis) using histomorphometric, ultrastructural and biochemical methods. Weak basophilia and cells with a fibroblastic phenotype were observed in the compression region at 7 days of age. On the other hand, a large area of intense tissue basophilia associated with a chondroblast‐like cell population was noted at the other ages. Collagen fibers exhibited a three‐dimensional network‐like arrangement at all ages. The number of connective tissue cells increased between 7 and 180 days of age and was reduced in older animals. The 180‐day‐old animals presented a well‐developed pericellular matrix rich in proteoglycans. The mean diameter of collagen fibrils increased from 7 to 180 days and was the same at 1,080 days. Glycosaminoglycan content was higher in 7‐day‐old animals. A higher amount of hydroxyproline was observed at 180 and 1,080 days. The swelling test showed a significant increase of wet weight in 7‐day‐old animals. In conclusion, the alterations that occur in the pressure‐bearing of bullfrog PLT are the result of physiological alterations of the animal with the maturation and aging. Microsc. Res. Tech. 77:797–805, 2014. © 2014 Wiley Periodicals, Inc.  相似文献   

3.
In some animals, such as mice and guinea pigs, a hormonally controlled mechanism increases the flexibility of the pubic symphysis and enhances the cervical remodeling necessary for safe delivery. Cervical ripening during pregnancy is associated with a paradoxical influx of leukocytes. However, the changes in cell metabolism during relaxation of the mouse pubic symphysis for delivery have not been extensively studied. In this work, we used light microscopy and transmission and scanning electron microcopy, as well as immunohistochemistry and Western blotting for MMP-8, to investigate the involvement of granulocytes or resident stromal cells in the relaxation of the virgin pubic symphysis during late pregnancy (days 18 and 19, before delivery) in vivo and in explanted joints. MMP-8 was studied because this collagenase is a hallmark for cervical ripening associated with the influx of granulocytes during late pregnancy. Extensive dissolution and disorganization of the extracellular matrix was seen around fibroblastic-like cells in late pregnancy. In contrast to the cervix (positive control), morphological and immunohistochemical analyses revealed that there was no characteristic cellular inflammatory response in the interpubic tissue. Staining for MMP-8 was observed in chondroid and fibroblastic-like cells of virgin and relaxed interpubic ligament, respectively. However, no granulocytes were seen during the extensive remodeling of the pubic joint in late pregnancy. These results indicate that constitutive stromal cells may have an important role in tissue relaxation during remodeling of the pubic symphysis in pregnancy.  相似文献   

4.
We have compared the biochemical expression of cholinergic enzymes with the morphological differentiation of efferent nerve fibers and endings in the cochlea of the postnatally developing mouse. Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) are present in the newborn cochlea at specific activities 63% and 25%, respectively, of their mature levels. The relative increases in ChAT, in AChE, and in its molecular forms over the newborn values start about day 4 and reach maturity by about day 10. The biochemical results correlate well with the massive presence of nerve fibers stained immunocytochemically for ChAT and AChE or enzymatically for AChE in the inner and outer hair cell regions. Ultrastructural studies, however, indicate the presence of only few vesiculated fibers and endings in the inner and outer hair cell regions. The appearance of large, cytologically mature endings occurs only toward the end of the third postnatal week. The discrepancy may be resolved in the electron microscope using the enzymatic staining for AChE. Labeling is seen on many nonvesiculated fibers and endings in the hair cell regions, suggesting that the majority of the efferent fibers in the perinatal organ may be biochemically differentiated but morphologically immature. The results may imply that the efferents to inner and outer hair cells develop earlier than indicated by previous ultrastructural studies. Moreover, the pattern of development suggests that in the cochlea, as in other tissues, the biochemical differentiation of the efferent innervation may precede the morphological maturation.  相似文献   

5.
Obstructive sleep apnea hypopnea syndrome (SAHS) is a complex disease of the upper respiratory airways. SAHS physiopathology is multifactorial in which airway compliance is a very important component. To evaluate the tissue changes in the palatopharyngeal muscle by morphometric, histochemical, immunohistochemical, and stereological quantification, with special attention to extracellular matrix associated with this muscle at the structural and ultrastructural levels. Thirty patients with SAHS were divided into groups of 10 according to disease severity: mild, moderate, and severe SAHS. In addition, the control group consisted of 10 patients. Fragments of palatopharyngeal muscle removed from patients with SAHS and tonsillectomies from patients in the control group were histopathologically submitted to light microscopy and transmission electron microscopy. Histopathological evaluations by light and transmission electron microscopes showed differences in analyzed groups, such as reduction of the muscle fiber diameter in patients with SAHS, taking disease severity into consideration. In contrast, stereological analysis showed a gradual increase of the collagen and elastic system fibers relative frequencies, proportionally to SAHS seriousness. MMP‐2 and MMP‐9 immunostaining also showed an increased reaction in the muscle fiber cytoplasm and endomisium during SAHS progression. The ultrastructural analysis showed that palatopharyngeal muscle fibers presented cytoplasmic residual corpuscles, a sign of early cell aging. In conclusion, the increase of tissue compliance in individuals with SAHS can be, in addition to other factors, consequence of diminished contractile activity of the muscle fibers, which exhibited clear signs of early senescence. Moreover, extracellular matrix components changes may contribute to muscle myopathy during SAHS progression. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.  相似文献   

6.
In order to further our understanding regarding the temporal and topographic ultrastructural aspects of the myocardium under thyrotoxicosis, thyroxine (T4; 25 and 100 microg/100 g bw) was administered to young rats 24 hours after birth until 15 days. The animals were then sacrificed, the hearts excised and weighed, and the ventricle tissue samples were then processed for confocal and transmission electron microscopy. At 48/72 hours and 1 week after initiation of T4 treatment with 100 microg/100 g bw, numerous lamellar bodies (probably formed by phospholipids) progressively accumulated in the heart. These bodies were observed in the cytosol, inside mitochondria and in the extracellular matrix. At 2 weeks of T4 treatment with 100 microg/100 g bw, lamellar bodies were virtually absent. Changes in cell shape, disorganization of intercellular junctions, and substantial myofibrillar disarray were observed in many cardiomyocytes. A gradient of myofibrillar disarray, which increased in abundance and intensity from the endocardium to the epicardium, was also observed. Immunocytochemical staining for desmin showed that the arrangement of this protein was disorganized in many cells of T4-treated rats as compared with normal ones, confirming ultrastructural data. The predominant appearance of myofibrillar disarray, associated with disorganization of cytoskeletal proteins in the deep myocardium, may be due to higher mechanical wall stress and consequent higher metabolic demand. Alternatively, differential sensitivity of cardiomyocytes to thyroid hormone in different areas is also a possibility.  相似文献   

7.
Round circomyarian fibres of leeches are peculiar helical muscles. The fibres are characterized by a lack of junctions, being separated by a thick extracellular matrix, and by scarce end-plates. Even so, the fibres grouped in units show the same degree of contraction. Biochemical, immunocytochemical and ultrastructural studies were performed in order: (a) to demonstrate the presence in the extracellular matrix of fibronectin, collagen type IV and laminin and in the cytoskeleton of desmin and α-actinin; (b) to show the possible link of extracellular matrix with the scaffold of intermediate filaments; (c) to evaluate how the extracellular matrix can play a role in the transduction of a signal during contraction–relaxation–superelongation phases.  相似文献   

8.
Implantation is one of the most regulated processes in human reproduction, by endocrine and immunological systems. Cytokines are involved in embryo-maternal communication and an impaired balance could result in pregnancy loss. Here we investigated the effect of interleukin 1-β on the activity of two important metalloproteinases (MMP-2 and MMP-9) that are involved in extracellular matrix remodeling as well as the secretion of leptin, one of the reproductive hormones actively regulating their activity and secretion. We found that IL-1β activates matrix metalloproteinase activity as well as increases leptin secretion. We propose that this interleukin, through the regulation of leptin, in turn activates matrix metalloproteinases which results in an increased cytotrophoblast invasion.  相似文献   

9.
The recent booming of multiphoton imaging of collagen fibrils by means of second harmonic generation microscopy generates the need for the development and automation of quantitative methods for image analysis. Standard approaches sequentially analyse two-dimensional (2D) slices to gain knowledge on the spatial arrangement and dimension of the fibrils, whereas the reconstructed three-dimensional (3D) image yields better information about these characteristics. In this work, a 3D analysis method is proposed for second harmonic generation images of collagen fibrils, based on a recently developed 3D fibre quantification method. This analysis uses operators from mathematical morphology. The fibril structure is scanned with a directional distance transform. Inertia moments of the directional distances yield the main fibre orientation, corresponding to the main inertia axis. The collaboration of directional distances and fibre orientation delivers a geometrical estimate of the fibre radius. The results include local maps as well as global distribution of orientation and radius of the fibrils over the 3D image. They also bring a segmentation of the image into foreground and background, as well as a classification of the foreground pixels into the preferred orientations. This accurate determination of the spatial arrangement of the fibrils within a 3D data set will be most relevant in biomedical applications. It brings the possibility to monitor remodelling of collagen tissues upon a variety of injuries and to guide tissues engineering because biomimetic 3D organizations and density are requested for better integration of implants.  相似文献   

10.
The use of non‐standard low‐temperature conditions in environmental scanning electron microscopy might be promising for the observation of coniferous tissues in their native state. This study is aimed to analyse and evaluate the method based on the principle of low‐temperature sample stabilization. We demonstrate that the upper mucous layer is sublimed and a microstructure of the sample surface can be observed with higher resolution at lower gas pressure conditions, thanks to a low‐temperature method. An influence of the low‐temperature method on sample stability was also studied. The results indicate that high‐moisture conditions are not suitable for this method and often cause the collapse of samples. The potential improvement of stability to beam damage has been demonstrated by long‐time observation at different operation parameters. We finally show high applicability of the low‐temperature method on different types of conifers and Oxalis acetosella. Microsc. Res. Tech., 78:13–21, 2015. © 2014 Wiley Periodicals, Inc.  相似文献   

11.
In order to investigate the ultrastructural three-dimensional relationship between extracellular matrices (ECM) and the plasma membranes of cultured cells, a freeze-etch replica method was devised. Bovine corneal endothelial cells were cultured on a Collodion film which covered a hole punched in a plastic coverslip, and were quickly frozen with a slammer with their basal surface facing a liquid nitrogen-cooled copper block. The cells were placed upside-down in a Balzers freeze-fracture machine and freeze-etched, and then platinum-carbon replicas were obtained. The structure of the ECM-plasma membrane interface was observed successfully and so this technique provides a new approach for investigating the ECM-plasma membrane (matrix-receptor) relationship.  相似文献   

12.
The remodeling of extracellular matrices by cells plays a defining role in developmental morphogenesis and wound healing, as well as in tissue engineering. Three-dimensional (3-D) type I collagen matrices have been used extensively as an in vitro model for studying cell-induced matrix reorganization at the macroscopic level. However, few studies have directly assessed the dynamic process of 3-D matrix remodeling at the cellular and subcellular level. We recently developed an experimental model for investigating cell-matrix mechanical interactions by plating green fluorescen protein (GFP)-zyxin transfected cells inside fibrillar collagen matrices and performing high-magnification time-lapse differential interference microscopy (DIC) and wide-field fluorescent imaging. In this study, we extend this experimental model by performing four-dimensional (4-D) reflected light and fluorescent confocal imaging (using either visible light or multiphoton excitation) of living corneal fibroblasts transfected to express GFP-zyxin or GFP-alpha-actinin, 18 h after plating inside 3-D collagen matrices. Reflected light confocal imaging allowed detailed visualization of the cells and the fibrillar collagen surrounding them. By overlaying maximum intensity projections of reflected light and GFP-zyxin or GFP-alpha-actinin images and generating stereo pair reconstructions, 3-D interactions between focal adhesions and collagen fibrils in living cells could be visualized directly. Focal adhesions were generally oriented parallel to the direction of collagen fibril alignment in front of the cell. Killing the cells induced relaxation of transient cell-induced tension on the matrix; however, significant permanent remodeling always remained. Time-lapse 3-D imaging demonstrated an active response to the Rho-kinase inhibitor Y-27632, as indicated by cell elongation, extracellular matrix relaxation, and extension of pseudopodial processes. It is interesting that, at higher cell densities, groups of collagen fibrils were compacted and aligned into straps between neighboring cells. Overall, the continued development and application of this new approach should provide important insights into the basic underlying biochemical and biomechanical regulatory mechanisms controlling matrix remodeling by corneal fibroblasts.  相似文献   

13.
Observation of heat‐deproteinized cortical bone specimens in incident light enabled the high definition documentation of the osteonal pattern of diaphyseal Haversian bone. This prompted a study to compare these images with those revealed by polarized light microscopy, carried out either on decalcified or thin, undecalcified, resin‐embedded sections. Different bone processing methods can reveal structural aspects of the intercellular matrix, depending on the light diffraction mode: birefringency in decalcified sections can be ascribed to the collagen fibrils orientation alone; in undecalcified sections, to both the ordered layout of collagen and the inorganic phase; in the heat‐deproteinized samples, exclusively to the hydroxyapatite crystals aggregation mode. The elemental chemical analysis documented low content of carbon and hydrogen, no detectable levels of nitrogen and significantly higher content of calcium and phosphorus in heat‐deproteinized samples, as compared with dehydrated controls. In both samples, the X‐ray diffraction (XRD) pattern did not show any significant difference in pattern of hydroxyapatite, with no peaks of any possible decomposition phases. Scanning electron microscopic (SEM) morphology of heat‐deproteinized samples could be documented with the fracturing technique facilitated by the bone brittleness. The structure of crystal aggregates, oriented in parallel and with marks of time periods, was documented. Comparative study of deproteinized and undecalcified samples showed that the matrix inorganic phase did not undergo a coarse grain thermal conversion until it reached 500°C, maintaining the original crystals structure and orientation. Incident light stereomicroscopy, combined with SEM analysis of deproteinized bone fractured surfaces, is a new enforceable technique which can be used in morphometric studies to improve the understanding of the osteonal dynamics. Microsc. Res. Tech. 79:691–699, 2016. © 2016 Wiley Periodicals, Inc.  相似文献   

14.
Doxazosin (DOX), an α‐adrenoceptor antagonist, induces the relaxation of smooth muscle cell tonus and reduces the clinical symptoms of benign prostatic hyperplasia (BPH). However, the effects of DOX in the prostate stromal microenvironment are not fully known. In a previous study, we showed that DOX treatment for 30 days increased deposition of collagen fibers in the three rat prostatic lobes. Herein, we investigated the effects of DOX on stromal cell ultrastructure and elastic fiber deposition. Adult Wistar rats were treated with DOX (25 mg/kg/day); and the ventral, dorsal, and anterior prostates were excised at 30 days of treatment. The prostatic lobes were submitted to histochemical and stereological‐morphometric analyze and transmission electron microscopy (TEM). Histochemical staining plus stereological analysis of the elastic fiber system showed that DOX‐treated prostatic lobes presented more elaunin and elastic fibers than controls, mainly in the ventral lobe. Ultrastructural analysis showed that fibroblasts and smooth muscle cells from DOX‐treated prostates presented active synthetic phenotypes, evidenced by enlarged rough endoplasmic reticulum and Golgi apparatus cisterns, and confirmed the observation of thickened elaunin fibers. Our findings suggest that, under α‐adrenergic blockade by DOX, the fibroblasts become more active and smooth muscle cells shift from a predominantly contractile to a more synthetic phenotype. The deposition of collagen and elastic system fibers in the prostatic stroma may counterbalance the absence of smooth muscle tone during α‐blockers treatment. Microsc. Res. Tech. 73:1036–1044, 2010. © 2010 Wiley‐Liss, Inc.  相似文献   

15.
A variety of microscopical techniques and molecular probes have been used to study the ultrastructure and composition of the cell surfaces of the conidia (i.e. spores) and infection structures produced by the hemibiotrophic fungal plant pathogen Colletotrichum lindemuthianum. The fungal conidium germinates to produce a germ-tube, the tip of which swells to produce a domed, melanized appressorium which adheres firmly to the plant surface. Penetration of the cuticle and cell wall is followed by the development of a biotrophic intracellular hypha, which is surrounded by an invagination of the host plasma membrane. Freeze-substitution of C. lindemuthianum germlings showed that conidia are coated with a dense layer of fibrillar material. This 'spore coat' contains irregularly shaped pores, giving it a reticular appearance. Negative staining of germlings revealed the presence of numerous long, flexuous fibres or fimbriae, protruding from the surfaces of germ-tubes and appressoria. Colloidal gold was used to visualize fungal extracellular proteins. The colloidal gold stained a fibrillar sheath around germ-tubes, whereas appressoria were surrounded by a halo, comprising an inner unstained region and a stained perimeter. The carbohydrate composition of the cell surfaces of the conidia and infection structures was studied by labelling cells with rhodamine- and fluorescein-conjugated lectins. The results showed that the extracellular matrices of germ-tubes and appressoria are very similar in composition, but differ from those of conidia and intracellular hyphae. Monoclonal antibodies have been prepared to germlings and infection structures of C. lindemuthianum and their use has provided further evidence that the extracellular matrices around germ-tubes and appressoria have several glycoproteins in common. The results also show that the cell surface of C. lindemuthianum becomes specialized during biotrophic development inside host cells.  相似文献   

16.
Migration, invasion and protease activity are essential for tumor progression and metastasis. Metastatic cells rely on invadopodia to degrade and invade extracellular matrix (ECM). Invadopodia are membrane protrusions with enzymes required for ECM degradation. These protrusions contain cortactin and membrane type 1 matrix metalloproteinase (MT1‐MMP) superimposed to areas of digested matrix. Here we characterized invadopodia in a cell line (CAC2) derived from human adenoid cystic carcinoma. We carried out fluorescent‐substrate degradation assay to assess in situ protease activity of CAC2 cells. Digestion spots in fluorescent substrate appear as black areas in green background. Cells were cultured on Matrigel‐gelatin‐FITC and fixed after 1 h and 3 h. CAC2 cells were double labeled to actin and cortactin. Cells were also double stained to actin and MT1‐MMP. Samples were studied by laser scanning confocal microscopy. In all time points CAC2 cells showed actin, cortactin, and MT1‐MMP colocalized with digestion spots in fluorescent substrate. We searched for other proteases involved in invadopodia activity. We have previously demonstrated that MMP9 influences adenoid cystic carcinoma behavior. This prompted us to investigate role played by MMP9 on invadopodia formation. CAC2 cells had MMP9 silenced by siRNA. After 1 h in fluorescent substrate, cells with silenced MMP9 showed clear decrease in matrix digestion compared with controls. No differences were found in cells with silenced MMP9 grown for 3 h on fluorescent substrate. Our results showed that CAC2 cells exhibit functional invadopodia containing cortactin and MT1‐MMP. Furthermore, MMP9 would be required in the initial steps of invadopodia formation. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.  相似文献   

17.
18.
The aim of this study was to assess the dynamics of osteoclast migration and the degradation of unmineralized extracellular matrix in an osteolytic metastasis by examining a well-standardized lung cancer metastasis model of nude mice. SBC-5 human lung small carcinoma cells were injected into the left cardiac ventricle of 6-week-old BALB/c nu/nu mice under anesthesia. At 25-30 days after injection, the animals were sacrificed and their femora and/or tibiae were removed for histochemical analyses. Metastatic lesions were shown to occupy a considerable area extending from the metaphyses to the bone marrow region. Tartrate resistant acid phosphatase (TRAPase)-positive osteoclasts were found in association with an alkaline phosphatase (ALPase)-positive osteoblastic layer lining the bone surface, but could also be localized in the ALPase-negative stromal tissues that border the tumor nodules. These stromal tissues were markedly positive for osteopontin, and contained a significant number of TRAPase-positive osteoclasts expressing immunoreactivity for CD44. We thus speculated that, mediating its affinity for CD44, osteopontin may serve to facilitate osteoclastic migration after their formation associated with ALPase-positive osteoblasts. We next examined the localization of cathepsin K and matrix metallo-proteinase-9 (MMP-9) in osteoclasts. Osteoclasts adjacent to the bone surfaces were positive for both proteins, whereas those in the stromal tissues in the tumor nests showed only MMP-9 immunoreactivity. Immunoelectron microscopy disclosed the presence of MMP-9 in the Golgi apparatus and in vesicular structures at the baso-lateral cytoplasmic region of the osteoclasts found in the stromal tissue. MMP-9-positive vesicular structures also contained fragmented extracellular materials. Thus, osteoclasts appear to either select an optimized function, namely secreting proteolytic enzymes from ruffled borders during bone resorption, or recognize the surrounding extracellular matrix by mediating osteopontin/CD44 interaction, and internalize the extracellular matrices. Microsc.  相似文献   

19.
The adaptations of the mouse uterus to pregnancy include extensive modifications of the cells and extracellular matrix of the endometrial connective tissue that surround the embryos. Around each implanted embryo this tissue redifferentiates into a transient structure called decidua, which is formed by polygonal cells joined by intercellular junctions. In the mouse, thick collagen fibrils with irregular profile appear in decidualized areas of the endometrium but not in the nondecidualized stroma and interimplantation sites. The fine organization of these thick fibrils has not yet been established. This work was addressed to understand the arrangement and fine structure of collagen fibrils of the decidua of pregnant mice during the periimplantation stage. Major modifications occurred in collagen fibrils that surrounded decidual cells: (1) the fibrils, which were arranged in parallel bundles in nonpregnant animals, became organized as baskets around decidual cells; (2) very thick collagen fibrils with very irregular profiles appeared around decidual cells. Analysis of replicas and serial sections suggests that the thick collagen fibrils form by the lateral aggregation of thinner fibrils to a central fibril resulting in very irregular profile observed in cross sections of thick fibrils. The sum of modifications of the collagen fibrils seem to represent an adaptation of the endometrium to better support the decidual cells while they hold the embryos during the beginning of their development. The deposition of thick collagen fibrils in the decidua may contribute to form a barrier that impedes leukocyte migration within the decidua, preventing immunological rejection of genetically dissimilar embryonic tissues.  相似文献   

20.
This article reviews the biochemical, physiological, and experimental data cumulated during the last decade on the Meissner and Pacinian corpuscles. It includes information about (i) the localization of molecules recently detected in sensory corpuscles; (ii) the unsolved problem of the accessory fibers in sensory corpuscles and the occurrence of myelin within them; (iii) the development of sensory corpuscles, especially their neuronal and growth factor dependency; (iv) the composition and functional significance of the extracellular matrix as an essential part of the mechanisms involved in the genesis of the stimuli generated in sensory corpuscles; (v) the molecular basis of mechanotransduction; (vi) a miscellaneous section containing sparse new data on the protein composition of sensory corpuscles, as well as in the proteins involved in live–death cell decisions; (vii) the changes in sensory corpuscles as a consequence of aging, the central, or peripheral nervous system injury; and finally, (viii) the special interest of Meissner corpuscles and Pacinian corpuscles for pathologists for the diagnosis of some peripheral neuropathies and neurodegenerative diseases. Microsc. Res. Tech., 2009. © 2008 Wiley‐Liss, Inc.  相似文献   

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