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1.
Native neurofilaments were submitted to cross-linking reactions with bifunctional reagents (DMA, DMS and DSS) and to chemical reactions with sterically bulky reagents such as EEDQ and DTAF , as well as a glutaraldehyde-activated gel. The 160K and 70K neurofilament proteins reacted slightly more than the 210K neurofilament protein with DMS and DSS. The accessibility of the three neurofilaments to the other chemical reagents was identical. These results were unexpected since neurofilament antibodies seem to react preferentially with 210K protein which is at the periphery of the filament, whereas the 70K protein, which is the backbone of the filament, is probably buried inside the filament. In the same way, it has been shown that the side of the 210K proteins are probably able to cross link the neurofilaments with non covalent and covalent bridges. Using different cross link reagents, we did not observe a characteristic reactivity of the 210K protein towards the different chemicals. We conclude that the three neurofilament proteins are equally exposed to the different sterically bulky reagent and that part of the polypeptide chain of the 70K and the 160K proteins are located at the outside of the filament.  相似文献   

2.
The proteoglycan (PG) on the surface of NMuMG mouse mammary epithelial cells consists of at least two functional domains, a membrane- intercalated domain which anchors the PG to the plasma membrane, and a trypsin-releasable ectodomain which bears both heparan and chondroitin sulfate chains. The ectodomain binds cells to collagen types I, III, and V, but not IV, and has been proposed to be a matrix receptor. Because heparin binds to the adhesive glycoproteins fibronectin, an interstitial matrix component, and laminin, a basal lamina component, we asked whether the cell surface PG also binds these molecules. Cells harvested with either trypsin or EDTA bound to fibronectin; binding of trypsin-released cells was inhibited by the peptide GRGDS but not by heparin, whereas binding of EDTA-released cells was inhibited only by a combination of GRDS and heparin, suggesting two distinct cell binding mechanisms. In the presence of GRGDS, the EDTA-released cells bound to fibronectin via the cell surface PG. Binding via the cell surface PG was to the COOH-terminal heparin binding domain of fibronectin. In contrast with the binding to fibronectin, EDTA-released cells did not bind to laminin under identical assay conditions. Liposomes containing the isolated intact cell surface PG mimic the binding of whole cells. These results indicate that the mammary epithelial cells have at least two distinct cell surface receptors for fibronectin: a trypsin- resistant molecule that binds cells to the sequence RGD and a trypsin- labile, heparan sulfate-rich PG that binds cells to the COOH-terminal heparin binding domain. Because the cell surface PG binds cells to the interstitial collagens (types I, III, and V) and to fibronectin, but not to basal lamina collagen (type IV) or laminin, we conclude that the cell surface PG is a receptor on epithelial cells specific for interstitial matrix components.  相似文献   

3.
We present a study on the effects of cross-linking on the adhesive properties of bio-inspired 3,4-dihydroxyphenylalanine (DOPA). DOPA has a unique catechol moiety found in adhesive proteins in marine organisms, such as mussels and polychaete, which results in strong adhesion in aquatic conditions. Incorporation of this functional group in synthetic polymers provides the basis for pressure-sensitive adhesives for use in a broad range of environments. A series of cross-linked DOPA-containing polymers were prepared by adding divinyl cross-linking agent ethylene glycol dimethacrylate (EGDMA) to monomer mixtures of dopamine methacrylamide (DMA) and 2-methoxyethyl acrylate (MEA). Samples were prepared using a solvent-free microwave-assisted polymerization reaction and compared to a similar series of cross-linked MEA materials. Cross-linking with EGDMA tunes the viscoelastic properties of the adhesive material and has the advantage of not reacting with the catechol group that is responsible for the excellent adhesive performance of this material. Adhesion strength was measured by uniaxial indentation tests, which indicated that 0.001 mol % of EGDMA-cross-linked copolymer showed the highest work of adhesion in dry conditions, but non-cross-linked DMA was the highest in wet conditions. The results suggest that there is an optimal cross-linking degree that displays the highest adhesion by balancing viscous and elastic behaviors of the polymer but this appears to depend on the conditions. This concentration of cross-linker is well below the theoretical percolation threshold, and we propose that subtle changes in polymer viscoelastic properties can result in significant improvements in adhesion of DOPA-based materials. The properties of lightly cross-linked poly(DMA-co-MEA) were investigated by measurement of the frequency dependence of the storage modulus (G') and loss modulus (G'). The frequency-dependence of G' and magnitude of G' showed gradual decreases with the fraction of EGDMA. Loosely cross-linked DMA copolymers, containing 0% and 0.001 mol % of EGDMA-cross-linked copolymers, displayed rheological behavior appropriate for pressure-sensitive adhesives characterized by a higher G' at high frequencies and lower G' at low frequencies. Our results indicate that dimethacrylate cross-linking of DMA copolymers can be used to enhance the adhesive properties of this unique material.  相似文献   

4.
Experiments are described which show that while the presence of calcium in the medium is required for the cells to maintain their adhesion, it is not necessary for the initial attachment of 3T3 cells to solid substrates. Cells are detached by treatment with urea at 4 degrees C suggesting that adhesion may involve hydrogen bonding between the cell surface and the substratum. Although most of the cell-bound calcium is removed by trypsin, the detaching effect of trypsinisation can be inhibited at low temperature suggesting that ionic calcium bridges are probably not directly involved in retaining the cells on the surface. Cells are made totally insensitive to removal by trypsin by prior washing with lanthanum. Our findings suggest that the external role of calcium in cell adhesion is exerted indirectly. We conclude that the cell presents to the exterior at least two physiochemical classes of molecule. One class composed of hydrogen bond-forming adhesive material (possible proteins) and another class of anti-adhesive molecules (possibly glycoproteins). These two components are somehow separated in the formation of adhesive 'plaques' and this process is process is apparently uninfluenced by the calcium concentration in the medium. However, the maintenance of the localised zones of adhesion is aided by factors which prevent their disruption by the intrusion into them of anti-adhesive molecules diffusing from adjacent regions of the cell membrane. These factors include cooling below the transition temperature of the membrane lipids and lateral cross-linking of non-adhesive elements by calcium. By contrast, conditions which reduce the stability of the separation of adhesive and non-adhesive surface components would be expected to diminish the overall adhesiveness of cells to the substratum.  相似文献   

5.
The adhesive properties of Chinese hamster V79 cells were analyzed and characterized by various cell dissociation treatments. The comparisons of aggregability among cells dissociated with EDTA, trypsin + Ca2+, and trypsin + EDTA, revealed that these cells have two adhesion mechanisms, a Ca2+-independent and a Ca2+-dependent one. The former did not depend on temperature, whereas the latter occurred only at physiological temperatures. Both mechanisms were trypsin sensitive, but the Ca2+- dependent one was protected by Ca2+ against trypsinization. In morphological studies, the Ca2+-independent adhesion appeared to be a simple agglutination or flocculation of cells, whereas the Ca2+- dependent adhesion seemed to be more physiological, being accompanied by cell deformation resulting in the increase of contact area between adjacent cells. Lactoperoxidase-catalyzed iodination of cell surface proteins revealed that several proteins are more intensely labeled in cells with Ca2+-independent adhesiveness than in cells without that property. It was also found that a cell surface protein with a molecular weight of approximately 150,000 is present only in cells with Ca2+-dependent adhesiveness. The iodination and trypsinization of this protein were protected by Ca2+, suggesting its reactivity to Ca2+. Possible mechanisms for each adhesion property are discussed, taking into account the correlation of these proteins with cell adhesiveness.  相似文献   

6.
Study of the interaction between germ cells and Sertoli cells in vitro   总被引:1,自引:0,他引:1  
The nature of membrane components involved in the binding between Sertoli cells and pachytene spermatocytes in culture and the metabolic requirements for the binding to occur have been studied. Mild proteolytic digestion of germ cells by trypsin completely inhibited adhesion of germ cells to somatic monolayer. Protein synthesis and glycosylation were required to restore the adhesive properties of trypsin-treated germ cells, showing that surface molecules involved in the binding are glycoproteins. Trypsinization of germ cells after labelling causes a great reduction of several bands which become detectable again after 12 h of recovery from trypsin digestion. Among these, two bands with apparent molecular weight (MW) of 78 000 and 51 000 could be candidate components in cell adhesion.  相似文献   

7.
Summary A new approach was investigated to study the interaction between integrins and actin via intracytoplasmic proteins. Because intracellular processes are hampered by the limiting plasma membrane, we developed an in vitro model with cells perforated by a bacterial toxin, streptolysin O. The specific conditions for the use of permeabilized cells to study the intramolecular associations occurring at adhesion plaques are described. The two cell types used, HUVEC and CHO, showed that the choice of the perforation method is of great importance. After perforation of cells in a monolayer, 75±10% of the cells remained adherent to a fibronectin substrate; after perforation of cells in suspension, only 25±10% of the cells readhered. Specific conditions were required however to maintain these adhesive properties up to 4 h: the presence of 1 mM Mg++ in the medium was crucial, and it was necessary to layer the cells on a specific coat rather than a substitute such as gelatin. Immunofluorescence investigations of actin, talin and vinculin, and Normarsky differential interference contrast microscopy showed retention of focal adhesion plaques in perforated cells. Moreover, in perforated cells antibodies directed against actin led to actin disorganization, showing that our model of perforated cells in a monolayer can give new insight to adhesion study.  相似文献   

8.
Cross-linking Phytochrome to its Receptor in situ using Imidoesters   总被引:1,自引:0,他引:1  
Phytochrome can be cross-linked to a particulate fraction usingimidoesters, namely dimethy adipimidate (DMA) and dimethyl suberimidate(DMS). DMS was more effective than DMA. Cross-linking of phytochrometo its in situ receptor effected by DMS occurred in red light-irradiatedcoleoptiles. If DMS cross-linking was carried out prior to redlight irradiation there was very little formation of particulatephytochrome. Phytochrome in the particulate fraction obtainedby in situ DMS cross-linking was totally resistant to the solubilizingeffect of washing with solutions of high salt concentrationand high pH and was indistinguishable spectro-scopically fromthe phytochrome in untreated coleoptiles. DMS cross-linkingof phytochrome to its assumed receptor in situ preferentiallyprotected it from destruction following red light irradiationand also prevented it from dissociating from its receptor followingR/FR1 irradiation when incubated subsequently in the dark. Thesecharacteristics of phytochrome in DMS-treated coleoptiles matchedthose observed using glutaraldehyde as the cross-linking reagent.It is therefore concluded that earlier results obtained usingglutaraldehyde are not peculiar to that reagent and can be duplicatedreadily using more defined bifunctional cross-linkers.  相似文献   

9.
Substrate-attached material (SAM) prepared from murine BALB/c 3T3 cells and various derivatives contains adhesion sites which pinch off from the cell surface during EGTA-mediated detachment but which remain bound to the serum-coated tissue culture substratum. SAM contains the related adhesive glycoproteins cold-insoluble globulin (CIG) (from serum in the medium) and fibronectin (synthesized by the cells) as detected by immune staining of electrophoretically separated proteins, using antibodies of defined specificity. Serum and SAM contain cross-linked multimers of serum-derived CIG (not disulfide-mediated) but not of cell-derived fibronectin; therefore, thiol-resistant cross-linking between CIG and fibronectin is not involved in adhesion of these cells. Immunofluorescence microscopy of SAM from sparse cultures reveals fibrillar pools containing cellular fibronectin, although most retraction fibers seen on EGTA-treated cells do not stain, even after treatment with non-ionic detergent. Very little specific staining can be detected in SAM prepared from dense cultures, although gel electrophoretic analysis reveals proportionately as much murine fibronectin as is found in SAM from sparse cultures. Hyaluronidase digestion of SAM has no effect on the immunofluorescent staining, while gentle trypsin digestion completely abolishes staining without removing all biochemically detectable fibronectin. We conclude that some of the fibronectin and CIG in adhesion sites is masked and unavailable for antibody binding and that multiple pools of fibronectin exist in this adhesive material.  相似文献   

10.
Gräbner R  Till U  Heller R 《Cytometry》2000,40(3):238-244
BACKGROUND: Endothelial cell adhesion molecules are involved in initiation and progression of vascular diseases. The purpose of this study was to determine conditions of fixation and dissociation of human umbilical vein endothelial cell (HUVEC) monolayers that permit a reliable flow cytometric determination of intracellular and surface content of E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). METHODS: TNFalpha-treated HUVEC monolayers were fixed with 0.5% formaldehyde at the end of the experimental incubation. Subsequently, either the monolayer was trypsinized and thereafter the cells were subjected to indirect fluorescence labeling or the monolayer was first labeled and then dissociated by trypsinization. Cell integrity was assessed by vimentin staining. Total adhesion molecule content was detected in saponin-permeabilized cells. RESULTS: HUVEC integrity was maintained when the fixation time of the monolayer did not exceed 5 min and trypsin/EDTA was used for dissociation. Surface adhesion molecules were partially hydrolyzed by trypsin when trypsinization preceded labeling but antibody binding protected adhesion molecules from degradation. VCAM-1 and E-selectin exhibited substantial trypsin-sensitive surface fractions but surface ICAM-1 was mainly trypsin resistant. Permeabilization with 0.06% saponin allowed the detection of considerable intracellular pools of the investigated adhesion molecules. CONCLUSIONS: The described method permits the reliable determination of surface and intracellular fractions of adhesion molecules in formaldehyde-fixed HUVEC monolayers and may be used for studies on the regulation of adhesion molecule expression.  相似文献   

11.
Peritoneal carcinomatosis involves a series of events including tumor cell interactions with mesothelial cells and the extracellular matrix (ECM). We have studied the adhesive and invasive properties of four human colorectal carcinoma cell lines (Co115, HT29, SW480, SW620) confronted in vitro with a human mesothelial cell monolayer or with the ECM proteins collagen IV, laminin-1, fibronectin, tenascin-C and vitronectin. Quantitation was achieved following staining of tumor cells with the calcein-AM fluorescent dye. We found that all four cell lines rapidly adhered to a mesothelial cell monolayer. This adhesion event was not inhibitable by anti-integrin and anti-CD44 antibodies. Following initial attachment, the SW480 and SW620 cells invaded the mesothelial cell monolayer more aggressively than HT29 and Col 15 cells. All cell lines adhered to ECM proteins with each one exhibiting an individual adhesion pattern. Adhesion to matrix was completely integrin-dependent. When tested in an invasion assay, HT29 and Co115 cells crossed Matrigel-coated filters while SW480 and SW620 cells did not. This invasion was inhibited by anti-β1 integrin antibodies. Taken together, our results demonstrate that the initial colorectal tumor cell—mesothelial cell interaction occurs through an integrin-independent mechanism while adhesion to matrix proteins and invasion through Matrigel are integrin-dependent events. Furthermore, the different invasive capacity of SW480 and SW620 versus HT29 and Co115 cells upon interaction with a mesothelial cell monolayer or Matrigel suggests that these two invasion events may be mediated by distinct mechanisms.  相似文献   

12.
Peritoneal carcinomatosis involves a series of events including tumor cell interactions with mesothelial cells and the extracellular matrix (ECM). We have studied the adhesive and invasive properties of four human colorectal carcinoma cell lines (Co115, HT29, SW480, SW620) confronted in vitro with a human mesothelial cell monolayer or with the ECM proteins collagen IV, laminin-1, fibronectin, tenascin-C and vitronectin. Quantitation was achieved following staining of tumor cells with the calcein-AM fluorescent dye. We found that all four cell lines rapidly adhered to a mesothelial cell monolayer. This adhesion event was not inhibitable by anti-integrin and anti-CD44 antibodies. Following initial attachment, the SW480 and SW620 cells invaded the mesothelial cell monolayer more aggressively than HT29 and Col 15 cells. All cell lines adhered to ECM proteins with each one exhibiting an individual adhesion pattern. Adhesion to matrix was completely integrin-dependent. When tested in an invasion assay, HT29 and Co115 cells crossed Matrigel-coated filters while SW480 and SW620 cells did not. This invasion was inhibited by anti-β1 integrin antibodies. Taken together, our results demonstrate that the initial colorectal tumor cell—mesothelial cell interaction occurs through an integrin-independent mechanism while adhesion to matrix proteins and invasion through Matrigel are integrin-dependent events. Furthermore, the different invasive capacity of SW480 and SW620 versus HT29 and Co115 cells upon interaction with a mesothelial cell monolayer or Matrigel suggests that these two invasion events may be mediated by distinct mechanisms.  相似文献   

13.
Treatment of baby hamster kidney cells with cytochalasin B or omission of divalent cations from the culture medium are conditions resulting in an inhibition of cell attachment at rest; however, these conditions do not result in inhibition of cell attachment in a centrifugal field. In marked contrast, treatment of cells with trypsin or with tranquilizers such as fluphenazine results in an inhibition of cell attachment at rest or in a centrifugal field. The evidence is interpreted to indicate that cell adhesion involves at least two biochemical processes: formation of the adhesive bond per se (inhibited by tranquilizers or trypsin) and a mechanical process of cell-to-substratum contact and/or spreading (inhibited by cytochalasin B or omission of divalent cations from the medium).  相似文献   

14.
The mechanical and adhesive properties of cancer cells significantly change during tumor progression. Here we assess the functional consequences of mismatched stiffness and adhesive properties between neighboring normal cells on cancer cell migration in an epithelial-like cell monolayer. Using an in vitro coculture system and live-cell imaging, we find that the speed of single, mechanically soft breast carcinoma cells is dramatically enhanced by surrounding stiff nontransformed cells compared with single cells or a monolayer of carcinoma cells. Soft tumor cells undergo a mode of pulsating migration that is distinct from conventional mesenchymal and amoeboid migration, whereby long-lived episodes of slow, random migration are interlaced with short-lived episodes of extremely fast, directed migration, whereas the surrounding stiff cells show little net migration. This bursty migration is induced by the intermittent, myosin II-mediated deformation of the soft nucleus of the cancer cell, which is induced by the transient crowding of the stiff nuclei of the surrounding nontransformed cells, whose movements depend directly on the cadherin-mediated mismatched adhesion between normal and cancer cells as well as α-catenin-based intercellular adhesion of the normal cells. These results suggest that a mechanical and adhesive mismatch between transformed and nontransformed cells in a cell monolayer can trigger enhanced pulsating migration. These results shed light on the role of stiff epithelial cells that neighbor individual cancer cells in early steps of cancer dissemination.  相似文献   

15.
In a study performed on transformed (SGS/3A) and normal syngeneic rat cells (FG/2) to identify the molecular mechanisms which regulate cell adhesion and contact inhibition in cell transformation, we investigated the effects of tumor promoters on cell to cell adhesion of rat fibroblasts. As tumor promoters we used 12-tetradecanoyl-phorbole-13-acetate (TPA) and the melittin, a polypeptide from bee venom, both substances capable of stimulating the neoplastic transformation. The intercellular adhesion assay consists in determining the percent of single cells labeled with 3H-L-leucine adhering to a confluent monolayer of unlabeled cells at different incubation times. The increase of cell-cell adhesion caused by TPA and melittin confirms what we have constantly observed in other experiments, i.e. that neoplastic cells SGS/3A always have a higher intercellular adhesion capacity than corresponding normal syngenic cells FG/2. Since one of the effects of the tumor promoters is also induction of a reversible alteration of the cytoskeleton, it is likely that their action on intercellular adhesion is regulated by a mechanism analogous to that proposed for explaining the increased intercellular adhesion observed after treatment with antimicrotubular compounds.  相似文献   

16.
Proteases have been used as a tool to investigate the role of surface molecules in fibronectin-mediated cell adhesion. Proteolytic digestion of membrane-proteins by pronase (1 mg/ml for 20 min at 37 degrees C) completely inhibited adhesion of baby hamster kidney (BHK) fibroblasts on fibronectin-coated plastic dishes. Various degrees of inhibition were also obtained after treatment with proteinase K, chymotrypsin, papain, subtilopeptidase A, and thermolysin. Protein synthesis was required to restore the adhesive properties of pronase-treated cells, showing the protein nature of the molecules involved in adhesion to fibronectin. A peculiar feature of these proteins was their resistance to cleavage by trypsin. After prolonged trypsin treatment (1 mg/ml for 20 min at 37 degrees C), cells adhered and spread on fibronectin-coated dishes, even when protein synthesis was inhibited by 4 microM cycloheximide. Under these conditions only three glycoproteins (gp) of molecular weight 130,000, 120,000, and 80,000 were left on the cell surface. These were precipitated by a rabbit antiserum against BHK cells that also inhibited adhesion of trypsin-treated cells. gp120 and gp80 were left at the cell surface after mild pronase digestion (0.2 mg/ml for 20 min at 37 degrees C), under conditions not affecting adhesion. These data suggest that these glycoproteins may be involved in fibronectin-mediated cell adhesion in some yet unknown way.  相似文献   

17.
Summary Chromaffin cells in the adrenal medulla are found in close proximity to capillary endothelial cells, thereby forming the classical endocrine complex. To examine the possible chemical basis of their interaction in more detail, we have grown bovine adrenal medullary endothelial (BAME) cells in monolayer cultures and added to them pheochromocytoma (PC12) cells, a chromaffin tumor cell line of rats. The PC12 cells were chosen because of the similarities they share with adrenal medullary chromaffin cells. PC12 cells rapidly attached to BAME cells cultures, their rate of adhesion being significantly enhanced over binding of PC12 cells to either uncoated plates or to monolayers of unrelated cell cultures. Consistent with this observation, we noted that the extracellular matrix (ECM) derived from the BAME cells did not enhance PC12 cell adhesion and did not promote neurite sprouting as previously described for ECM derived from corneal endothelial cells. The specific adhesion between PC12 and BAME cells could be abolished by cell surface extracts derived from these two cells but not by extracts derived from unrelated cell types. This activity was heat-labile, sensitive to trypsin and, to a lesser extent, to neuraminidase. We therefore conclude that PC12 cells may interact with BAME cells by specific proteinaceous adhesive factors associated with their plasma membranes. These interactions might represent the formative role of cell-cell contacts in the organization of the developing adrenal gland.Abbreviations BAME bovine adrenal medullary endothelial cells - DMEM Dulbecco's modified essential medium - ECM extracellular matrix - EMEM Eagle's modified essential medium - FCS fetal calf serum - PBS phosphate-buffered saline - PC12 rat pheochromocytoma cells  相似文献   

18.
The spores of Bacillus subtilis show remarkable resistance to many environmental stresses, due in part to the presence of an outer proteinaceous structure known as the spore coat. GerQ is a spore coat protein essential for the presence of CwlJ, an enzyme involved in the hydrolysis of the cortex during spore germination, in the spore coat. Here we show that GerQ is cross-linked into higher-molecular-mass forms due in large part to a transglutaminase. GerQ is the only substrate for this transglutaminase identified to date. In addition, we show that cross-linking of GerQ into high-molecular-mass forms occurs only very late in sporulation, after mother cell lysis. These findings, as well as studies of GerQ cross-linking in mutant strains where spore coat assembly is perturbed, lead us to suggest that coat proteins must assemble first and that their cross-linking follows as a final step in the spore coat formation pathway.  相似文献   

19.
K Handa  Y Igarashi  M Nisar  S Hakomori 《Biochemistry》1991,30(50):11682-11686
GMP-140 (CD62 or PADGEM), a member of the selectin family, is a membrane glycoprotein in secretory granules of platelets and endothelial cells. When these cells are activated by agonists such as thrombin or AMP, GMP-140 is rapidly redistributed to the cell surface. The carbohydrate epitope defined by GMP-140 was identified as sialosyl-Le(x) (as for ELAM-1), which may play an essential role in adhesion of leukocytes or tumor cells on endothelial cells, through aggregation with platelets. Redistribution of GMP-140 from alpha-granules of platelets to the cell surface, induced by thrombin and PMA, was strongly inhibited by preincubation of platelets with N,N-dimethylsphingosine (DMS) or N,N,N-trimethylsphingosine (TMS) at 10-20 microM concentration for a brief period (5 min). Inhibition of GMP-140 redistribution to the cell surface by DMS or TMS was also detected by a cell adhesion assay using HL60 cells, which highly express sialosyl-Le(x); i.e., HL60 cells adhered on platelets activated by thrombin or PMA but not on platelets which were briefly preincubated with DMS or TMS followed by activation. The inhibitory effect of DMS or TMS on GMP-140 redistribution is not due to cytotoxicity, since the TMS-treated platelets were fully capable of aggregating in the presence of ristocetin. Sphingosine (SPN) and protein kinase C inhibitors such as H-7 and calphostin C showed weaker inhibitory activity than DMS and TMS. Our results indicate that both DMS and TMS could be useful reagents to inhibit cell surface expression of crucial selectins which promote adhesion of Le(x-) or sialosyl-Le(x)-expressing cells with platelets and endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

20.
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