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1.
Summary The aim of this study was to identify the G6PDH-active sinusoidal cells in the rat liver described by Rieder et al. (1978). Because of their number and distribution in the liver parenchyma, endothelial cells and pit cells could be excluded. Fat-storing cells were specifically marked by vital staining with vitamin A and identified by fluorescence microscopy. Kupffer cells could be detected after vital staining with carmine. Both staining methods allowed a subsequent incubation for the demonstration of G6PDH activity in the same unfixed cryostat section. Whereas more than 80% of the fluorescent particles were found outside the enzyme-positive cells, all G6PDH-active cells contained carmine particles. After counting the G6PDH-active cells, an estimation of 0.217 × 108 cells/g liver tissue was obtained. The results indicate that high G6PDH activity is common to all Kupffer cells, and is therefore a highly specific marker enzyme for this class of sinusoidal liver cells.The essential parts of this study will be presented as an Inaugural-Dissertation to the Medical Faculty of the University of Freiburg by W. HosemannSupported by a grant from the SFB 46 (Molgrudent)  相似文献   

2.
The endocytosis of latex particles (0.33, 0.46 and 0.80 micron in diameter) in the sinusoidal endothelial and Kupffer cells of the rat liver was studied electron microscopically. When the liver was perfused with serum-free oxygenated Krebs Ringer bicarbonate, latex particles of all three sizes were taken up by the endothelial cells. After a 10-min perfusion, particles were incorporated by the luminal cell surface of the perikarya or of the thick portion of the endothelial cells. A large patch of bristle coat was surrounding the ingested particle. The number of ingested particles in the endothelial cells, however, was much less than in the Kupffer cells. In in vivo experiments, no endocytosis of the latex particles was observed in the endothelial cells. In the Kupffer cells, particles were engulfed by the ruffled membranes or sank into the cytoplasm without a large patch of the bristle coat both in the perfusion system and in vivo. These observations show that at least 0.80 micron latex particles are taken up by the bristle-coated membranes in the sinusoidal endothelial cells of the perfused liver. The endocytic mechanism for latex particles in the endothelial cells is different from that of the Kupffer cells.  相似文献   

3.
S Yokota  H Tsuji  K Kato 《Histochemistry》1985,82(2):141-148
Light and electron microscopic localization of cathepsin D in rat liver was investigated by post-embedding immunoenzyme and protein A-gold techniques. By light microscopy, cytoplasmic granules of parenchymal cells and Kupffer cells were stained for cathepsin D. Weak staining was also noted in sinusoidal endothelial cells. In the parenchymal cells many of positive granules located around bile canaliculi. In the Kupffer cells and the endothelial cells, diffuse staining was noted in the cytoplasm in addition to granular staining. By electron microscopy, gold particles representing the antigenic sites for cathepsin D were seen in typical secondary lysosomes and some multivesicular bodies of the parenchymal cells and Kupffer cells. The lysosomes of the endothelial cells and fat-storing cells were weakly labeled. Quantitative analysis of the labeling density in the lysosomes of these three types of cells demonstrated that the lysosomes of parenchymal cells and Kupffer cells are main containers of cathepsin D in rat liver. The results suggest that cathepsin D functions in the intracellular digestive system of parenchymal cells and Kupffer cells but not so much in that of the endothelial cells.  相似文献   

4.
Summary Light and electron microscopic localization of cathepsin D in rat liver was investigated by post-embedding immunoenzyme and protein A-gold techniques. By light microscopy, cytoplasmic granules of parenchymal cells and Kupffer cells were stained for cathepsin D. Weak staining was also noted in sinusoidal endothelial cells. In the parenchymal cells many of positive granules located around bile canaliculi. In the Kupffer cells and the endothelial cells, diffuse staining was noted in the cytoplasm in addition to granular staining. By electron microscopy, gold particles representing the antigenic sites for cathepsin D were seen in typical secondary lysosomes and some multivesicular bodies of the parenchymal cells and Kupffer cells. The lysosomes of the endothelial cells and fat-storing cells were weakly labeled. Quantitative analysis of the labeling density in the lysosomes of these three types of cells demonstrated that the lysosomes of parenchymal cells and Kupffer cells are main containers of cathepsin D in rat liver. The results suggest that cathepsin D functions in the intracellular digestive system of parenchymal cells and Kupffer cells but not so much in that of the endothelial cells.  相似文献   

5.
Beta-glucuronidase and N-AS-D-chloroacetate esterase cytochemistry have been applied to rat liver sinusoidal endothelial cells and Kupffer cells. Both staining procedures allowed a clear-cut differentiation of either cell type. Kupffer cells which had been stained with beta-glucuronidase showed a positive reaction, whereas sinusoidal endothelial cells were completely negative. If the chloroacetate reaction was used, the former stained diffusely while the latter showed a characteristic granular staining pattern. Identity and purity of sinusoidal endothelial cells and Kupffer cells was validated by transmission and scanning electron microscopy as well as by the pattern of released eicosanoids which is characteristic for either cell type. These two staining techniques are a valuable addition to the peroxidase reaction commonly applied for differentiation.  相似文献   

6.
Beta-glucuronidase and N-AS-D-chloroacetate esterase cytochemistry have been applied to rat liver sinusoidal endothelial cells and Kupffer cells. Both staining procedures allowed a clear-cut differentiation of either cell type. Kupffer cells which had been stained with beta-glucuronidase showed a positive reaction, whereas sinusoidal endothelial cells were completely negative. If the chloroacetate reaction was used, the former stained diffusely while the latter showed a characteristic granular staining pattern. Identity and purity of sinusoidal endothelial cells and Kupffer cells was validated by transmission and scanning electron microscopy as well as by the pattern of released eicosanoids which is characteristic for either cell type. These two staining techniques are a valuable addition to the peroxidase reaction commonly applied for differentiation.  相似文献   

7.
Formaldehyde treated albumin (F-HSA) was found to consist of a monomeric and a polymeric fraction. Both fractions were primarily endocytosed by rat liver sinusoidal cells. However, immunohistochemical staining of endocytosed material showed that the relative contribution of the endothelial and Kupffer cells in uptake of the monomer and the polymer differed significantly, with the monomer mainly having an endothelial cell- and the polymer predominantly having a Kupffer cell pattern of distribution. To directly confirm these heterogeneous patterns, we injected in vivo the 125I-labeled F-HSA fractions and isolated the endothelial and Kupffer cells by centrifugal elutriation. 73.7% of the monomeric F-HSA was found in endothelial cells and only 14.9% was found in Kupffer cells. In contrast, the polymeric F-HSA (1500 kD) was mainly endocytosed by Kupffer cells (71%), whereas the endothelial cells contributed only for 24% in hepatic uptake. In vivo studies and isolated perfused rat liver experiments showed that endocytosis of both monomer and polymer was inhibited by co-administration of polyinosinic acid, a well known inhibitor for scavenger receptors, indicating that these receptors on endothelial and Kupffer cells are mainly involved in this uptake process.  相似文献   

8.
Morphological characteristics of the interaction of low density lipoproteins (LDL) and acetylated low density lipoproteins (AcLDL) with rat liver cells are described. These liver cell types are mainly responsible for the catabolism of these lipoproteins in vivo. Isolated rat liver Kupffer, endothelial, and parenchymal cells were incubated with LDL or AcLDL conjugated to 20 nm colloidal gold. LDL was mainly internalized by Kupffer cells, whereas AcLDL was predominantly found in endothelial cells. Kupffer and endothelial cells displayed different morphological characteristics in the processing of these lipoproteins. Kupffer cells bound LDL at uncoated regions of the plasma membrane often at the base of pseudopodia, and internalized the particles via small smooth vesicles. These uptake characteristics differ from the classical LDL uptake pathway, as described for other cell types, and may be related to the unique recognition properties of the receptor of Kupffer cells as observed in biochemical studies. Liver endothelial cells bound AcLDL in coated pits, followed by rapid uptake. Uptake proceeded through small coated vesicles, and after 5 min of incubation large (600-1200 nm) electron-lucent vacuoles (endosomes) with AcLDL-gold particles arranged along the membrane region were present. The endosomes were often associated closely with the cell membrane which might enable direct recycling of AcLDL receptors. These observations might explain the high efficiency of these cells in the processing of modified LDL in vivo.  相似文献   

9.
We studied the simultaneous binding of galactose and mannose-exposing ligands in sinusoidal rat liver cells during development and aging. The galactose-specific receptors were visualized using 17 nm diameter colloidal gold particles coupled with Lactosylated bovine serum albumine (LacBSA), while mannose-specific receptors were localized by means of 5 nm diameter particles adsorbed with mannan. We observed the presence of four different classes of Kupffer cells in relation to the ligands bound. The percentage of each group of Kupffer cells varied in relation to the age of the subject from which the sample was taken. There were few double-labelled cells in the livers from newborn rats, with numbers increasing with age to adulthood, and decreasing again in the older animals. Cells without labelling were in the majority after birth, but they decreased in number up to adulthood and increased again during subsequent aging. The numbers of single-labelled cells did not change significantly during liver maturation. We hypothesize that the exposition of galactose and mannose-specific receptorial systems is regulated by developmental conditions.  相似文献   

10.
To determine whether hepatic sinusoidal cells contain glucagon receptors and, if so, to study the significance of the receptors in the cells, binding of [125I]-glucagon to nonparenchymal cells (mainly endothelial cells and Kupffer cells) isolated from mouse liver was examined by quantitative autoradiography and biochemical methods. Furthermore, the pathway of intracellular transport of colloidal gold-labeled glucagon (AuG) was examined in vivo. Autoradiographic and biochemical results demonstrated many glucagon receptors in both endothelial cells and Kupffer cells, and more receptors being present in endothelial cells than in Kupffer cells. In vivo, endothelial cells internalized AuG particles into coated vesicles via coated pits and transported the particles to endosomes, lysosomes, and abluminal plasma membrane. Therefore, receptor-mediated transcytosis of AuG occurs in endothelial cells. The number of particles present on the abluminal plasma membrane was constant if the amount of injected AuG increased. Therefore, the magnitude of receptor-mediated transcytosis of AuG appears to be regulated by endothelial cells. Kupffer cells internalized the ligand into cytoplasmic tubular structures via plasma membrane invaginations and transported the ligand exclusively to endosomes and lysosomes, suggesting that the ligand is degraded by Kupffer cells.  相似文献   

11.
We studied the kinetics of hepatic uptake of liposomes during serum-free recirculating perfusion of rat livers. Liposomes consisted of phosphatidylcholine, cholesterol and phosphatidylserine in a 6:4:0 or a 3:4:3 molar ratio and were radiolabelled with [3H]cholesteryl oleyl ether. The negatively charged liposomes were taken up to a 10-fold higher extent than the neutral ones. Hepatic uptake of fluorescently labelled liposomes was examined by fluorescence microscopy. The neutral liposomes displayed a typical Kupffer cell distribution pattern, in addition to weak diffuse staining of the parenchyma, while the negatively charged liposomes showed a characteristic sinusoidal lining pattern, consistent with an endothelial localization. In addition, scattered Kupffer cell staining was distinguished as well as diffuse parenchymal fluorescence. The mainly endothelial localisation of the negatively charged liposomes was confirmed by determining radioactivity in endothelial and Kupffer cells isolated following a 1-h perfusion. Perfusion in the presence of polyinosinic acid, an inhibitor of scavenger receptor activity, reduced the rate of uptake of the negatively charged liposomes twofold, indicating the involvement of this receptor in the elimination mechanism. These results are compatible with earlier in vitro studies on liposome uptake by isolated endothelial cells and Kupffer cells, which showed that in the absence of serum also endothelial cells in situ are able to take up massive amounts of negatively charged liposomes. The present results emphasize that the high in vitro endothelial cell uptake in the absence of serum from earlier observations was not an artifact induced by the cell isolation procedure.  相似文献   

12.
The surface ultrastructure of Kupffer cells in the rat liver has been studied by scanning electron microscopy (SEM). The results demonstrate that Kupffer cells are both significantly different and clearly distinct from endothelial cells. Kupffer cells have neither pores (and/or "sieve plates") nor fenestrations, all of which are present in endothelial cells. They possess a stellate shape, and only indirectly, with slender and irregular evaginations, contribute to the lining of the sinusoidal wall. Furthermore, the luminal surface in some areas contains a large population of short microvilli, microphicae and invaginations. These elements form a kind of microlabyrinth which may correpond to the "worm-like" structures described by transmission electron microscopy (TEM). In the present study, transition forms between endothelial and Kupffer cells were never found. On the contrary, considering the highly fenestrated nature of the endothelial cells, the Kupffer cells may, by ameboid movements, easily cross the overlapping barrier of the sinusoid and protrude into the lumen. Thus, acting as activated macrophages, the Kupffer cells might function to prevent the entrance of foreign material into the tissues of the liver through the fragile and highly fenestrated endothelium. Finally, the topographical reconstruction of the sinusoid by correlated SEM and TEM studies demonstrates the Kupffer cells, with their protruding cytoplasm and ability to extend into the lumen of the sinusoid, may actually change the caliber of the vessel, and thus function as a "sphincter" which causes a temporary arrest of the blood flow when the diameter of the sinusoidal lumen is reduced.  相似文献   

13.
Summary In the present study we have localized neutral phosphatase, acid phosphatase, alkaline phosphatase and 5 nucleotidase in the sinusoidal cells of rat liver using enzyme cytochemistry at light and electron microscopical level.Neutral phosphatase was present in the endoplasmic reticulum and nuclear envelope of parenchymal cells and of sinusoidal endothelial, Kupffer and fat-storing cells. The intensity of the neutral phosphatase reaction was stronger in sinusoidal than in parenchymal cells. Sinusoidal cells were devoid of cytochemically demonstrable alkaline phosphatase. Abundant acid phosphatase was present in the many lysosomes of endothelial and Kupffer cells. Substanually less acid phosphatase-positive lysosomes were found in fat-storing cells. 5 nucleotidase was present on the cell membrane of fat-storing cells, on 90% of all Kupffer cells and on the microvilli of parenchymal cells.We have further shown that combined staining for 5 nucleotidase and for endogenous peroxidase, offers a histochemical tool to discriminate between the three main sinusoidal cell types in normal rat liver.  相似文献   

14.
We studied the kinetics of hepatic uptake of liposomes during serum-free recirculating perfusion of rat livers. Liposomes consisted of phosphatidylcholine, cholesterol and phosphatidylserine in a 6:4:0 or a 3:4:3 molar ratio and were radiolabelled with [3H]cholesteryl oleyl ether. The negatively charged liposomes were taken up to a 10-fold higher extent than the neutral ones. Hepatic uptake of fluorescently labelled liposomes was examined by fluorescence microscopy. The neutral liposomes displayed a typical Kupffer cell distribution pattern, in addition to weak diffuse staining of the parenchyma, while the negatively charged liposomes showed a characteristic sinusoidal lining pattern, consistent with an endothelial localization. In addition, scattered Kupffer cell staining was distinguished as well as diffuse parenchymal fluorescence. The mainly endothelial localisation of the negatively charged liposomes was confirmed by determining radioactivity in endothelial and Kupffer cells isolated following a 1-h perfusion. Perfusion in the presence of polyinosinic acid, an inhibitor of scavenger receptor activity, reduced the rate of uptake of the negatively charged liposomes twofold, indicating the involvement of this receptor in the elimination mechanism. These results are compatible with earlier in vitro studies on liposome uptake by isolated endothelial cells and Kupffer cells, which showed that in the absence of serum also endothelial cells in situ are able to take up massive amounts of negatively charged liposomes. The present results emphasize that the high in vitro endothelial cell uptake in the absence of serum from earlier observations was not an artifact induced by the cell isolation procedure.  相似文献   

15.
Endocytosis of [125I]iodixanol was studied in vivo and in vitro in rat liver cells to determine fluid phase endocytic activity in different liver cells (hepatocytes, Kupffer cells and endothelial cells). The Kupffer cells were more active in the uptake of [l25I]iodixanol than parenchymal cells or endothelial cells. Inhibition of endocytic uptake via clathrin-coated pits (by potassium depletion and hypertonic medium) reduced uptake of [125I]iodixanol much more in Kupffer cells and endothelial cells than in hepatocytes. To gain further information about the importance of clathrin-mediated fluid phase endocytosis, the expression of proteins known to be components of the endocytic machinery was investigated. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, endothelial cells and Kupffer cells were found to express approximately fourfold more rab4, rab5 and rab7 than parenchymal cells, while clathrin was expressed at a higher level in endothelial cells than in Kupffer cells and hepatocytes. Using electron microscopy it was shown that liver endothelial cells contained approximately twice as many coated pits per membrane unit than the parenchymal and Kupffer cells, thus confirming the immunoblotting results concerning clathrin expression. Electron microscopy on isolated liver cells following fluid phase uptake of horseradish peroxidase (HRP) showed that HRP-containing organelles had a different morphology in the different cell types: In the liver endothelial cells HRP was in small, tubular endosomes, while in Kupffer cells HRP was mainly found in larger structures, reminiscent of macropinosomes. Parenchymal cells contained HRP in small vacuolar endosomes with a punctuated distribution. In conclusion, we find that the Kupffer cells and the endothelial cells have a higher pinocytic activity than the hepatocytes. The hepatocytes do, however, account for most of the total hepatic uptake. The fluid phase endocytosis in liver endothelial cells depends mainly on clathrin-mediated endocytosis, while the parenchymal cells have additional clathrin-independent mechanisms that may play an important role in the uptake of plasma membrane components. In the Kupffer cells the major uptake of fluid phase markers seems to take place via a macropinocytic mechanism.  相似文献   

16.
Betaine, taurine, and inositol participate as osmolytes in liver cell volume homeostasis and interfere with cell function. In this study we investigated whether osmolytes are also released from the intact liver independent of osmolarity changes. In the perfused rat liver, phagocytosis of carbon particles led to a four- to fivefold stimulation of taurine efflux into the effluent perfusate above basal release rates. This taurine release was inhibited by 70-80% by the anion exchange inhibitor DIDS or by pretreatment of the rats with gadolinium chloride. Administration of vasopressin, cAMP, extracellular ATP, and glucagon also increased release of betaine and/or taurine, whereas insulin, extracellular UTP, and adenosine were without effect. In isolated liver cells, it was shown that parenchymal cells and sinusoidal endothelial cells, but not Kupffer cells and hepatic stellate cells, release osmolytes upon hormone stimulation. This may be caused by a lack of hormone receptor expression in these cells, because single-cell fluorescence measurements revealed an increase of intracellular calcium concentration in response to vasopressin and glucagon in parenchymal cells and sinusoidal endothelial cells but not in Kupffer cells and hepatic stellate cells. The data show that Kupffer cells release osmolytes during phagocytosis via DIDS-sensitive anion channels. This mechanism may be used to compensate for the increase in cell volume induced by the ingestion of phagocytosable material. The physiological significance of hormone-induced osmolyte release remains to be evaluated.  相似文献   

17.
The purpose of this study was to determine the cellular distribution and degradation in rat liver following intravenous injection of superparamagnetic iron oxide nanoparticles used for magnetic resonance imaging (NC100150 Injection). Relaxometric and spectrophotometric methods were used to determine the concentration of the iron oxide nanoparticles and their degradation products in isolated rat liver parenchymal, endothelial and Kupffer cell fractions. An isolated cell phantom was also constructed to quantify the effect of the degradation products on the loss of MR signal in terms of decreased transverse relaxation times, T2*. The results of this study show that iron oxide nanoparticles found in the NC100150 Injection were taken up and distributed equally in both liver endothelial and Kupffer cells following a single 5 mg Fe/kg body wt. bolus injection in rats. Whereas endothelial and Kupffer cells exhibited similar rates of uptake and degradation, liver parenchymal cells did not take up the NC100150 Injection iron oxide particles. Light-microscopy methods did, however, indicate an increased iron load, presumably as ferritin/hemosiderin, within the hepatocytes 24 h post injection. The study also confirmed that compartmentalisation of ferritin/hemosiderin may cause a significant decrease in the MRI signal intensity of the liver. In conclusion, the combined results of this study imply that the prolonged presence of breakdown product in the liver may cause a prolonged imaging effect (in terms of signal loss) for a time period that significantly exceeds the half-life of NC100150 Injection iron oxide nanoparticles in liver.  相似文献   

18.
Tenascin is a major glycoprotein constituent of the extracellular matrix with a strong affinity to fibronectin; its distribution is believed to be temporarily and spatially limited. Tenascin gene expression is increased during wound healing processes. As repair mechanisms in chronic liver diseases resemble wound healing we studied tenascin gene expression in rat liver and in isolated rat liver cells. In normal rat liver a tenascin specific antiserum stains sinusoidal cells with fiber-like prolongations, which at the same time are desmin-positive (ITO-cells). In the CCl4-acutely-damaged liver a strong tenascin staining is detected in cells located among the mononuclear cells of the inflammatory infiltrates in the areas of necrosis and in cells of the sinusoids. In CCl4-chronically-damaged liver a strong tenascin staining is demonstrable in the connective tissue septa. In both cases, many of the tenascin-positive cells can be identified as desmin-positive by means of the double-staining fluorescence technique. The wall of larger vessels is always tensacin-negative. The staining pattern obtained with a fibronectin-specific antiserum is somewhat comparable with that of tenascin but the vessel wall was positive. hepatocytes, Kupffer cells, ITO-cells and endothelial cells were isolated from rat liver and studied for their capacity to express the tenascin gene. Biosynthetically labeled tenascin was immunoprecipated from supernatants and cell lysates obtained from cultured ITO-cells and to a much lesser extent from intracellular lysates obtained from endothelial cells; its synthesis in ITO-cells increased during the time in culture. Tenascin was also identified immuno-cytochemically in increasing amount in ITO-cells in culture.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

19.
We had hypothesized that preclustered arrangement of galactose-specific receptor activity on rat liver macrophages enables these cells to internalize multivalent, particulate ligands in contrast to the clearance of molecules mediated by statistically distributed receptors on hepatocytes. We now took advantage of the nonclustered receptor distribution in newborn rat liver macrophages to study the in vivo clearance of particulate ligands. Gold particles 5, 17, and 50 nm in diameter (Au5, Au17, Au50), coated with lactosylated bovine serum albumin (LacBSA), were injected into the vena cava and livers were perfusion fixed after allowing for binding and uptake for 3 min. In sinusoidal cells from rats 15 days old LacBSA-Au5 and LacBSA-Au17 were taken up by endothelial cells and all sizes by liver macrophages. In newborn rat liver no LacBSA-Au50 or LacBSA-Au17 was retained in liver macrophages. Uptake of LacBSA-Au5 by sinusoidal cells was significant. LacBSA-Au17 was taken up in significant amounts by endothelial cells of newborn rats which correlates to the findings that galactose-specific binding sites on endothelial cells were found to localize as clusters over coated pits irrespective of age. These results demonstrate the crucial role of clustered receptors in binding and uptake of larger particulate ligands via this lectin-like binding activity.  相似文献   

20.
Tenascin is a major glycoprotein constituent of the extracellular matrix with a strong affinity to fibronectin; its distribution is believed to be temporarily and spatially limited. Tenascin gene expression is increased during wound healing processes. As repair mechanisms in chronic liver diseases resemble wound healing we studied tenascin gene expression in rat liver and in isolated rat liver cells. In normal rat liver a tenascin specific antiserum stains sinusoidal cells with fiber-like prolongations, which at the same time are desmin-positive (ITO-cells). In the CCl4-acutely-damaged liver a strong tenascin staining is detected in cells located among the mononuclear cells of the inflammatory infiltrates in the areas of necrosis and in cells of the sinusoids. In CG4-chronically-damaged liver a strong tenascin staining is demonstrable in the connective tissue septa. In both cases, many of the tenascin-positive cells can be identified as desmin-positive by means of the double-staining fluorescence technique. The wall of larger vessels is always tensacin-negative. The staining pattern obtained with a fibronectin-specific antiserum is somewhat comparable with that of tenascin but the vessel wall was positive. Hepatocytes, Kupffer cells, ITO-cells and endothelial cells were isolated from rat liver and studied for their capacity to express the tenascin gene. Biosynthetically labeled tenascin was immunoprecipated from supernatants and cell lysates obtained from cultured ITO-cells and to a much lesser extent from intracellular lysates obtained from endothelial cells; its synthesis in ITO-cells increased during the time in culture. Tenascin was also identified immuno-cytochemically in increasing amount in ITO-cells in culture. We conclude that ITO-cells may play a major role in tenascin synthesis during liver fibrogenesis. Some of these results were presented at the Annual Meeting of the American Association Study of the Liver, Chicago, USA, 1990. G.R. holds a Hermann and Lilly Schilling professorship  相似文献   

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