Objective: To study the vitronectin expression in human spermato-zoa and its role in fertilization. Methods: Spermatozoa from 14 fer-tile and 8 infertile men with normal semen data were studied. Follow-ing recovery of...Objective: To study the vitronectin expression in human spermato-zoa and its role in fertilization. Methods: Spermatozoa from 14 fer-tile and 8 infertile men with normal semen data were studied. Follow-ing recovery of motile populations by swim-up, spermatozoa were ca-pacitated and immunostained with rabbit anti-human Vn polyclonal an-tibody and goat anti-rabbit IgG-FTTC second antibody. The percentageof spermatozoa expressing Vn was determined using a FAScan flowcytometer. Meanwhile, the fertilizing ability of capacitated spermato-zoa was determined with human spermatozoa zona-free hamster eggpenetration assay (SPA). Results: The mean ± s proportion ofspermatozoa expressing Vn of fertile men was 21.24% ± 11.70% and3.64±3.27% for infertile men (P<0.05). The penetration rate ofSPA in the fertile group was > 10%, but that in the infertile group ,< 10%. There is a correlation between positive sperm Vn expressionand percentage of eggs penetrated (r=0.476). Conclusion: Theseresults indicate the expression of Vn in human capacitated spermatozoaand its correlation with the fertilizing ability. The abnormal expressionof Vn on human spermatozoa may be one of the unexplained infertilereasons. (Reprod Contracep 2001; 21: 24-28)展开更多
AIM:To explore an xeno-free and defined coating substrate suitable for the culture of H9 human embryonic stem cell-derived retinal pigment epithelial(hES-RPE)cells in vitro,and compare the behaviors and functions of h...AIM:To explore an xeno-free and defined coating substrate suitable for the culture of H9 human embryonic stem cell-derived retinal pigment epithelial(hES-RPE)cells in vitro,and compare the behaviors and functions of h ESRPE cells on two culture substrates,laminin521(LN-521)and truncated recombinant human vitronectin(VTN-N).METHODS:hES-RPE cells were used in the experiment.The abilities of LN-521 and VTN-N at different concentrations to adhere to hES-RPE cells were compared with a high-content imaging system.Quantitative real-time polymerase chain reaction was used to evaluate RPE-specific gene expression levels midway(day 10)and at the end(day 20)of the time course.Cell polarity was observed by immunofluorescent staining for apical and basal markers of the RPE.The phagocytic ability of hES-RPE cells was identified by flow cytometry and immunofluorescence.RESULTS:The cell adhesion assay showed that the ability of LN-521 to adhere to hES-RPE cells was dosedependent.With increasing coating concentration,an increasing number of cells attached to the surface of LN-521-coated wells.In contrast,VTN-N presented a strong adhesive ability even at a low concentration.The optimal concentration of LN-521 and VTN-N required to coat and adhesion to hES-RPE cells were 2 and 0.25μg/cm^(2),respectively.Furthermore,both LN-521 and VTN-N could facilitate adoption of the desired cobblestone cellular morphology with tight junction and showed polarity by the hES-RPE cells.However,hES-RPE cells cultivated in VTN-N had a greater phagocytic ability,and it took less time for these hES-RPE cells to mature.CONCLUSION:VTN-N is a more suitable coating substrate for cultivating hES-RPE cells.展开更多
Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor (serpin) superfamily of proteins, circulates in a complex with vitronectin. Furthermore, these two proteins are co-localized in the ...Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor (serpin) superfamily of proteins, circulates in a complex with vitronectin. Furthermore, these two proteins are co-localized in the extracellular matrix (ECM) in many different pathophysiological conditions. Though PAI-1 is a well-characterized inhibitor of serine proteases, recent emphasis has also focused on its protease-independent functions. Vitronectin, a multi-domain protein that binds a wide variety of ligands and proteins, exists in the circulation in a preferred monomeric state, while in the extracellular matrix it exists as a multimer resulting from an altered conformation. Though the mechanism for the conformational alterations and compartmentalization in tissues is unknown, there are a number of biomolecules including PAI-1 that appear to cause such changes. Experimental analysis has established that PAI-1 induces association of vitronectin to higher-order species in a concentration-dependent fashion [1]. This report extends our investigations into the mechanism of the interaction between vitronectin and PAI-1 to explore the physiological relevance of these higher-order complexes for cellular adhesion and migration. In this study, we evaluate the effects of the pericellular microenvironment on the functions of the multimeric complexes in a variety of relevant biological settings. Our findings underscore the importance of the variability of components within this microenvironment, including different receptors and ECM components, in governing the way in which the vitronectin/PAI-1 complex mediates cell-matrix interactions.展开更多
Objective:This study was aimed at assessing the dynamics of vitronectin (VN), laminin (LN), and heparan sulfate/heparin (HS/HP) content changes during experimental burn healing. Methods:VN, LN, and HS/HP were isolated...Objective:This study was aimed at assessing the dynamics of vitronectin (VN), laminin (LN), and heparan sulfate/heparin (HS/HP) content changes during experimental burn healing. Methods:VN, LN, and HS/HP were isolated and purified from normal and injured skin of domestic pigs, on the 3rd, 5th, 10th, 15th, and 21st days following thermal damage. The wounds were treated with apitherapeutic agent (propolis), silver sulfadiazine (SSD), physiological salt solution, and propolis vehicle. VN and LN were quantified using an immunoenzymatic assay and HS/HP was estimated by densitometric analysis. Results:Propolis treatment stimulated significant increases in VN, LN, and HS/HP contents during the initial phase of study, followed by a reduction in the estimated extracellular matrix molecules. Similar patterns, although less extreme, were observed after treatment with SSD. Conclusions:The beneficial effects of propolis on experimental wounds make it a potential apitherapeutic agent in topical burn management.展开更多
Biomaterial surface chemistry engenders profound consequences on cell adhesion and the ultimate tissue response by adsorbing proteins from extracellular matrix,where vitronectin(Vn)is involved as one of the crucial me...Biomaterial surface chemistry engenders profound consequences on cell adhesion and the ultimate tissue response by adsorbing proteins from extracellular matrix,where vitronectin(Vn)is involved as one of the crucial mediator proteins.Deciphering the adsorption behaviors of Vn in molecular scale provides a useful account of how to design biomaterial surfaces.But the details of structural dynamics and consequential biological effect remain elusive.Herein,both experimental and computational approaches were applied to delineate the conformational and orientational evolution of Vn during adsorption onto self-assembled monolayers(SAMs)terminating with-COOH,-NH2,-CH3 and-OH.To unravel the interplay between cell binding and the charge and wettability of material surface,somatomedin-B(SMB)domain of Vn holding the RGD cell-binding motif was employed in molecular dynamics(MD)simulations,with orientation initialized by Monte Carlo(MC)method.Experimental evidences including protein adsorption,cell adhesion and integrin gene expressions were thoroughly investigated.The adsorption of Vn on different surface chemistries showed very complex profiles.Cell adhesion was enabled on all Vn-adsorbed surfaces but with distinct mechanisms mostly determined by conformational change induced reorientation.Higher amount of Vn was observed on negatively charged surface(COOH)and hydrophobic surface(CH3).However,advantageous orientations defined by RGD loop conditions were only obtained on the charged surfaces(COOH and NH2).Specifically,COOH surface straightened up the Vn molecules and accumulated them into a higher density,whereas CH3 surface squashed Vn and stacked them into higher density multilayer by tracking adsorption but with the RGD loops restrained.These findings may have a broad implication on the understanding of Vn functionality and would help develop new strategies for designing advanced biomaterials.展开更多
文摘Objective: To study the vitronectin expression in human spermato-zoa and its role in fertilization. Methods: Spermatozoa from 14 fer-tile and 8 infertile men with normal semen data were studied. Follow-ing recovery of motile populations by swim-up, spermatozoa were ca-pacitated and immunostained with rabbit anti-human Vn polyclonal an-tibody and goat anti-rabbit IgG-FTTC second antibody. The percentageof spermatozoa expressing Vn was determined using a FAScan flowcytometer. Meanwhile, the fertilizing ability of capacitated spermato-zoa was determined with human spermatozoa zona-free hamster eggpenetration assay (SPA). Results: The mean ± s proportion ofspermatozoa expressing Vn of fertile men was 21.24% ± 11.70% and3.64±3.27% for infertile men (P<0.05). The penetration rate ofSPA in the fertile group was > 10%, but that in the infertile group ,< 10%. There is a correlation between positive sperm Vn expressionand percentage of eggs penetrated (r=0.476). Conclusion: Theseresults indicate the expression of Vn in human capacitated spermatozoaand its correlation with the fertilizing ability. The abnormal expressionof Vn on human spermatozoa may be one of the unexplained infertilereasons. (Reprod Contracep 2001; 21: 24-28)
基金Supported by the National Natural Science Foundation of China(No.81730026No.81970816)+2 种基金the National Key R&D Program(No.2017YFA0105301)Science and Technology Commission of Shanghai Municipality(No.20Z11900400No.19495800700)。
文摘AIM:To explore an xeno-free and defined coating substrate suitable for the culture of H9 human embryonic stem cell-derived retinal pigment epithelial(hES-RPE)cells in vitro,and compare the behaviors and functions of h ESRPE cells on two culture substrates,laminin521(LN-521)and truncated recombinant human vitronectin(VTN-N).METHODS:hES-RPE cells were used in the experiment.The abilities of LN-521 and VTN-N at different concentrations to adhere to hES-RPE cells were compared with a high-content imaging system.Quantitative real-time polymerase chain reaction was used to evaluate RPE-specific gene expression levels midway(day 10)and at the end(day 20)of the time course.Cell polarity was observed by immunofluorescent staining for apical and basal markers of the RPE.The phagocytic ability of hES-RPE cells was identified by flow cytometry and immunofluorescence.RESULTS:The cell adhesion assay showed that the ability of LN-521 to adhere to hES-RPE cells was dosedependent.With increasing coating concentration,an increasing number of cells attached to the surface of LN-521-coated wells.In contrast,VTN-N presented a strong adhesive ability even at a low concentration.The optimal concentration of LN-521 and VTN-N required to coat and adhesion to hES-RPE cells were 2 and 0.25μg/cm^(2),respectively.Furthermore,both LN-521 and VTN-N could facilitate adoption of the desired cobblestone cellular morphology with tight junction and showed polarity by the hES-RPE cells.However,hES-RPE cells cultivated in VTN-N had a greater phagocytic ability,and it took less time for these hES-RPE cells to mature.CONCLUSION:VTN-N is a more suitable coating substrate for cultivating hES-RPE cells.
文摘Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor (serpin) superfamily of proteins, circulates in a complex with vitronectin. Furthermore, these two proteins are co-localized in the extracellular matrix (ECM) in many different pathophysiological conditions. Though PAI-1 is a well-characterized inhibitor of serine proteases, recent emphasis has also focused on its protease-independent functions. Vitronectin, a multi-domain protein that binds a wide variety of ligands and proteins, exists in the circulation in a preferred monomeric state, while in the extracellular matrix it exists as a multimer resulting from an altered conformation. Though the mechanism for the conformational alterations and compartmentalization in tissues is unknown, there are a number of biomolecules including PAI-1 that appear to cause such changes. Experimental analysis has established that PAI-1 induces association of vitronectin to higher-order species in a concentration-dependent fashion [1]. This report extends our investigations into the mechanism of the interaction between vitronectin and PAI-1 to explore the physiological relevance of these higher-order complexes for cellular adhesion and migration. In this study, we evaluate the effects of the pericellular microenvironment on the functions of the multimeric complexes in a variety of relevant biological settings. Our findings underscore the importance of the variability of components within this microenvironment, including different receptors and ECM components, in governing the way in which the vitronectin/PAI-1 complex mediates cell-matrix interactions.
基金Project (KNW-2-138/09) supported by the Medical University of Silesia, Poland
文摘Objective:This study was aimed at assessing the dynamics of vitronectin (VN), laminin (LN), and heparan sulfate/heparin (HS/HP) content changes during experimental burn healing. Methods:VN, LN, and HS/HP were isolated and purified from normal and injured skin of domestic pigs, on the 3rd, 5th, 10th, 15th, and 21st days following thermal damage. The wounds were treated with apitherapeutic agent (propolis), silver sulfadiazine (SSD), physiological salt solution, and propolis vehicle. VN and LN were quantified using an immunoenzymatic assay and HS/HP was estimated by densitometric analysis. Results:Propolis treatment stimulated significant increases in VN, LN, and HS/HP contents during the initial phase of study, followed by a reduction in the estimated extracellular matrix molecules. Similar patterns, although less extreme, were observed after treatment with SSD. Conclusions:The beneficial effects of propolis on experimental wounds make it a potential apitherapeutic agent in topical burn management.
基金financially supported by the National Key R&D Program of China(2017YFC1105000)Science and Technology Planning Project of Guangdong Province(2017B030314008)+5 种基金National Natural Science Foundation of China(51572087,31700823)Shenzhen Science and Technology Innovation Committee(JCYJ20170818160503855)Outstanding Scholar Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory(2018GZR110102001)GDST-NWO science industry cooperation programme Chemistry(2018A050501006)Natural Science Foundation of Guangdong Province of China(2020A1515011354)the 111 Project(B13039).
文摘Biomaterial surface chemistry engenders profound consequences on cell adhesion and the ultimate tissue response by adsorbing proteins from extracellular matrix,where vitronectin(Vn)is involved as one of the crucial mediator proteins.Deciphering the adsorption behaviors of Vn in molecular scale provides a useful account of how to design biomaterial surfaces.But the details of structural dynamics and consequential biological effect remain elusive.Herein,both experimental and computational approaches were applied to delineate the conformational and orientational evolution of Vn during adsorption onto self-assembled monolayers(SAMs)terminating with-COOH,-NH2,-CH3 and-OH.To unravel the interplay between cell binding and the charge and wettability of material surface,somatomedin-B(SMB)domain of Vn holding the RGD cell-binding motif was employed in molecular dynamics(MD)simulations,with orientation initialized by Monte Carlo(MC)method.Experimental evidences including protein adsorption,cell adhesion and integrin gene expressions were thoroughly investigated.The adsorption of Vn on different surface chemistries showed very complex profiles.Cell adhesion was enabled on all Vn-adsorbed surfaces but with distinct mechanisms mostly determined by conformational change induced reorientation.Higher amount of Vn was observed on negatively charged surface(COOH)and hydrophobic surface(CH3).However,advantageous orientations defined by RGD loop conditions were only obtained on the charged surfaces(COOH and NH2).Specifically,COOH surface straightened up the Vn molecules and accumulated them into a higher density,whereas CH3 surface squashed Vn and stacked them into higher density multilayer by tracking adsorption but with the RGD loops restrained.These findings may have a broad implication on the understanding of Vn functionality and would help develop new strategies for designing advanced biomaterials.
