ABSTRACT
To establish vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) as secretary biomarkers for cell growth on topographic substrates, we have evaluated the secretion and expression of these 2 factors by SH-SY5Y human neuroblastoma cells on poly-L-lactide (PLLA) micropillar arrayed topographic substrates. We fabricated topographic substrates with UV lithography, silicon etching and polydimethylsiloxane-based replica molding, and interfaced SH-SY5Y human neuroblastoma cells with both the topographic substrates and PLLA flat substrates. Cell morphology and spreading were examined with scanning electron microscopy. The secretion and mRNA expression of VEGF and IL-8 were evaluated with enzyme linked immunosorbent assay (ELISA) and real time qPCR, respectively, 24 hours after cell plating. We successfully achieved 4 topographic substrates with a nominal pillar diameter of 2 microm and 4 microm, and a nominal pillar spacing of 2 microm and 7 microm. We found that the secretion and mRNA expression of VEGF and/or IL-8 by SH-SY5Y cells on 2-2 microm (pillar diameter-spacing), 4-2 microm and 4-7 microm topographic substrates were upregulated in comparison to those by cells on PLLA flat substrate, 24 hours after cell plating. Furthermore, both cytokines were even more substantially upregulated on the 2-7 microm substrate than on the other 3 topographic substrates. Compared to those on PLLA flat substrate, cells on topographic substrates showed significant changes in morphology (spreading area, perimeter and roundness), and the increase in the secretion and mRNA expression of VEGF and IL-8 was accompanied with a decrease in cell spreading areas. These results provided evidence that pillar arrayed topography was an important microenvironmental factor in affecting VEGF and IL-8 expression or secretion, and VEGF and IL-8 might serve as important secretary biomarkers for growth on topographic substrates by SH-SY5Y cells.
Subject(s)
Humans , Biomarkers , Cell Line , Cell Proliferation , Cellular Microenvironment , Interleukin-8 , Genetics , Bodily Secretions , Neuroblastoma , Bodily Secretions , Polyesters , Chemistry , RNA, Messenger , Genetics , Vascular Endothelial Growth Factor A , Genetics , Bodily SecretionsABSTRACT
The present paper was aimed to explore the effect of Shuxuetong on the membrane viscoelasticity of erythrocyte taken from the acute phase patients suffering from chronic pulmonary heart disease. The membrane viscoelasticity of erythrocyte was taken from the acute phase patients suffering from chronic pulmonary heart disease. The changes of membrane viscoelasticity of erythrocyte after treated with shuxuetong were detected by micropipette aspiration technique. The results showed that the Shuxuetong of certain concentration could cause the decrease of membrane elastic modulus and viscous coefficients in acute phase patients suffering from chronic pulmonary heart disease. The study offers experimental evidences that the comprehensive treatment of pulmonary heart disease should involve the drug or measure to improve the erythrocyte deformability.
Subject(s)
Aged , Female , Humans , Male , Middle Aged , Blood Viscosity , Chronic Disease , Drugs, Chinese Herbal , Therapeutic Uses , Elasticity , Erythrocyte Deformability , Physiology , Erythrocyte Membrane , Physiology , Erythrocytes , Physiology , Phytotherapy , Pulmonary Heart Disease , Blood , Drug TherapyABSTRACT
UV photolithography and hydrofluoric acid wet etching were used to produce silicon master molds and polydimethylsiloxane (PMDS)-based soft lithography was adopted to fabricate three-dimensional poly(lactic-co-glycolic acid) (PLGA) and PDMS microwell patterns with high aspect ratio and channel connection. Nine microwell patterns were thus obtained with different structural dimensions. Patterns were treated with oxygen plasma etching and polylysine coating to enhance hydrophilicity and cell compatibility for subsequent culture of C17. 2 neural stem cells. With proliferation during the culture, C17. 2 cells gradually distributed within the microwells, showing an obviously three-dimensional (3-D) growth behavior. The presence of channel structures greatly favored the 3-D growth of C17. 2 neural stem cells on the microwell patterns. Multi-layered scanning with confocal microscopy and 3-D rendering after carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) staining showed that most C17. 2 cells grew within a range of 30 to 90 microm from the microwell bottom. Immunofluorescence staining indicated that C17. 2 cells within 3-D microwell patterns were uniformly nestin-positive on day 2 after cell plating. It could well be concluded that the microwell patterns thus fabricated were suitable for the 3-D culture and subsequent differentiation of C17. 2 neural stem cells. And the cells can be maintained with uniform stemness properties while cultured in these microwell patterns.
Subject(s)
Cell Culture Techniques , Methods , Dimethylpolysiloxanes , Chemistry , Imaging, Three-Dimensional , Intermediate Filament Proteins , Metabolism , Lactic Acid , Chemistry , Microscopy, Confocal , Nerve Tissue Proteins , Metabolism , Nestin , Neural Stem Cells , Cell Biology , Polyglycolic Acid , ChemistryABSTRACT
The membrane viscoelasticity of erythrocyte taken from both normal subjects and patients with cor pulmonale during acute exacerbation was investigated using a micropipette aspiration technique. Experimental results were analysed with vogit viscoelaticity model based on pioneering theory of Chein et al. The results showed that the erythrocyte membrane elastic moduli ((6.970 +/- 1.050) x 10(-3) dyn/cm) and viscous coefficients ((0.936 +/- 0.242) x 10(-4) dyn x s/cm) of the cor pulmonale patients was significantly higher than those of the normal subjects ((5.203 +/- 1.051) X 10(-3) dyn/cm, (0.620 +/- 0.053) x 10(-4) dyn x s/cm). The membrane elastic moduli, viscous coefficients, rigidity of erythrocyte, and viscosity were all increased. It may be the important subcellular mechanism to cause the decrease of erythrocyte deformability and hyperviscosity of blood in these patients.
Subject(s)
Humans , Blood Viscosity , Elasticity , Erythrocyte Deformability , Physiology , Erythrocyte Membrane , Physiology , Erythrocytes , Physiology , Models, Biological , Pulmonary Heart Disease , BloodABSTRACT
The Modified silk fibroin membranes were prepared by mixing the aqueous solutions of both silk fibroin and chitosan with the use of oxidized glucose aldehyde as a crosslinking agent. It was characterized by FTIR, DSC, measurements of membrane-potential and mechanical properties, the water swelling ratios and permeability coefficient for model drug 5-Fu in the different pH buffer solutions. It was shown that there were some strong hydrogen bond interaction and good compatibility between silk fibroin and chitosan molecules in the modified silk fibroin films. The isoelectric point of modified fibroin film was about pH 5.35, but that of natural fibroin film was around pH 4.5. It was also found that the mechanical properties of modified fibroin films were much better than those of fibroin itself. Its tensile strength and breaking elongation were greatly enhanced with the increase of chitosan content and their maximum values were as high as 71.4-72.7 MPa and 2.96%-3.82% respectively, at the composition of 40 wt%-60 wt% chitosan. Its coefficient of permeability decreased firstly and then increased slowly with the change of the pH value of solutions from pH 5 to pH 9, and the minimum coefficient of permeability was observed when pH=7.
Subject(s)
Biocompatible Materials , Chemistry , Chitosan , Chemistry , Cross-Linking Reagents , Delayed-Action Preparations , Drug Carriers , Fibrin Fibrinogen Degradation Products , Fibroins , Chemistry , Hydrogen-Ion Concentration , Membranes , Silk , ChemistryABSTRACT
A micropipette technique was adopted to investigate the effect of blockade of integrin betal on adhesion of hepatocellular carcinoma (HCC) cells onto type IV collagen (Col IV) coated surfaces and pseudopod protrusion of HCC cells in response to Col IV stimulation. Adhesion strength was expressed as an adhesion force, which was defined as the product of the cross sectional area and critical negative pressure needed to detach single cell away from the substrate. Chemotactic pseudopod protrusion of an HCC cell was evaluated using a dual-pipette set-up, in which two pipettes filled with Col IV solution were positional in close contact with the same cell and pseudopod protrusion into each pipette was viewed dynamically and recorded with a tape recorder. The lengths of pseudopods were measured and plotted against time to obtain a pseudopod growth curve. The integrin beta1 subunit on the surfaces of HCC cells was analyzed by flow cytometry. The results showed that the adhesion forces for HCC cells adhering on 5 microg/ml Col IV coated surfaces were 932 +/- 134 (x 10(-10) N, n = 60). Upon treatment of HCC cells with Anti-CD29 in a protein concentration of 5 microg/ml and 10 microg/ml, the value decreased significantly to 449 +/- 119 (x 10(-10) N, n = 60) and 220 +/- 78 (x 10(-10) N, n = 55), respectively. In dual pipette chemotaxis experiment, when the two pipettes were filled with Col IV in an identical concentration of 600 microg/ml, pseudopods extended from the HCC cell into each of the pipettes nearly symmetrically, i.e., with nearly identical maximum pseudopod length and similar pseudopod growth curves. Upon addition of Anti-CD29 to one of the pipettes in a protein concentration of 20 microg/ml, pseudopod protrusion was blocked nearly completely while protrusion into the opposite pipette became more evidently, with larger maximum length. Expression of integrin beta1 was up to 95.78% to cells chosen in the experiment. These results suggested that integrin beta1 subunit was important constituent receptor subunit for mediating HCC cell adhesion and chemotactic pseudopod protrusion to Col IV.
Subject(s)
Humans , Antibodies, Monoclonal , Pharmacology , Carcinoma, Hepatocellular , Metabolism , Pathology , Cell Adhesion , Chemotaxis , Allergy and Immunology , Collagen Type IV , Metabolism , Integrin beta1 , Allergy and Immunology , Liver Neoplasms , Metabolism , Pathology , Neoplasm Invasiveness , Receptors, Very Late Antigen , Allergy and Immunology , Tumor Cells, CulturedABSTRACT
In this study we examined the in vitro characteristics of tenocyte adhesion to biologically-modified surface of polymer. Polylactic-co-glycolic acid (PLGA) 85/15 films were prepared by a solvent-casting technique. Each film was adhered onto the bottom of a chamber. The film was precoated with poly-D-lysine (PDL), and then coated with serum-free F12 medium containing various concentrations of fibronectin (FN), type I collagen (CN I), and insulin-like growth factor1 (IGF-1). The monoclonal antibodies (to FN and to CN I) with various dilutions were used to inhibit attachment of tenocytes to surface precoated with FN or CN I. Human embryonic tendon cells (HETCs) and transformed human embryonic tendon cells (THETCs) were used as the seeding cells. The system used for the measurement of adhesion force was the micropipette aspiration experiment system. The micropipette was manipulated to aspirate a small portion of the tenocyte body by using a small aspiration pressure. Then the pipette was pulled away from the adhesion area by micromanipulation. The minimum force required to detach the tenocyte from the substrate was defined as the adhesion force. The results showed that modification of FN or CN I by precoating significantly enhanced attachment of tenocytes to surface of polymer (P < 0.05). As antibodies to FN or CN I were added to a polymer film precoated with FN or CN I, the adhesion force decreased significantly (P < 0.05). We concluded that the specific adhesion forces of tenocytes to extracellular matrix adhesion proteins (FN and CN I) had coordinated action and showed good dependence on their precoating concentrations, and were inhibited by the antibodies to these adhesion proteins. Films precoated with IGF-1 strongly accelerated the adhesion of tenocytes to polymer. These results indicate that the specific adhesion of tenocytes to polymer can be promoted by coating extracellular matrix adhesive proteins and insulin-like growth factor1. It is of great importance to construct tissue-engineered tendon.
Subject(s)
Humans , Biocompatible Materials , Chemistry , Cell Adhesion , Physiology , Cells, Cultured , Extracellular Matrix Proteins , Pharmacology , Growth Substances , Pharmacology , Lactic Acid , Chemistry , Polyglycolic Acid , Chemistry , Polylysine , Pharmacology , Polymers , Chemistry , Tendons , Cell Biology , Embryology , Physiology , Tissue EngineeringABSTRACT
The viscoelastic properties of hepatocellular carcinoma (HCC) cells were measured by means of micropipette aspiration technique. Further,the authors studied the changes in viscoelastic coefficients by treating with colchicine and cytochalasin D. The results showed that the elastic coefficients of HCC cells were obviously higher than the corresponding value of hepatocytes. By treating with colchicine, the effects on viscoelastic properties of HCC cells were obviously different in ways and extents from those on viscoelastic properties of hepatocytes.,but the viscoelastic properties of hepatocytes by treated with cytochalasin D had the same trend of decreasing as those of HCC cells. These results represent the change in cytoskeleton structure and function among hepatocytes and HCC cells, this change might affect tumor cells invasion and metastasis.
ABSTRACT
The viscoelastic properties of hepatocellular carcinoma (HCC) cells were measured by means of micropipette aspiration technique. Further,the authors studied the changes in viscoelastic coefficients by treating with colchicine and cytochalasin D. The results showed that the elastic coefficients of HCC cells were obviously higher than the corresponding value of hepatocytes. By treating with colchicine, the effects on viscoelastic properties of HCC cells were obviously different in ways and extents from those on viscoelastic properties of hepatocytes.,but the viscoelastic properties of hepatocytes by treated with cytochalasin D had the same trend of decreasing as those of HCC cells. These results represent the change in cytoskeleton structure and function among hepatocytes and HCC cells, this change might affect tumor cells invasion and metastasis.