ABSTRACT
Objective To develop a small-diameter tissue-engineered blood vessels which possesses normal blood vessels physiological structure, good biocompatibility, and mechanical properties. And it was evaluated by mechanical and biological of national standard of medi-cal transfusion material. Methods The bio-derived material were regarded as the ground substance, and it was evaluated by mechanical and biological of national standard after composite modification. Results The axial and radial tensile stress of the blood vessel was 23. 14 N and 36. 79 N respectively, and it was greater than the standard 7. 5N. The tensile rate of the axial and radial was 95. 19% and 80. 24% respec-tively, which were higher than the standard value 20%. The suture strength of the blood vessel was 13. 71 N, which was conform to the me-chanical requirement. Mainly used blood vessels or its extracts to detect the pH of the blood vessels is in the scope of control deionized water pH (7. 5 ± 1. 5);the hemolysis rate was 1. 3972% which was less than 5%;the whole blood coagulation time was 50% longer than the con-trol level, and there was no stimulation after intradermal injection. Conclusion With bio-derived material as the ground substance and com-positely modified, this kind od blood vessels is conform to the mechanical and biological of national standard, and it has the potential of clini-cal application which could play an important role in the replacement therapy of small-diameter vascular xenografts.
ABSTRACT
This investigation was aimed to explore whether over-expression of 27heme oxygenase-1 (HO-1) could protect bone marrow mesenchymal stem cells(BMSCs)against injury induced by high-concentration glucose. We cultured BMSCs in high-concentration glucose medium, and up-regulated or inhibited HO-1 expression in BMSCs through its agonist or inhibitor. We detected the ability of BMSCs proliferation and secretion respectively by MTT and enzyme-linked immunosorbnent assay (ELISA). Then we detected the effect of BMSCs conditions medium on proliferation and migration of human umbilical vein endothelial cells (HUVECs) through scratch experiments and transwell assay. It was found that HO-1 over-expression could not only promote BMSCs proliferation, but also promote secretion of vascular endothelial growth factor (VEGF), and could further accelerate the proliferation and migration of HUVECs. It could be well concluded that HO-1-over-expressing BMSCs can not only inhibit damage induced by high-concentration glucose, but can promote the proliferation and migration of vascular endothelial cells through paracrine as well. The result indicated that HO-1-over-expressing BMSCs played an important role in the treatment of diabetic vascular complication.
Subject(s)
Humans , Cell Movement , Cell Proliferation , Cells, Cultured , Culture Media, Conditioned , Pharmacology , Glucose , Toxicity , Heme Oxygenase-1 , Metabolism , Human Umbilical Vein Endothelial Cells , Cell Biology , Mesenchymal Stem Cells , Cell Biology , Up-Regulation , Vascular Endothelial Growth Factor A , MetabolismABSTRACT
This experiment was aimed to create A20 gene site-specific zinc finger DNA-binding protein. The sequence of A20 gene promoter was analyzed with bioinformatics means and submitted to ZF Tools Server at TSRI. Using the database of the web site, we determined the A20 gene valid target sites and designed the amino acid sequence of zinc finger protein predicted to be bound to the target site. And then, the structure of the protein sequence was analyzed and homology was modeled with various bioinformatics means. Based on the characteristic of this protein, the prokaryotic expression vector pTYB11-ZFP was constructed and expressed. Thus, the artificial zinc finger protein that recognized A20 specific sequence was designed, and expressed in Escherichia coli. The results indicate that it is feasible to design engineered artificial Zinc finger proteins by means of bioinformatics.
Subject(s)
Humans , Amino Acid Sequence , Base Sequence , DNA-Binding Proteins , Chemistry , Genetics , Molecular Sequence Data , Protein Binding , Protein Engineering , Methods , Transcription Factors , Chemistry , Genetics , Zinc Fingers , GeneticsABSTRACT
To investigate the biomechanical behavior of human intestines. The tensile test human intestine was performed with the electronic tension machine in this paper. The results indicate that the exponential relationship for the stress-strain of the human intestine was obtained, and the exponential coefficient a of each segment of the intestine is almost the same although the constant C is different. It also shows that the relative rate of stretch length of each segment intestines is different in longitudinal and circumferential directions. And the incremental elastic modulus of colon is less than those of small intestine. It is considered that the colon can be more easily deformed. The experimental results provide the theoretic basis for research on intestinal endoscopic microrobot.
Subject(s)
Humans , Biomechanical Phenomena , Colon, Transverse , Physiology , Elasticity , In Vitro Techniques , Intestine, Small , Physiology , Stress, Mechanical , Tensile StrengthABSTRACT
In this study, we prepared the acellular bone matrix of the inbred-line Banna mini-pig by using tissue engineering method and evaluated its possible application in bone tissue engineering. Histological analysis, xenoantigen expression and biomechanical measurement were performed on the matrix. HE staining and scanning electron microscopy showed the cellular components were almost removed. Immunohischemical result demonstrated that the xenoantigen, alpha-gal,was also eliminated. There was no statistically significant difference between the acellular bone matrix group and control group. The acellular bone matrix can provide appropriate space structure and strength for grafts. In conclusion, our data suggest that acellular bone matrix is a new kind of ideal bone scaffold material.
Subject(s)
Animals , Female , Male , Antigens, Heterophile , Biomechanical Phenomena , Bone Matrix , Allergy and Immunology , Stress, Mechanical , Swine , Swine, Miniature , Tissue Engineering , alpha-GalactosidaseABSTRACT
This study was aimed to examine the effectiveness of a gene transfer of human TGFbeta1 gene into endothelial cells and to determine whether TGFbeta1 increases ECM expression of endothelial cells. With the help of DOTAP, endothelial cells were transfected with pMAMneoTGFbeta1. The positive cell clones were selected with G418. The stable transfection and expression of TGFbeta1 in the endothelial cells were determined by immunofluorescence analysis. The expression levels of collagen I and fibronectin in the transfected and untransfected endothelial cells were determined by Western blot. The adhesion force between endothelial cells and matrix was determined by a micropipette technical system. The results showed abundant TGFbeta1 stable expression in the endothelial cells. TGFbeta1 gene was noted to increase collagen I and fibronectin expression and increase the adhesion between endothelial cells and matrix. These findings indicated that TGFbeta1 can be used in vascular tissue engineering for the enhancement of endothelial cells adhesion.
Subject(s)
Humans , Cell Adhesion , Cells, Cultured , Collagen Type I , Genetics , Endothelium, Vascular , Cell Biology , Physiology , Extracellular Matrix , Metabolism , Physiology , Fibronectins , Genetics , Tissue Engineering , Transforming Growth Factor beta , Genetics , Umbilical Veins , Cell BiologyABSTRACT
To explore the human smooth muscle cells seeding in blood vessel of minor pig after trypsin treatment and provide data for xenotransplantation and for using pig vessel in tissue engineering. HE and silver stain were used for checking the smooth muscle cells seeding in acellular blood vessel. The results showed that the smooth muscle cells seeding succeeded and the smooth muscle cells were in normal morphological distribution. These demonstrate that the pig aorta can be used for smooth muscle cells seeding, and hence for constructing new vascular grafts.
Subject(s)
Animals , Female , Humans , Male , Bioprosthesis , Blood Vessel Prosthesis , Blood Vessels , Cell Biology , Cell Separation , Cell Transplantation , Muscle, Smooth, Vascular , Cell Biology , Swine , Swine, Miniature , Tissue Engineering , Methods , Transplantation, HeterologousABSTRACT
This study sought to explore the change of the major histocompatibility complex (MHC) antigen expression and the endothelization of blood vessel in minor pig after trypsin treatment, and to provide data for xenotransplantation and pig vessel for use in tissue engineering. Western blot assays were conducted for detecting the expression of MHC xenoantigens. Scanning electron microscopy was used for checking the endothelization of decellularized blood vessel. The results showed that MHC antigen is not expressed after trypsin treatment. The endothelization is accomplished. The endothelial cells have normal morphological distribution. These demonstrate that the antigen of pig aorta is significantly decreased and it can be used for constructing new vascular grafts.
Subject(s)
Animals , Female , Male , Bioprosthesis , Blood Vessel Prosthesis , Cells, Cultured , Endothelial Cells , Cell Biology , Major Histocompatibility Complex , Physiology , Swine , Swine, Miniature , Tissue Engineering , Methods , Tissue ScaffoldsABSTRACT
This study was conducted to examine the effectiveness of a gene transfer of human TGF beta 1 gene into smooth muscle cells and whether the TGF beta 1 can increase elastin expression of smooth muscle cells. With the help of DOTAP, smooth muscle cells were transfected with pMAMneoTGF beta 1. The positive cell clones were selected with G418. The stable transfection and expression of TGF beta 1 in the smooth muscle cells were determined by immunofluorescence analysis. The expression of elastin in the transfected and untransfected cells were determined by in situ hybridization. The adhesion force between smooth muscle cells and matrix was detected by micropipette system. The results showed abundant TGF beta 1 stable expression in smooth muscle cells. TGF beta 1 gene can increase two-three times elastin expression and increase the adhesion between smooth muscle cells and matrix. TGF beta 1 can be used in vascular tissue engineering to increase smooth muscle cells adhesion.
Subject(s)
Humans , Cell Adhesion , Cells, Cultured , Elastin , In Situ Hybridization , Muscle, Smooth, Vascular , Cell Biology , Metabolism , Transfection , Transforming Growth Factor beta , Genetics , Physiology , Transforming Growth Factor beta1ABSTRACT
Objective To investigate the effects of zinc finger protein gene A20 on the inhibition of lipopolysaccharide (LPS) induced interleukin 8 (IL 8) expression in endothelial cells. Methods Plasmid pcDNA3.1EHA20 was transfected into human umbilical vein endothelial cells (HUVECs) by DOTAP method. The positive cell clones were screened with G418. The stable transfection and expression of A20 in HUVECs were determined by immunofluorescent analysis. IL 8 expression was detected by sandwich ELISA with double monoclonal antibodies. Results High expression of A20 gene in HUVECs transfected with pCDNA3.1EHA20 was confirmed by immunofluorescent analysis. IL 8 expression increased in LPS induced endothelial cells. A20 gene could inhibit more than 70% LPS induced IL 8 expression ( P
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Objective To construct the recombinant adenovirus of mouse Osf2/Cbfal gene and to observe its ability to infect NIH3T3 fibroblasts. Methods The Osf2/Cbfal gene fragment was cloned into the shuttle plasmid pAdTrack-CMV to form the transfer vector by the method of homogenous recombination in bacteria. Then the recombinant adenovirus was transfected into NIH3T3 cells using Lipofectine DOTAP, The target gene was detected by poly-merase chain reaction (PCR). The titer and its infection rate were determined using the green fluorescent protein (GFP) expression in the shuttle plasmid. Results Restriction endonuclease and PCR analyses confirmed that the Osf2/Cbfal gene was successfully inserted into the adenovirus vector. The titer of the recombinant adenovirus was 1.6?1012 pfu/ml. The adenovirus had a strong effect on NIH3T3 cells. Conclusion The recombinant adenovirus containing Osf2/Cbfal gene was successfully constructed by the method of homogenous recombination in bacteria.
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To explore the changes of the antigen expression and the biomechanical characteristics of blood vessel in Banna little ear pig before and after trypsin treatment, and provide data for xenotransplantation and pig vessel using for tissue engineering. Geometric morphology and microstructure of pig cartoid artery were stuided quantitatively by histologic method and computer image analysis. The relationship between pressure and diameter was observed at different period of time before and after trypsin treatment. Affinity-immunohistochemistry assay was conducted to detect the expression of xenoantigens (alpha-Gal). The results showed that alpha-Gal antigen is only expressed in vascular endothelial cellsouly. There is no significant difference in blood vessel compliance. These demonstrate that the antigenicity of pig carotid artery is significantly reduced, however, the mechanical characteristics did not change significantly. We suppose that pig vessels treated by trypsin can be used as the substrate material for vascular tissue engineering.
Subject(s)
Animals , Female , Male , Animals, Inbred Strains , Antigens, Heterophile , Blood Vessels , Physiology , Stress, Mechanical , Swine , Tissue Engineering , Trypsin , PharmacologyABSTRACT
Objective Previous studies have demonstrated that LPS can induce endothelial cell activation and the expression of E-selectin. In this study, we examined whether A20 gene could inhibit the expression of E-selectin in endothelial cells induced by LPS. Methods With the help of DOTAP, endothelial cells were transfected with pCDNA3.1 EHA20. The postive cell clones were selected with G418.The stable transfection and expression of A20 in the endothelial cells were determined by immunofluorescence analysis. The E-selectin expression was checked by immunofluorescence, Western blot and in situ hybridization. Results Abundant A20 stable expression in endothelial cells transfected with pCDNA3.1 EHA20 was confirmed by immunofluorescence analysis. E-selectin expression increased in LPS-inducible endothelial cells. A20 gene inhibited 90% LPS-inducible E-selectin expression(P
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Objective To observe the effectiveness of transferring human A20 gene into endothelial cells. Methods The shuttle plasmid pCAGGSEHA20 was constructed using gene cloning and recombined technique. Endothelial cells were transfected with pCAGGSEHA20 and pMAMneo by DOTAP. The postive cell clones were selected with G418. The stable transfection and expression of A20 in the endothelial cells were determined by in situ hybridization and immunohistochemical analysis. Results The two fragments digested from pCAGGSEHA20 by EcoRⅠ represented 4 6?kb and 2 3?kb by electrophoresis, which were confirmed to be the carrier and the A20 gene fragments inserted originally. The above results indicate that the construction of pCAGGSEHA20 was successful. Abundant A20 stable expression in endothelial cells transfected with pCAGGSEHA20 was confirmed by in situ hybridization and immunohistochemical analysis.Conclusion By means of the DOTAP, hA20 gene can be transferred and stably expressed in endothelial cells.