This study was designed to explore the immune reconstitution efficacy of human thymic slices transplanted into renal capsule,subcutaneous or muscle of nude mice,and further explore the optimal location of heterotopic transplantation.The thymus tissue discarded from congenital heart disease patients was made into 0.5-1 mm thick tissue sections and cultured in vitro to remove immune cells.H&E staining and immunohistochemical staining were used to assess the residual tissue structure and cell types in thymic slices,while quantitative PCR methods were used to assess the function of residual cells in thymic slices.Then thymic slices were transplanted into the renal capsule,subcutaneous or muscle of nude mice,and the immune reconstitution efficacy was compared by flow cytometry and histology.Data showed that after 14 days of culture in vitro,the clearance rate of T lymphocytes in the thymic slices was more than 90%,and the epithelial cell network structure of the tissue was intact,while a large number of macrophages,dendritic cells and endothelial cells remained.Quantitative PCR results showed that the gene expression levels of epithelial cell markers and secreted cytokines in cultured thymic slices could be effectively maintained.Flow cytometry showed that at 16 weeks after transplantation,the proportion of T cells in peripheral blood of mice in different transplantation groups were significantly increased,whereas the proportion of T cells in muscle group was the highest.In situ histological examination showed that the regeneration of thymus tissue was detected at all three transplant sites.In addition,the graft detection rate was 40%in the renal capsule group,60%in the subcutaneous group and 100%in the musclegroup.In conclusion,the human thymic slices cultured in vitro for 14 days retain a complete thymic matrix microenvironment.Transplantation of human thymic slices can effectively reconstruct the ratio of T cells in nude mice,and the muscle is the most effective transplantation site.

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.
Cell Adhesion ; Cells, Cultured ; Collagen Type I ; biosynthesis ; genetics ; Endothelium, Vascular ; cytology ; physiology ; Extracellular Matrix ; metabolism ; physiology ; Fibronectins ; biosynthesis ; genetics ; Humans ; Tissue Engineering ; Transforming Growth Factor beta ; biosynthesis ; genetics ; Umbilical Veins ; cytology

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.
Animals ; Bioprosthesis ; Blood Vessel Prosthesis ; Blood Vessels ; cytology ; Cell Separation ; Cell Transplantation ; Female ; Humans ; Male ; Muscle, Smooth, Vascular ; cytology ; Swine ; Swine, Miniature ; Tissue Engineering ; methods ; Transplantation, Heterologous

BACKGROUND: Changes of elastic fibers in middle cerebral artery(MCA)is close related withthe aged cerebrovascular disease.OBJECTIVE: To observe the changes of elastic fibers of MCA in different aging rats.DESIGN: A descriptive and controlled study based on experimental animals.SETTING: Department of anatomy and central laboratory in a university.MATERIALS: Totally 36 healthy Wistar rats with either gender, weighing 200 - 280 g, were selected from the Animal Institute of the third medical military university of Chongqing[certification SCXX (army) 2002-007].INTERVENTIONS: Changes of elastic fibers of MCA of different aging rats were observed with light microscope, transmission electron microscope and image analysis system. MAIN OUTCOME MEASURES: ①) Major outcome: changes of elastic lamella in MCA of different aging rats; ②) Secondary outcome: ultramicrostructural changes of internal lamella under the transmission electron microscope. RESULTS: With the increase of age, the folded extent and quantity of internal elastic lamella were decreased, and the content of elastic fibers were also decreased significantly( P ＜ 0.01 ). However, the ratio of collagen fibers to elastic fibers was increased significantly( P ＜ 0.01 ) . In the aging group above 24 months, the internal elastic lamina thinned, delaminated and disrupted, and the lipid deposited in it. Endothelial cells and smooth muscle cells passed through the internal elastic lamina. CONCLUSION: Changes of elastic fibers may be related with the increased susceptibility to the cerebrovascular disease in aged people.

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.
Animals ; Bioprosthesis ; Blood Vessel Prosthesis ; Cells, Cultured ; Endothelial Cells ; cytology ; Female ; Major Histocompatibility Complex ; physiology ; Male ; Swine ; Swine, Miniature ; Tissue Engineering ; methods ; Tissue Scaffolds

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.
Cell Adhesion ; Cells, Cultured ; Elastin ; biosynthesis ; Humans ; In Situ Hybridization ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Transfection ; Transforming Growth Factor beta ; biosynthesis ; genetics ; physiology ; Transforming Growth Factor beta1

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

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.
Animals ; Animals, Inbred Strains ; Antigens, Heterophile ; biosynthesis ; Blood Vessels ; drug effects ; physiology ; Female ; Male ; Stress, Mechanical ; Swine ; Tissue Engineering ; Trypsin ; pharmacology

Objective To isolate the bone marrow mesenchymal stem cells(MSCs) from the GFP-expressing mouse, and to study the osteoblastic differentation of the cells. Methods MSCs were isolated by density gradient centrifugation, then the clutrued cells were induced to osteoblastic differentiation using the conditional medium. We detectd the expression of GFP and MSCs differentiation into osteoblasts by histochemistry and immunochemistry. Results The MSCs maintained the expression of GFP during expanded and induced process. After induced for 10 days, lots of alkaline phosphatase and osteoclcin staining positive cells were observed.Conclusion The MSCs of GFP-expressing mouse were successfully isolated and differentiated into ostoblasts. It may be valuable for tracing the seeding cells in tissue engineering bone.

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