ABSTRACT
<p><b>OBJECTIVE</b>To examine the differentiation of 5-azacytidine-induced bone marrow stromal stem cells (BMSCs) into cardiomyocyte-like cells and hepatocyte growth factor (HGF) gene expression after HGF gene transfection.</p><p><b>METHODS</b>5-azacytidine was used to induce beagle dog BMSCs to differentiate into cardiomyocyte-like cells. Morphological observation and immunohistochemistry were performed to detect the expression of the markers of cardiomyocyte-like cells including beta-myosin heavy chain (beta-MHC) and alpha-sarcomeric actin. The cells were then transfected with Ad-HGF, and the mRNA and protein expressions of HGF gene were detected by RT-PCR and ELISA, respectively.</p><p><b>RESULTS</b>After 4 weeks of induction with 5-azacytidine, the BMSCs differentiated into cardiomyocyte-like cells. The expressions of HGF at the mRNA and protein levels were confirmed in the cells after transfection with Ad-HGF. The peak HGF protein secretion was 10(3) ng/ml at 48 h after transfection.</p><p><b>CONCLUSION</b>Ad-HGF can efficiently transfect BMSCs induced with 5-azacytidine, and this result provides basic experimental evidence for biotherapy of ischemic heart disease using BMSCs.</p>
Subject(s)
Animals , Dogs , Azacitidine , Pharmacology , Bone Marrow Cells , Cell Biology , Metabolism , Cell Differentiation , Cells, Cultured , Gene Expression , Hepatocyte Growth Factor , Genetics , Metabolism , Mesenchymal Stem Cells , Cell Biology , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To evaluate the therapeutic effect of vascular endothelial growth factor (VEGF) and tumor necrosis factor receptor (TNFR) on avascular necrosis of the femoral head in rabbits.</p><p><b>METHODS</b>Avascular necrosis of the femoral head was induced in 26 New Zealand white rabbits by injections of horse serum and prednisolone. The rabbits were then divided into VEGF/TNFR treatment group, VEGF treatment group, and untreated model group, with another 4 normal rabbits as the normal control group. In the two treatment groups, the therapeutic agents were injected percutaneously into the femoral head. Enzyme-linked immunosorbent assay was performed to determine the concentration of TNF-alpha in rabbit serum followed by pathological examination of the changes in the bone tissues, bone marrow hematopoietic tissue and the blood vessels in the femoral head.</p><p><b>RESULTS</b>Compared with the model group, the rabbits with both VEGF and TNFR treatment showed decreased serum concentration of TNF-alpha with obvious new vessel formation, decreased empty bone lacunae in the femoral head and hematopoietic tissue proliferation in the bone marrow cavity.</p><p><b>CONCLUSION</b>Percutaneous injection of VEGF and TNFR into the femoral head can significantly enhance bone tissue angiogenesis and ameliorate osteonecrosis in rabbits with experimental femoral head necrosis.</p>
Subject(s)
Animals , Female , Male , Rabbits , Drug Therapy, Combination , Femur Head Necrosis , Drug Therapy , Random Allocation , Receptors, Tumor Necrosis Factor , Therapeutic Uses , Tumor Necrosis Factor-alpha , Blood , Vascular Endothelial Growth Factor A , Therapeutic UsesABSTRACT
<p><b>OBJECTIVE</b>To study the activities of interleukin (IL)-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (hIL-2/mGM-CSF).</p><p><b>METHODS</b>SOE PCR was used to change the linker of the fusion protein for higher activities. The fusion protein was expressed in Escherichia coli (E. coli) BL21 (DE3) in inclusion body (IB) form. After IB was extracted and clarified, it was denatured and purified by affinity chromatography. The protein was refolded by dilution in a L-arginine refolding buffer and refined by anion chromatography. The protein activity was detected by cytokine-dependent cell proliferation assay.</p><p><b>RESULTS</b>The expression of hIL-2/mGM-CSF in E. coli yielded approximately 20 mg protein /L culture and the purity was about 90%. The specific activities of IL-2 and GM-CSF were 5.4 x 10(6) IU/mg and 7.1 x 10(6) IU/mg, respectively.</p><p><b>CONCLUSION</b>This research provides important information about the anti-tumor activity of hIL-2/mGM-CSF in vivo, thus facilitating future clinical research on hIL-2/mGM-CSF used in immune therapy.</p>
Subject(s)
Animals , Humans , Mice , Arginine , Chemistry , Genetics , Metabolism , Base Sequence , Biological Assay , Cell Proliferation , Chromatography, Affinity , Chromatography, Ion Exchange , Cytokines , Metabolism , Escherichia coli , Genetics , Gene Expression , Granulocyte-Macrophage Colony-Stimulating Factor , Chemistry , Genetics , Metabolism , Interleukin-2 , Chemistry , Genetics , Metabolism , Molecular Sequence Data , Protein Folding , Protein Renaturation , Recombinant Fusion Proteins , Chemistry , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To study the CDR3 spectratyping and clonal expansion of T cells in the peripheral blood mononuclear cells (PBMCs) of patients with beta-mediterranean anemia patient undergoing allogeneic peripheral blood stem cell (PBSC) transplantation.</p><p><b>METHODS</b>The total RNA was isolated from the PBMCs of a nine-year-old boy with beta-mediterranean anemia before and after PBSC transplantation, and at the time of occurrence of graft-versus-host disease (GVHD). The CDR3 length was analyzed using immunoscope technique, and the characteristics of the T cell receptor (TCR) on the T cells with clonal expansion were examined by gene sequencing.</p><p><b>RESULTS</b>The 24 TCR BV CDR3 repertoire showed Gaussian distribution in the PBMCs isolated before the transplantation, and some of the TCR BV family CDR3 showed skewing in the PBMCs isolated 23 days after transplantation and at the onset of GVHD (28 days after transplantation), suggesting the clonal expansion of the donor PBSCs.</p><p><b>CONCLUSION</b>The host PBMCs show muti-clonal expansion 23 days after PBSC transplantation, and in the stage of GVHD, some of the TCR BV family T cells show significant monoclonal expansion. Analysis of TCR CDR3 spectratyping and the molecular characteristics of specific TCR may help evaluate the immune reconstitution following the transplantation and indicate specific treatment for potential GVHD.</p>
Subject(s)
Child , Humans , Male , Complementarity Determining Regions , Genetics , Allergy and Immunology , Graft vs Host Disease , Allergy and Immunology , Peripheral Blood Stem Cell Transplantation , Methods , Receptors, Antigen, T-Cell, alpha-beta , Genetics , Allergy and Immunology , beta-Thalassemia , Allergy and Immunology , General SurgeryABSTRACT
<p><b>OBJECTIVE</b>To assess the feasibility of recombinant type 1 adeno-associated virus (rAAV1) as a vector for gene therapy of corneal neovascularization.</p><p><b>METHODS</b>The rAAV1 vector carrying enhanced green fluorescence protein (EGFP) gene (rAAV1-EGFP) was transfected into ECV304 cells at different multiplicities of infection (MOI=5 x 10(3), 5 x 10(4), 5 x 10(5)). EGFP expression in the cells was observed under inverted fluorescence microscope, and the EGFP-positive cell percentage determined by flow cytometry. MTT assay was used to assess the proliferation of the transfected cells.</p><p><b>RESULTS</b>The cells with rAAV1-EGFP transfection at MOI of 5 x 10(5) began to exhibit GFP expression 2 days after transfection, and the fluorescence intensity increased with the MOI used for transfection. GFP expression reached the maximum on day 7, at the point of which the transduction efficiency of rAAV1-EGFP in ECV304 cells was 45.90%, 58.56% and 68.31% corresponding to MOIs of 5 x 10(3), 5 x 10(4), and 5 x 10(5), respectively. MTT assay did not reveal significant difference in the absorbance between the transfected cells and the control cells at 72 and 96 h after transfection.</p><p><b>CONCLUSION</b>arAAV1-EGFP gene can be stably and efficiently expressed in ECV304 cells without causing cell growth inhibition, suggesting the potential of rAAV1 as a safe and efficient vector for gene therapy of corneal neovascularization.</p>
Subject(s)
Humans , Cell Line , Corneal Neovascularization , Therapeutics , Dependovirus , Genetics , Endothelial Cells , Cell Biology , Metabolism , Genetic Therapy , Methods , Genetic Vectors , Green Fluorescent Proteins , Genetics , Metabolism , Microscopy, Fluorescence , Recombinant Fusion Proteins , Genetics , Metabolism , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To clone the full-length Rcet3 gene, a novel gene related to family 2 cystatins, from mouse testis or other tissues.</p><p><b>METHODS</b>Rcet3 gene was cloned using digital differential display (DDD) and RT-PCR was performed for cloning the full-length Rcet3 gene from adult mouse testis cDNA library with sequence analysis.</p><p><b>RESULTS</b>Rcet3 cDNA was 610 bp in length, consisting of 4 exons to encode a protein with 140 amino acid residues. The encoded protein contained a potential signal peptide and a cystatin domain, but lacked critical consensus site important for cysteine protease inhibition. These characteristics could be seen in the Cres subgroup related to the family 2 cystatins. Rcet3 was specifically expressed in adult mouse testis, epididymis and the cerebrum, but at higher levels in the testis than in the epididymis and cerebrum.</p><p><b>CONCLUSION</b>Rcet3 may be a new member of Cres subgroup of family 2 cystatins.</p>
Subject(s)
Animals , Male , Mice , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Cystatins , Genetics , DNA, Complementary , Chemistry , Genetics , Gene Expression Profiling , Mice, Inbred C57BL , Molecular Sequence Data , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Testis , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To analyze the drift of the complementarity-determining region 3 (CDR3) of T cell receptor beta (TCRbeta) chain variable region in T cells of healthy volunteers cultured with interleukin-2 (IL-2).</p><p><b>METHODS</b>T cells were isolated from the peripheral blood and cultured in vitro in the presence of IL-2. The non-specific killing effect of the cells was analyzed by LDH releasing assay, and the distribution of TCRbeta chain CDR3 in healthy volunteers by immunoscope spectratyping method to evaluate the clonality of the T cells.</p><p><b>RESULTS</b>The results showed Gaussian distribution of TCR Vbeta gene CDR3 in healthy volunteers. The T cell cultured with IL-2, however, displayed some anomalous and oligoclonal expansion in different TCR Vbeta families without killing effect against nasophargngal carcinoma cell line CNE2.</p><p><b>CONCLUSION</b>IL-2 may affect TCRbeta chain CDR3 distribution in T cells cultured in vitro.</p>
Subject(s)
Humans , Cells, Cultured , Complementarity Determining Regions , Genetics , Genetic Drift , Interleukin-2 , Metabolism , Leukocytes, Mononuclear , Metabolism , Receptors, Antigen, T-Cell, alpha-beta , Genetics , T-Lymphocytes , Allergy and Immunology , MetabolismABSTRACT
Vascular endothelial growth factor 121 (VEGF(121)) was expressed as inclusion bodies by recombinant Escherichia coli. High concentrations of both biomass (46 g dry cell/L) and VEGF(121) inclusion bodies (4.5 g/L) were obtained by applying a high-cell-density culture. After the inclusion bodies were washed and dissolved, VEGF(121) was refolded at 0.2 mg/ml by ultrafiltration in refolding buffer with a yield of 81%. Renatured VEGF(121) was purified by anion chromatography and Sephacry S-100 chromatography with purity higher than 95% and final purification yield of 31%. The purified VEGF(121) could stimulate the proliferation of human umbilical vein endothelial cells as demonstrated by a biological activity assay.
Subject(s)
Humans , Cell Line , Cell Proliferation , Dose-Response Relationship, Drug , Endothelial Cells , Cell Biology , Escherichia coli , Genetics , Metabolism , Inclusion Bodies , Metabolism , Protein Folding , Recombinant Proteins , Chemistry , Vascular Endothelial Growth Factor A , Genetics , PharmacologyABSTRACT
<p><b>OBJECTIVE</b>To observe the pathological changes in the blood vessels in rabbit femoral head with glucocorticoid-induced necrosis and investigate the pathogenesis of glucocorticoid-induced osteonecrosis.</p><p><b>METHODS</b>Twenty New Zealand white rabbits were randomly divided into two groups, namely group A. which was injected with horse serum and prednisone and group B as the control group. Chinese ink was injected into the femoral cavity of the rabbits to observe the blood vessels in the femoral head under optical microscope and the femoral head was examined histopathologically.</p><p><b>RESULTS</b>Compared with the normal control group, the rabbits in group A had significantly decreased number of perfused vessels, which was featured by defective perfusion, osteocytie pyknosis or necrosis, increase of empty ostoocyte lacunae and fat cells, decrease of hematopoietic tissue, and blood vessel occlusion.</p><p><b>CONCLUSION</b>Vascular occlusion and vasculitis due to glucocorticoid treatment may cause avascular necrosis of the femoral head.</p>
Subject(s)
Animals , Female , Male , Rabbits , Blood Vessels , Pathology , Femur Head , Pathology , Femur Head Necrosis , Pathology , Prednisolone , Random Allocation , Vasculitis , PathologyABSTRACT
The GFP(green fluorescence protein)-streptavidin(SA) bi-functional fusion protein was generated and characterized in order to demonstrate novel platform for efficiently and durably modifying the cell surface with SA-tagged bi-functional proteins.The GFP-SA/pET24d construct was generated and expressed in BL21(DE3) host bacteria at the high level.The recombinant protein GFP-SA was purified through the Ni-NTA affinity chromatography,and then refolded.After biotinylation B16 tumor cells were modified with GFP-SA bi-functional fusion protein and then subjected to fluorescent microscopy and FACS analysis.The effect of surface modification on the viability and growth of B16.F10 tumor cells was evaluated by MTT staining.The GFP-SA recombinant fusion protein was expressed in BL21(DE3) at about 20 % of total bacterial proteins.The GFP-SA bi-functional fusion protein exhibited the bi-functionality,i.e.,SA-mediated high-affinity binding to biotinylated cell surfaces and GFP-emitted green fluorescence.The cell surface modification with GFP-SA bi-functional fusion protein did not affect the viability and growth of the modified B16.F10 tumor cells significantly.The GFP-SA bi-functional fusion protein was obtained and could be displayed efficiently on the surface of the biotinylated B16.F10 tumor cells through the specific and tight interaction between streptavidin and biotin,thus can be used as good trace protein and experimental control in the development of surface-modified tumor vaccine.