OBJECTIVETo study the expression of histone acetyltransferases (HATs) and histone deacetylases (HDACs) in children with tetralogy of Fallot (TOF), and to investigate the role of histone acetylation and acetylation-related enzymes in the pathogenesis of TOF.

METHODSMyocardial tissue samples in the TOF group were obtained from 46 children with TOF who underwent radical operation, and myocardial tissue samples in the control group were obtained from 16 children who suffered accidental deaths and had no cardiac anomalies as shown by autopsy. The acetylation of H3K9, H3K18 and H3K27 was evaluated by immunohistochemistry. The mRNA expression of HATs and HDACs in the myocardium was measured by real-time PCR. The correlation between mRNA expression of HATs and HDACs and histone acetylation was analyzed.

RESULTSCompared with the control group, the TOF group showed significantly increased acetylation of H3K9 (P=0.0165) and significantly decreased acetylation of H3K18 (P=0.0048) and H3K27 (P=0.0084). As to 4 HATs and 6 HDACs, the mRNA expression of EP300 and CBP was significantly higher in the TOF group than in the control group (P=0.025; P=0.017), and there was no significant difference in the mRNA expression of other HATs and HDACs between the two groups. The correlation analysis revealed a positive correlation between H3K9 acetylation and mRNA expression of EP300 (r=0.71, P<0.01) and CBP (r=0.72, P<0.01).

CONCLUSIONSUpregulated mRNA expression of EP300 and CBP may be associated with increased H3K9 acetylation, suggesting that EP300 and CBP might affect cardiac development by regulating H3K9 acetylation.

Acetylation ; E1A-Associated p300 Protein ; genetics ; Female ; Histone Acetyltransferases ; genetics ; Histone Deacetylases ; genetics ; Histones ; metabolism ; Humans ; Infant ; Male ; Myocardium ; metabolism ; Peptide Fragments ; genetics ; RNA, Messenger ; analysis ; Sialoglycoproteins ; genetics ; Tetralogy of Fallot ; metabolism

OBJECTIVETo regenerate dentin-pulp complex by tissue engineering with human stem cells from apical papilla cells (SCAP) as the seed cells.

METHODSSCAP was separated from from normal human impacted third molars with immature roots by outgrowth culture. The cells were then cultured in the differentiation medium for 3 weeks or in normal medium for 60 days, and analyzed for mineralization potential by Alizarin red staining. The osteo/odontogenic markers including alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OC) and dentin sialoprotein (DSP) were investigated by immunofluorescence staining and reverse transcription-polymerase chain reaction. The co-cultured mixture of SCAP and HA/TCP, or HA/TCP alone was implanted subcutaneously on the back of nude mice for 8 weeks, and the implants were collected and examined by HE and immunohistochemical staining.

RESULTSRound alizarin red-positive nodules formed in the isolated cells after cell culture in the differentiation medium for 3 weeks or in normal medium for 60 days with positive staining for osteo/odontogenic markers. SCAP with HA/TCP could regenerate pulp-dentin complex-like tissue in nude mice. The cells near the dentin-like tissue were positive for DSP. No mineral tissue was found in mice receiving HA/TCP implantation.

CONCLUSIONSSCAP may serve as a promising seed cell for dentin-pulp complex tissue engineering.

Adolescent ; Adult ; Alkaline Phosphatase ; analysis ; Animals ; Cell Culture Techniques ; Cell Differentiation ; Coculture Techniques ; Dental Papilla ; cytology ; Dental Pulp ; cytology ; Extracellular Matrix Proteins ; analysis ; Female ; Humans ; Integrin-Binding Sialoprotein ; analysis ; Mice ; Mice, Nude ; Odontogenesis ; physiology ; Osteocalcin ; analysis ; Phosphoproteins ; analysis ; Sialoglycoproteins ; analysis ; Stem Cells ; chemistry ; physiology ; Tissue Engineering ; methods ; Young Adult

OBJECTIVE: Supplementation with vitamin E is able to protect bone against free radical-induced elevation of bone-resorbing cytokines. We examined gene expression by microarray analysis during the differentiation of human mesenchymal stem cells treated with vitamin E into osteoblasts in vitro. METHODS: Human bone marrow stem cells were cultured in osteogenic differentiation medium and vitamin E was added. A colorimetric immunoassay for the quantification of cell proliferation was used to measure osteoblast differentiation. Gene expression was analyzed using a microarray technique. We also used a real time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: It was found that vitamin E enhanced cell proliferation when compared to cells cultured in media without vitamin E. We focused on 68 genes which are related to osteogenesis and osteoclastogenesis. Alkaline phosphatase, transforming growth factor-beta 1, fibroblast growth factor receptor 1, matrix metalloproteinase 2, muscle segment homeobox 2, bone morphogenetic protein 1, biglycan, vascular endothelial growth factor B, dentin sialophosphoprotein, cartilage oligomeric matrix protein, runt-related transcription factor 2, fibroblast growth factor receptor 3, and SMAD2 were upregulated > 2-fold compared to the control. Conversely, osteopetrosis-associated transmembrane protein 1, microphthalmia-associated transcription factor, and epidermal growth factor receptor were downregulated > 2-fold compared to the control. Vitamin E produced a 1.5-fold increase in the expression of runt-related transcription factor 2 and transforming growth factor-beta 1 as determined by real time RT-PCR. CONCLUSION: Vitamin E had a positive effect on the gene expressions regarding osteogenic differentiation of mesenchymal stem cells.
Alkaline Phosphatase ; Biglycan ; Bone Marrow ; Bone Morphogenetic Protein 1 ; Cartilage ; Cell Proliferation ; Cytokines ; Dentin ; Durapatite ; Extracellular Matrix Proteins ; Gene Expression ; Genes, Homeobox ; Glycoproteins ; Humans ; Immunoassay ; Matrix Metalloproteinase 2 ; Mesenchymal Stromal Cells ; Microarray Analysis ; Microphthalmia-Associated Transcription Factor ; Muscles ; Osteoblasts ; Osteogenesis ; Phosphoproteins ; Receptor, Epidermal Growth Factor ; Receptor, Fibroblast Growth Factor, Type 1 ; Receptor, Fibroblast Growth Factor, Type 3 ; Sialoglycoproteins ; Stem Cells ; Transcription Factors ; Vascular Endothelial Growth Factor B ; Vitamin E ; Vitamins

BACKGROUNDHuman adipose tissue-derived stromal cells (hADSCs) can be induced to differentiate along an osteoblastic lineage under stimulation of dexamethasone (DEX). Recent studies, however, have questioned the efficacy of glucocorticoids such as DEX in mediating the osteogenesis process of skeletal progenitor cells and processed lipoaspirate cells. Is it possible to find a substitute for DEX? Therefore, this study was designed to investigate osteogenic capacity and regulating mechanisms for osteoblastic differentiation of hADSCs by comparing osteogenic media (OM) containing either 1, 25-dihydroxyvitamin D(3) (VD) or DEX and determine if VD was an ideal substitute for DEX as an induction agent for the osteogenesis of hADSCs.

METHODSOsteogenic differentiation of hADSCs was induced by osteogenic medium (OM) containing either 10 nmol/L VD or 100 nmol/L DEX. Differentiation of hADSCs into osteoblastic lineage was identified by alkaline phosphatase (ALP) staining, von Kossa staining, and reverse transcription-polymerase chain reaction assays for mRNA expression of osteogenesis-related genes such as type I collagen (COL I), bone sialoprotein (BSP), osteocalcin (OC), bone morphogenetic protein (BMP)-2, BMP-4, BMP-6, BMP-7, runt-related transcription factor 2/core binding factor alpha1 (Runx2/Cbfa1), osterix (Osx), and LIM mineralization protein-1 (LMP-1).

RESULTSvon Kossa staining revealed that the differentiated cells induced by both VD and DEX were mineralized in vitro. They also expressed osteoblast-related markers, such as ALP, COL I, BSP, and OC. Runx2/Cbfa1, Osx, BMP-6, and LMP-1 were upregulated during VD and DEX-induced hADSC osteoblastic differentiation, but BMP-4, BMP-7 were not. BMP-2 was only expressed in VD-induced differentiated cells.

CONCLUSIONSVD or DEX-induced hADSCs differentiate toward the osteoblastic lineage in vitro. Runx2/Cbfa1, Osx, BMP-2, BMP-6, and LMP-1 are involved in regulating osteoblastic differentiation of hADSCs, but BMP-4, BMP-7 are not. VD, but not DEX, induces expression of BMP-2 during osteogenic induction of hADSCs. VD is an ideal substitute for DEX for osteogenic induction of hADSCs.

Adaptor Proteins, Signal Transducing ; Adipose Tissue ; cytology ; Adult ; Calcitriol ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Collagen Type I ; genetics ; Cytoskeletal Proteins ; Dexamethasone ; pharmacology ; Humans ; Integrin-Binding Sialoprotein ; Intracellular Signaling Peptides and Proteins ; genetics ; LIM Domain Proteins ; Middle Aged ; Osteoblasts ; cytology ; drug effects ; Osteocalcin ; genetics ; Osteogenesis ; drug effects ; RNA, Messenger ; analysis ; Sialoglycoproteins ; genetics ; Stromal Cells ; cytology ; drug effects

OBJECTIVETo clone and sequence the upstream of mouse dentin sialophosphoprotein promoter.

METHODSGenomic DNA was obtained from Balb/c mouse blood. The upstream of mouse dentin sialophosphoprotein promoter segments was obtained by PCR. Then the segments were inserted into T-vector. The plasmids were identified by digestion with the restriction enzyme analysis. The positive clone was sequenced and compared with Genebank.

RESULTSThe upstream of mouse dentin sialophosphoprotein promoter was divided into three sequences and three different target segments with 997 bp, 1004 bp and 674 bp in length were obtained. After identified, sequenced and compared with Genebank, the sequences of the segments were consistent with those displayed on Genebank by 99%.

CONCLUSIONThe clone of the upstream of mouse dentin sialophosphoprotein promoter was successful. This work will help to study the regulation of DSPP expression.

Animals ; Cloning, Molecular ; Extracellular Matrix Proteins ; genetics ; Mice ; Mice, Inbred BALB C ; Phosphoproteins ; genetics ; Promoter Regions, Genetic ; Sequence Analysis, DNA ; Sialoglycoproteins ; genetics

BACKGROUNDImmune rejection is the main reason of grafts failure after corneal transplantation. This study was to determine whether interleukin-1 receptor antagonist (IL-1ra) eye drops could prolong corneal allografts survival in high-risk corneal orthotopic allotransplantation in rat model and to study the effect of IL-1ra on the expression of CD1-positive cells in the grafts.

METHODSFor all experiments, the Sprague-Dawley (SD) rats' corneas were transplanted into Wistar rats' eyes. High-risk transplants included those that had been sutured into Wistar recipient beds with corneal neovascularization induced by placement of three interrupted sutures in the host cornea 7 days earlier. All the animals were divided, in a masked fashion, into three treatment groups and one control group. Each treatment group received IL-1ra eye drops of different concentrations (1 mg/ml, 3 mg/ml, or 5 mg/ml, respectively) four times a day for 30 days. The control group received 0.9% normal saline (NS) eye drops in the same way as the treatment groups. All allografts were evaluated for signs of rejection from the first day after surgery. Ten days later, corneal specimens were processed to examine the expression of CD1-positive cells and histopathological changes.

RESULTSThe survival time of the transplants was 5.80 +/- 0.79, 5.89 +/- 1.05, 6.78 +/- 0.83, and 9.00 +/- 2.36 days respectively in the control or three treatment groups. Compared with the control group, 1 mg/ml IL-1ra eye drop did not prolong the survival time of the allografts (t = 0.210, P > 0.05). However, 3 mg/ml and 5 mg/ml IL-1ra eye drop did prolong the survival time of the grafts (t >or= 2.627, P < 0.05), with the latter showing more obvious effect. Immunohistochemical examinations showed a significant decrease in inflammatory cell and CD1-positive cell infiltration in IL-1ra treated groups compared with the control group.

CONCLUSIONSIL-1ra can promote corneal allograft survival in a dose-dependant manner by reducing the infiltration of CD1-positive cells in high-risk corneal transplantation.

Animals ; Antigens, CD1 ; analysis ; Cornea ; pathology ; Corneal Transplantation ; Female ; Graft Survival ; drug effects ; Immunohistochemistry ; Interleukin 1 Receptor Antagonist Protein ; Ophthalmic Solutions ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Risk ; Sialoglycoproteins ; administration & dosage ; Transplantation, Homologous

OBJECTIVETo test the bovine cementoblasts (CBs) cementum-forming ability in vivo.

METHODSRoot fragments of newborn bovine freshly extracted mandibular incisor were cultured routinely and 4th-5th passages of CBs were harvested. CBs were then cultured in the medium supplemented with 50 mg/L alpha-ascorbic acid and 10 mmol/l beta-glycerolphosphate to form a thick layer as tissue engineering scaffold for cementum formation. Collagen membrane was used as control scaffold. 2 x 10(6) cells were attached to the CBs-made carrier as well as collagen membrane scaffolds and transplanted subcutaneously into immunodeficient mice. Transplants were harvested at 7th week. Histological sections were stained with HE, alizarin red S and van Kossa methods as well as monoclonal Ab against bovine cementum attachment protein (CAP).

RESULTSCBs-made scaffold supported more cementum-like tissue (CLT) formation than collagen-made scaffold. The CLT formed on CBs scaffold was partly calcified with embedded cells. Uncalcified cementoid-like material could be seen on the surface and was encircled by cubical CB-like cells. The CLT was also positive to CAP and van Kossa staining.

CONCLUSIONSThese results suggest that the bovine CBs can form cementum-like tissue. The cell-made carrier is a better scaffold than collagen membrane.

Alkaline Phosphatase ; analysis ; Animals ; Bone Transplantation ; methods ; Cattle ; Cell Adhesion Molecules ; analysis ; Cells, Cultured ; Dental Cementum ; chemistry ; cytology ; transplantation ; Immunohistochemistry ; Integrin-Binding Sialoprotein ; Male ; Mice ; Mice, Nude ; Osteocalcin ; analysis ; Osteonectin ; analysis ; Sialoglycoproteins ; analysis ; Tissue Engineering ; methods ; Transplantation, Heterologous

BACKGROUND: The human immune response to Mycobacterium tuberculosis is mediated by macrophages and T-lymphocytes. The alveolar macrophage phagocyting mycobacterium produces interleukin (IL) -1 as an inflammatory mediator, and IL-8 as a cytokine for leukocyte recruitment and granuloma formation. Interleukin-1 receptor antagonist (IL-1ra) is an internal antagonist of IL-1. METHODS: Plasma levels of IL-1ra and IL-8 and other serologic markers were measured in 18 patients with active tuberculosis before treatment and after 2 months and 6 months of treatment. RESULTS: During treatment with antituberculous medication, patients showed significant changes in hemoglobin, hematocrit, white blood cells (WBC), platelet, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), ferritin and plasma IL-1ra. After 2 months of treatment, ESR and CRP diminished significantly; after 6 months, hemoglobin increased while WBC, platelet, ESR, CRP and ferritin decreased significantly compared to their pre-treatment levels. There were two groups: patients with delayed therapeutic responses, and patients with early responses. At each point of observation, the former group of patients showed lower body weight and lower levels of hemoglobin and hematocrit, and higher levels of WBC, platelet, ESR, IL-8 and IL-1ra than the latter group. During the course of the treatment, we observed considerable differences in body weight, body mass index, hemoglobin, hematocrit, WBC and platelet counts, ESR, CRP and ferritin in both the early-response and delayed-response groups. CONCLUSION: We believe that the plasma concentrations of IL-1ra and IL-8, which showed different peaks during the course of treatment, reflected their different functions and patterns of secretion. Moreover the concentrations did not seem as sensitive as other inflammatory markers to evaluate disease activity during antituberculosis treatment. However, IL-1ra can be considered a marker for disease activity and response to treatment.
Adult ; Aged ; Aged, 80 and over ; Antitubercular Agents/therapeutic use ; Biological Markers/*blood ; C-Reactive Protein/analysis ; Comparative Study ; Female ; Human ; Interleukin-8/*blood ; Male ; Middle Aged ; Receptors, Interleukin-1/*antagonists & inhibitors ; Sialoglycoproteins/*blood ; Tuberculosis, Pulmonary/*blood/*drug therapy

OBJECTIVETo investigate the osteoblast-like functional characteristics exhibited by human periodontal ligament cells (hPDLCs) under mechanical force.

METHODSHuman PDLCs cultured in vitro were stretched by mechanical force. Radioimmunoassay (RIA) was used to measure the expression of secreting alkaline phosphotase (ALP) and osteocalcin (OCN). The non-secreting ALP, OCN and osteopontin (OPN) in cells were determined by immunohistochemistry.

RESULTSIt exhibited increasing of ALP secreted into conditional media, and in the 24 hour period there were two peaks which appeared at the 2nd and 4th hour and the 24th hour (P < 0.01). While in the late of the 24 hours, expression of OCN in conditional media increased (P < 0.05).

CONCLUSIONMechanical force induces hPDLCs to differentiate into functional osteoblast-like cells and plays a role in bone remodeling.

Alkaline Phosphatase ; metabolism ; Cells, Cultured ; Humans ; Osteocalcin ; analysis ; Osteoclasts ; physiology ; Osteopontin ; Periodontal Ligament ; cytology ; Sialoglycoproteins ; analysis ; Stress, Mechanical

OBJECTIVETo investigate the possible association of osteopontin (OPN) with liver metastasis in colorectal cancer.

METHODSRNA was quantitated by RT-PCR from colorectal cancer and surrounding normal tissues, also from liver metastatic tissues and normal liver tissues. ISH was utilized to detect OPN mRNA in colorectal cancer and liver metastatic tissues.

RESULTSIn 44 cases, the expressions of OPN mRNA in colorectal cancer tissues were higher than those in the surrounding normal tissues (t = 4.89, P = 0.000). The expression of OPN mRNA in colorectal cancer was as high as 18.2%, but in liver metastasis was 80% (chi(2) = 22.42, P = 0.000). In ISH, OPN mRNA located in the cytoplasm of colorectal cancer cells.

CONCLUSIONSThe expressions of OPN mRNA were the highest in liver metastatic tissues, moderate in colorectal cancer tissues, and lowest in surrounding normal tissues, indicating that OPN is a potential marker for liver metastasis of colorectal cancer.

Adult ; Aged ; Aged, 80 and over ; Colon ; metabolism ; Colorectal Neoplasms ; metabolism ; pathology ; Female ; Humans ; In Situ Hybridization ; Liver ; metabolism ; Liver Neoplasms ; metabolism ; secondary ; Male ; Middle Aged ; Neoplasm Staging ; Osteopontin ; RNA, Messenger ; analysis ; Sialoglycoproteins ; genetics