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1.
Article in Chinese | WPRIM | ID: wpr-879535

ABSTRACT

OBJECTIVE@#To compare the mRNA level of cell proliferation-related genes Twist1, SIRT1, FGF2 and TGF-β3 in placenta mesenchymal stem cells (PA-MSCs), umbilical cord mensenchymals (UC-MSCs) and dental pulp mesenchymal stem cells (DP-MSCs).@*METHODS@#The morphology of various passages of PA-MSCs, UC-MSCs and DP-MSCs were observed by microscopy. Proliferation and promoting ability of the three cell lines were detected with the MTT method. Real-time PCR (RT-PCR) was used to determine the mRNA levels of Twist1, SIRT1, FGF2, TGF-β3.@*RESULTS@#The morphology of UC-MSCs and DP-MSCs was different from that of PA-MSCs. Proliferation ability and promoting ability of the PA-MSCs was superior to that of UC-MSCs and DP-MSCs. In PA-MSCs, expression level of Twist1 and TGF-β3 was the highest and FGF2 was the lowest. SIRT1 was highly expressed in UC-MSCs. With the cell subcultured, different expression levels of Twist1, SIRT1, FGF2, TGF-β3 was observed in PA-MSCs, UC-MSCs and DP-MSCs.@*CONCLUSION@#Up-regulated expression of the Twist1, SIRT1 and TGF-β3 genes can promote proliferation of PA-MSCs, UC-MSCs and DP-MSCs, whilst TGF-β3 may inhibit these. The regulatory effect of Twist1, SIRT1, FGF2 and TGF-β3 genes on PA-MSCs, UC-MSCs and DP-MSCs are different.


Subject(s)
Cell Differentiation , Cell Proliferation/genetics , Cells, Cultured , Dental Pulp/cytology , Female , Fibroblast Growth Factor 2/genetics , Humans , Mesenchymal Stem Cells/cytology , Nuclear Proteins/genetics , Placenta/cytology , Pregnancy , Sirtuin 1/genetics , Transforming Growth Factor beta3/genetics , Twist-Related Protein 1/genetics , Umbilical Cord/cytology
2.
J. appl. oral sci ; 26: e20170231, 2018. graf
Article in English | LILACS, BBO | ID: biblio-893679

ABSTRACT

Abstract We previously reported that elevated extracellular calcium (Ca2+) levels increase bone morphogenetic protein 2 expression in human dental pulp (hDP) cells. However, it is unknown whether extracellular Ca2+ affects the expression of other growth factors such as fibroblast growth factor 2 (FGF2). Objective: The present study aimed to examine the effect of extracellular Ca2+ on FGF2 gene expression in hDP and immortalized mouse dental papilla (mDP) cells. Materials and Methods: Cells were stimulated with 10 mM CaCl2 in the presence or absence of cell signaling inhibitors. FGF2 gene expression was assessed using real-time polymerase chain reaction. The phosphorylation status of signaling molecules was examined by Western blotting. Results: Extracellular Ca2+ increased FGF2 gene expression in mDP and hDP cells. Gene expression of the calcium-sensing receptor and G protein-coupled receptor family C group 6 member A, both of which are extracellular Ca2+ sensors, was not detected. Ca2+-mediated Fgf2 expression was reduced by pretreatment with the protein kinase A (PKA) inhibitor H-89 or extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 but not by pretreatment with the protein kinase C inhibitor GF-109203X or p38 inhibitor SB203580. Extracellular Ca2+ increased PKA activity and ERK1/2 phosphorylation. Ca2+-induced PKA activity decreased by pretreatment with PD98059. Conclusions: These findings indicate that elevated extracellular Ca2+ levels led to increased Fgf2 expression through ERK1/2 and PKA in mDP cells and that this mechanism may be useful for designing regenerative therapies for dentin.


Subject(s)
Animals , Mice , Gene Expression/drug effects , Calcium/pharmacology , Fibroblast Growth Factor 2/drug effects , Cyclic AMP-Dependent Protein Kinases/drug effects , Mitogen-Activated Protein Kinase 1/drug effects , Dental Papilla/drug effects , Mitogen-Activated Protein Kinase 3/drug effects , Time Factors , Calcium Chloride/pharmacology , Enzyme-Linked Immunosorbent Assay , Cells, Cultured , Blotting, Western , Reproducibility of Results , Fibroblast Growth Factor 2/analysis , Fibroblast Growth Factor 2/genetics , Cyclic AMP-Dependent Protein Kinases/analysis , Mitogen-Activated Protein Kinase 1/analysis , MAP Kinase Signaling System/drug effects , Mitogen-Activated Protein Kinase 3/analysis , Real-Time Polymerase Chain Reaction
3.
Article in English | WPRIM | ID: wpr-50915

ABSTRACT

MicroRNAs (miRNAs) regulate the proliferation and metastasis of cancer cells. Here, we showed that miR-152 was downregulated in non-small-cell lung cancer (NSCLC) tissues and cell lines. Overexpression of miR-152 suppressed cell proliferation and colony formation and also limited migration and invasion. Fibroblast growth factor 2 (FGF2) was confirmed as a direct target of miR-152. FGF2 knockdown suppressed cell proliferation, colony formation, migration and invasion, whereas FGF2 overexpression partially reversed the suppressive effect of miR-152. Furthermore, the presence of miR-152 was inversely correlated with FGF2 in NSCLC tissues. Overall, this study demonstrated that miR-152 suppressed the proliferation and invasion of NSCLC cells by downregulating FGF2. These findings provide novel insights with potential therapeutic applications for the treatment of NSCLC.


Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Cell Movement , Cell Proliferation , Female , Fibroblast Growth Factor 2/genetics , Gene Expression Regulation, Neoplastic , Humans , Lung/metabolism , Lung Neoplasms/genetics , Male , MicroRNAs/genetics , Middle Aged , Neoplasm Invasiveness/genetics
4.
Article in English | WPRIM | ID: wpr-56433

ABSTRACT

The present study was conducted to investigate the effect of silymarin on experimental liver toxication induced by Fumonisin B1 (FB1) in BALB/c mice. The mice were divided into six groups (n = 15). Group 1 served as the control. Group 2 was the silymarin control (100 mg/kg by gavage). Groups 3 and 4 were treated with FB1 (Group 3, 1.5 mg/kg FB1, intraperitoneally; and Group 4, 4.5 mg/kg FB1). Group 5 received FB1 (1.5 mg/kg) and silymarin (100 mg/kg), and Group 6 was given a higher dose of FB1 (4.5 mg/kg FB1) with silymarin (100 mg/kg). Silymarin treatment significantly decreased (p < 0.0001) the apoptotic rate. FB1 administration significantly increased (p < 0.0001) proliferating cell nuclear antigen and Ki-67 expression. Furthermore, FB1 elevated the levels of caspase-8 and tumor necrosis factor-alpha mediators while silymarin significantly reduced (p < 0.0001) the expression of these factors. Vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) expressions were significantly elevated in Group 4 (p < 0.0001). Silymarin administration alleviated increased VEGF and FGF-2 expression levels (p < 0.0001). In conclusion, silymarin ameliorated toxic liver damage caused by FB1 in BALB/c mice.


Subject(s)
Animals , Antioxidants/pharmacology , Apoptosis/drug effects , Cell Proliferation/drug effects , Female , Fibroblast Growth Factor 2/genetics , Fumonisins/toxicity , Gene Expression Regulation/drug effects , Hepatocytes/drug effects , Ki-67 Antigen/metabolism , Liver/drug effects , Mice , Mice, Inbred BALB C , Mycotoxins/toxicity , Neovascularization, Physiologic/drug effects , Proliferating Cell Nuclear Antigen/metabolism , Silymarin/pharmacology , Tumor Necrosis Factor-alpha/metabolism , Vascular Endothelial Growth Factor A/genetics
5.
Article in English | WPRIM | ID: wpr-187635

ABSTRACT

Gene transfer of basic fibroblast growth factor (bFGF) has been shown to induce significant endothelial migration and angiogenesis in ischemic disease models. Here, we investigate what factors are secreted from skeletal muscle cells (SkMCs) transfected with bFGF gene and whether they participate in endothelial cell migration. We constructed replication-defective adenovirus vectors containing the human bFGF gene (Ad/bFGF) or a control LacZ gene (Ad/LacZ) and obtained conditioned media, bFGF-CM and LacZ-CM, from SkMCs infected by Ad/bFGF or Ad/LacZ, respectively. Cell migration significantly increased in HUVECs incubated with bFGF-CM compared to cells incubated with LacZ-CM. Interestingly, HUVEC migration in response to bFGF-CM was only partially blocked by the addition of bFGF-neutralizing antibody, suggesting that bFGF-CM contains other factors that stimulate endothelial cell migration. Several proteins, matrix metalloproteinase-1 (MMP-1), plasminogen activator inhibitor-1 (PAI-1), and cathepsin L, increased in bFGF-CM compared to LacZ-CM; based on 1-dimensional gel electrophoresis and mass spectrometry. Their increased mRNA and protein levels were confirmed by RT-PCR and immunoblot analysis. The recombinant human bFGF protein induced MMP-1, PAI-1, and cathepsin L expression in SkMCs. Endothelial cell migration was reduced in groups treated with bFGF-CM containing neutralizing antibodies against MMP-1 or PAI-1. In particular, HUVECs treated with bFGF-CM containing cell-impermeable cathepsin L inhibitor showed the most significant decrease in cell migration. Cathepsin L protein directly promotes endothelial cell migration through the JNK pathway. These results indicate that cathepsin L released from SkMCs transfected with the bFGF gene can promote endothelial cell migration.


Subject(s)
Antibodies, Neutralizing/immunology , Cathepsin L/genetics , Cell Movement , Cells, Cultured , Comet Assay , Dependovirus/genetics , Endothelial Cells/cytology , Fibroblast Growth Factor 2/genetics , Gene Transfer Techniques , Humans , Immunoblotting , JNK Mitogen-Activated Protein Kinases , Lac Operon/genetics , Mass Spectrometry , Matrix Metalloproteinase 1/biosynthesis , Muscle, Skeletal/metabolism , Neovascularization, Physiologic , Plasminogen Activator Inhibitor 1/biosynthesis , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction
6.
Article in English | WPRIM | ID: wpr-122147

ABSTRACT

Platelet-rich plasma (PRP) contains growth factors that promote tissue regeneration. Previously, we showed that heparin-conjugated fibrin (HCF) exerts the sustained release of growth factors with affinity for heparin. Here, we hypothesize that treatment of skin wound with a mixture of PRP and HCF exerts sustained release of several growth factors contained in PRP and promotes skin wound healing. The release of fibroblast growth factor 2, platelet-derived growth factor-BB, and vascular endothelial growth factor contained in PRP from HCF was sustained for a longer period than those from PRP, calcium-activated PRP (C-PRP), or a mixture of fibrin and PRP (F-PRP). Treatment of full-thickness skin wounds in mice with HCF-PRP resulted in much faster wound closure as well as dermal and epidermal regeneration at day 12 compared to treatment with either C-PRP or F-PRP. Enhanced skin regeneration observed in HCF-PRP group may have been at least partially due to enhanced angiogenesis in the wound beds. Therefore, this method could be useful for skin wound treatment.


Subject(s)
Animals , Blotting, Western , Cell Proliferation , Dermis/cytology , Female , Fibrin/metabolism , Fibroblast Growth Factor 2/genetics , Heparin/metabolism , Immunoenzyme Techniques , Intercellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Inbred BALB C , Platelet-Rich Plasma/metabolism , Proto-Oncogene Proteins c-sis/genetics , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Regeneration , Skin/cytology , Vascular Endothelial Growth Factor A/genetics , Wound Healing/physiology
7.
Article in English | WPRIM | ID: wpr-200108

ABSTRACT

Although human telomerase catalytic subunit (TERT) has several cellular functions including telomere homeostasis, genomic stability, cell proliferation, and tumorigenesis, the molecular mechanism underlying anti-apoptosis regulated by TERT remains to be elucidated. Here, we show that ectopic expression of TERT in spontaneously immortalized human fetal fibroblast (HFFS) cells, which are a telomerase- and p53-positive, leads to increases of cell proliferation and transformation, as well as a resistance to DNA damage response and inactivation of p53 function. We found that TERT and a mutant TERT (no telomerase activity) induce expression of basic fibroblast growth factor (bFGF), and ectopic expression of bFGF also allows cells to be resistant to DNA-damaging response and to suppress activation of p53 function under DNA-damaging induction. Furthermore, loss of TERT or bFGF markedly increases a p53 activity and DNA-damage sensitivity in HFFS, HeLa and U87MG cells. Therefore, our findings indicate that a novel TERT-bFGF axis accelerates the inactivation of p53 and consequent increase of resistance to DNA-damage response.


Subject(s)
Apoptosis , Catalytic Domain , Cell Line, Transformed , Cell Proliferation , DNA Damage , Fetus/cytology , Fibroblast Growth Factor 2/genetics , Fibroblasts/cytology , Gene Expression Regulation, Neoplastic , HeLa Cells , Humans , RNA, Messenger/genetics , Telomerase/deficiency , Tumor Suppressor Protein p53/metabolism
8.
Article in English | WPRIM | ID: wpr-193635

ABSTRACT

The study investigated the effects of adenovirus-mediated gene transfection of basic fibroblast growth factor (bFGF), bFGF combined with interleukin-1 receptor antagonist protein (IL-Ra) and/or insulin-like growth factor-1 (IGF-1) both in human osteoarthritis (OA) chondrocytes and rabbits OA model. Human OA chondrocytes were delivered by adenovirus-mediated bFGF, IL-Ra and IGF-1 vectors, respectively. Chondrocyte proliferation, glycosaminoglycan (GAG) content, expression of type II collagen, ADAMTS-5, MMP-13, MMP-3 and TIMP-1 were determined. Rabbit OA model was induced by anterior cruciate ligament transaction (ACLT) in knees. Adenoviral vectors encoding human bFGF, IL-Ra and IGF-1 were injected intraarticularly into the knee joints after ACLT. The effects of adenovirus- mediated gene transfection on rabbit OA were evaluated. In vitro, the transfected genes were expressed in cell supernatant of human OA chondrocytes. AdbFGF group significantly promoted chondrocyte proliferation, and increased GAG and type II collagen synthesis than in the OA group. As two or three genes were transfected in different combinations, there was significant enhancement on the GAG content, type II collagen synthesis, and TIMP-1 levels, while ADAMTS-5, MMP-13, and MMP-3 levels were reduced. In vivo, the transfected genes were expressed in synovial fluid of rabbits. Intraarticular delivery of bFGF enhanced the expression of type II collagen in cartilage and decreased cartilage Mankin score compared with the OA control group (P = 0.047; P < 0.01, respectively). Multiple-gene transfection in different combinations showed better results than bFGF transfection alone. This study suggests that bFGF gene transfection is effective in treating experimental OA. Multiple gene transfection has better biologic effects on OA.


Subject(s)
Adenoviridae/genetics , Animals , Chondrocytes/drug effects , Collagen Type II/genetics , Fibroblast Growth Factor 2/genetics , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Humans , Insulin-Like Growth Factor I/genetics , Interleukin 1 Receptor Antagonist Protein/genetics , Interleukin-1/genetics , Matrix Metalloproteinase 13/genetics , Matrix Metalloproteinase 3/genetics , Osteoarthritis/therapy , Rabbits , Tissue Inhibitor of Metalloproteinase-1/genetics , Transfection
9.
Article in English | WPRIM | ID: wpr-207075

ABSTRACT

We investigated expression profiles and biological effects of the naked DNA vectors in the heart. To this end, naked DNA vector was injected into the apex of the beating rat heart after thorocotomy. When the expression of LacZ reporter was examined by reverse transcription-PCR and histochemical staining for b-galactosidase, LacZ expression was detected only in the heart, suggesting limited dissemination of the injected vector in vivo. Even within the heart, LacZ expression was limited to the injection area (apex). Similar observations were made with other transgenes such as VEGF and basic fibroblast growth factor (bFGF), where 77% and 69% of the total transgene exprssion were detected in the heart segments containing the apex. Although VEGF and bFGF expressions were detected until 2 weeks after DNA injection, the highest levels of VEGF and bFGF were observed on day 5 and day 1, respectively. The optimal doses of the vectors were 10 mg and 25 mg for the VEGF and bFGF vectors, respectively. Interestingly, injection of bFGF vector led to 50% increase in the level of endogenous murine VEGF expression. Consistent with this finding, the number of vessels that stained positive for alpha-smooth muscle actin was increased in the bFGF vector-injected heart. These results suggest that simple injection of naked DNA vector may be sufficient to induce significant angiogenesis in the myocardium and that naked DNA gene therapy may be a feasible approach for the treatment of ischemic heart disease.


Subject(s)
Animals , Coronary Vessels/metabolism , DNA/genetics , Fibroblast Growth Factor 2/genetics , Gene Expression Profiling/methods , Gene Expression Regulation , Genes, Reporter/genetics , Genetic Vectors/genetics , Lac Operon/genetics , Male , Myocardium/metabolism , Rats , Rats, Sprague-Dawley , Time Factors , Transgenes/genetics , Vascular Endothelial Growth Factor A/genetics
10.
Article in English | WPRIM | ID: wpr-634038

ABSTRACT

To construct basic fibroblast growth factor (bFGF) eukaryotic expression vector and to evaluate the possibility of bFGF gene therapy in orthopedic disease, the pCD-rbFGF recombinant plasmid was constructed by cloning rat basic fibroblast growth factor (bFGF) cDNA into an eukaryotic expression vector, pcDNA3. Rat osteoblasts were transfected with pCD-rbFGF plasmid by lopofectin mediated gene transfer, the transient expression was detected by streptavidin-biotin-enzyme complex (SABC) method. It was observed that the expression of rat bFGF gene was detected 72 h after transfected distinctly. Basic fibroblast growth factor gene therapy is a method of potential for a wide array of orthopedic diseases.


Subject(s)
Cells, Cultured , DNA, Complementary/genetics , Escherichia coli/genetics , Eukaryotic Cells/metabolism , Fibroblast Growth Factor 2/biosynthesis , Fibroblast Growth Factor 2/genetics , Gene Transfer Techniques , Osteoblasts/cytology , Osteoblasts/metabolism , Plasmids , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Transfection , Transformation, Genetic
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