RESUMO
Down's syndrome is one of the most common human congenital genetic diseases and affected patients have increased risk of periodontal disease. To examine involvement of the disease with periodontal disease development, we established immortalized periodontal ligament cells obtained from a Down's syndrome patient by use of SV40T-Ag and hTERT gene transfection. Expressions of SV40T-Ag and hTERT were observed in periodontal ligament cell-derived immortalized cells established from healthy (STPDL) and Down's syndrome patient (STPDLDS) samples. Primary cultured periodontal ligament cells obtained from a healthy subject (pPDL) had a limited number of population doublings (< 40), while STPDL and STPDLDS cells continued to grow with more than 80 population doublings. Primary cultured periodontal ligament cells obtained from the patient showed a chromosome pattern characteristic of Down's syndrome with trisomy 21, whereas STPDLDS samples showed a large number of abnormal chromosomes in those results. Gene expression analysis revealed that expression of DSCR-1 in STPDLDS is greater than that in STPDL. These results suggest that the newly established STPDLDS cell line may be a useful tool for study of periodontal disease in Down's syndrome patients.
Assuntos
Antígenos Transformantes de Poliomavirus/genética , Síndrome de Down , Fragmentos de Peptídeos/genética , Ligamento Periodontal/citologia , Telomerase/genética , Transfecção/métodos , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Síndrome de Down/genética , Expressão Gênica , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Doenças PeriodontaisRESUMO
Dental pulp cells (DPCs), including dental pulp (DP) stem cells, play a role in dentine repair under certain conditions caused by bacterial infections associated with caries, tooth fracture and injury. Mesenchymal stem cells (MSCs) have also been shown to be involved in this process of repair. However, the mechanisms through which MSCs are recruited to the DP have not yet been elucidated. Therefore, the aim of the present in vitro study was to investigate whether stromal cell-derived factor 1α (SDF1)-C-X-C chemokine receptor type 4 (CXCR4) signaling is involved in tissue repair in the DP of deciduous teeth. A single-cell clone from DPCs (SDP11) and UE7T-13 cells were used as pulp cells and MSCs, respectively. The MG-63 and HuO9 cells, two osteosarcoma cell lines, were used as positive control cells. Reverse transcription polymerase chain reaction (RT-PCR) revealed that all cell lines (SDP11, UE7T-13 MG-63 and HuO9) were positive for both SDF1 and CXCR4 mRNA expression. Moreover, immunocytochemical analysis indicated that SDF1 and CXCR4 proteins were expressed in the SDP11 and UE7T-13 cells. SDF1 was also detected in the cell lysates (CLs) and conditioned medium (CM) collected from the SDP11 and UE7T-13 cells, and AMD3100, a specific antagonist of CXCR4, inhibited the migration of the UE7T-13 cells; this migration was induced by treatment with CM, which was collected from the SDP11 cells. In addition, real-time PCR showed that the expression of SDF1 in the SDP11 cells was inhibited by treatment with 20 ng/ml fibroblast growth factor (FGF)-2, and exposure to AZD4547, an inhibitor of the FGF receptor, blocked this inhibition. Collectively, these data suggest that SDF1 produced by DP plays an important role in homeostasis, repair and regeneration via the recruitment of MSCs.
Assuntos
Movimento Celular , Quimiocina CXCL12/metabolismo , Polpa Dentária/citologia , Células-Tronco Mesenquimais/citologia , Receptores CXCR4/metabolismo , Dente Decíduo/citologia , Linhagem Celular , Células Cultivadas , Quimiocina CXCL12/análise , Quimiocina CXCL12/genética , Criança , Polpa Dentária/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/metabolismo , Receptores CXCR4/análise , Receptores CXCR4/genética , Transdução de Sinais , Dente Decíduo/metabolismoRESUMO
Stromal cell-derived factor (SDF)-1α has been reported to play a crucial role in stem cell homing and recruitment to injured sites. However, no information is available about its role in periodontal tissues. The aim of this in vitro study was to investigate the effects of basic fibroblast growth factor (FGF-2) and transforming growth factor (TGF)-ß1 on SDF-1α expression in immortalized periodontal ligament (PDL) cells derived from deciduous teeth (SH9 cells). Real-time PCR and western blot analyses showed that SDF-1α mRNA expression in SH9 cells was markedly inhibited by FGF-2 treatment for 48 h. SU5402, which directly interacts with the catalytic domain of the FGF receptor 1 (FGFR1) and suppresses its phosphorylation, inhibited the FGF-2-related decrease in SDF-1α expression. These results suggest that FGF-2 signaling via the FGFR1 pathway inhibits SDF-1α expression. Conversely, SDF-1α expression in SH9 cells was increased by TGF-ß1 treatment for 12 h. Western blot analysis showed that this treatment induced Smad2/3 phosphorylation. A time-course experiment showed that SDF-1α expression levels reached a maximum 12 h after the TGF-ß1 treatment and returned to basal levels by 48 h. Real-time PCR analysis showed that Smad7 mRNA expression peaked by 6 h after TGF-ß1 treatment. Since Smad7 siRNA downregulated Smad7 expression by approximately 2.5-fold compared with the negative control siRNA, the induction of SDF-1α expression was prolonged. Furthermore, treatment of SH9 cells with TGF-ß1 for 12 h induced transwell migration of UE7T-13 cells, which are mesenchymal stem cells derived from human bone marrow. Therefore, SDF-1α may play an important role in stem and progenitor cell recruitment and homing to injured sites in the periodontal ligament, and regulation of SDF-1α expression may be a useful tool in cell-based therapy for periodontal tissue regeneration.