RESUMEN
Sirtuins are recently redefined as a family of nicotinamide adenine dinucleotide (NAD)-dependent deacylases. Sirtuins in mammals including human have seven members, which are SIRT1-7. Compared to other sirtuin members, not much study is focused on mitochondrial sirtuins (SIRT3-5). In mitochondrial sirtuins, SIRT4 was the last of less well-understood mitochondrial sirtuins especially for its robust enzymatic activity. This makes SIRT4 become the last puzzle of mitochondrial sirtuins, and thus brings some obstacles for studying SIRT4 biological functions or developing SIRT4 modulators. In this review, we will summarize and discuss the current findings for substrates, biological functions and possible enzymatic activities of SIRT4. The purpose of this review is to facilitate in discovering the robust enzymatic activity of SIRT4 and eventually finish this last puzzle of mitochondrial sirtuins.
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Mitocondrias/metabolismo , Sirtuinas/metabolismo , Animales , Humanos , Estructura Molecular , Sirtuinas/químicaRESUMEN
OBJECTIVE: To investigate the role of phosphatase of regenerating liver-3 (PRL-3) in the 17ß-estradiol (E2)- and interleukin 6 (IL-6)-induced migration of endometrial stromal cells (ESCs) from ectopic endometrium. METHODS: Ectopic endometrial tissues were collected from patients with endometriosis, and PRL-3 expression in ectopic and eutopic endometrium was examined by immunohistochemistry. Endometrial stromal cells isolated from ectopic endometrium were treated with E2, progesterone (P), IL-6, or sodium orthovanadate (Sov) to inhibit PRL-3. Total RNA and protein were extracted from ESCs after treatment for quantitative real-time polymerase chain reaction and Western blot analyses. Cell migration was assessed using a scratch wound assay. RESULTS: Phosphatase of regenerating liver 3 protein was highly expressed in the endometrial glandular cells (EGCs) and ESCs in ectopic endometrium, whereas its weak expression was observed only in EGCs in eutopic endometrium. Both E2 and IL-6 treatment significantly increased PRL-3 messenger RNA and protein expression, and P treatment significantly inhibited PRL-3 expression. However, E2-induced PRL-3 expression in ESCs from ectopic endometrium was significantly blocked by IL-6 antibody. Moreover, E2- and IL-6-enhanced cell migration was completely abrogated by Sov treatment. Furthermore, Sov treatment could significantly promote PTEN expression but inhibit E2- and IL-6-induced p-AKT activation. CONCLUSION: Phosphatase of regenerating liver 3 plays a key role in the E2- and IL-6-induced migration of ESCs from ectopic endometrium, a process that is involved in the PTEN-AKT signaling pathway.
RESUMEN
Exosomes are nanosized small membrane microvesicles of endocytic origin secreted by most cell types. Exosomes, through its carrying protein or RNA from derived cells, affect gene regulation networks or epigenetic reorganization of receptor cell, and then modulate the physiological processes of cells. Studies have shown that external exosomes secreted by breast cancer cells or other cells play an important role in the development of tumor, including cell migration, cell differentiation and the immune response, etc. In this article, the latest studies were summarized to provide an overview of current understanding of exosomes in breast cancer.
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Neoplasias de la Mama , Exosomas , Movimiento Celular , Humanos , ARNRESUMEN
OBJECTIVE: Successful transplantation of avulsed teeth is to restore the attachment and regenerate the periodontal support. Different strategies have been applied in treatment from modification of teeth storage, antibiotic usage to peridontium tissue replacement. We developed a novel periodontal ligament cell-sheet delivery system to apply on delayed replanted teeth in promoting periodontal healing in a canine model. DESIGN: Autologous periodontal ligament (PDL) fibroblasts were isolated from extracted premolars of beagle dog. The cell-sheets were fabricated using normal culture dish after stimulation of extracellular matrix formation. Teeth were surgically extracted and attached soft tissues were removed. After root canal treatment, the root of teeth were wrapped by the PDL cell-sheets and replanted back to prior socket accordingly whilst teeth without cell sheets as a control. Eight weeks after surgery, the animals were sacrificed and decalcified specimens were prepared. Regeneration of periodontal tissue was evaluated through histology assay. RESULTS: Multi-layered PDL cell-sheet could be attached on tooth root and most cells on sheet-tooth constructs were viable before replantation. Minimum clinical signs of inflammation were observed in experiment. PDL cell-sheets group show significant higher occurrence of favourable healing (88.4%) than control group with low healing (5.3%). Periodontal ligament and cememtum tissue regeneration was observed in the experimental group, and the regenerated tissues showed high collagen type III, type I and fibronectin expression. CONCLUSION: The periodontal ligament cell-sheets fabricated through normal cell culture dish has a potential for regeneration of periodontal ligament and may become a novel therapy for avulsed teeth replantation.
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Regeneración Tisular Guiada Periodontal/métodos , Ligamento Periodontal/trasplante , Avulsión de Diente/cirugía , Reimplante Dental/métodos , Animales , Células Cultivadas , Perros , Fibroblastos/trasplante , Ligamento Periodontal/citología , Periodoncio/fisiología , Periodoncio/cirugía , Ingeniería de Tejidos , Cicatrización de HeridasRESUMEN
Osteosarcomas are the most prevalent primary bone tumors found in pediatric patients. To understand their molecular etiology, cell culture models are used to define disease mechanisms under controlled conditions. Many osteosarcoma cell lines (e.g., SAOS-2, U2OS, MG63) are derived from Caucasian patients. However, patients exhibit individual and ethnic differences in their responsiveness to irradiation and chemotherapy. This motivated the establishment of osteosarcoma cell lines (OS1, OS2, OS3) from three ethnically Chinese patients. OS1 cells, derived from a pre-chemotherapeutic tumor in the femur of a 6-year-old female, were examined for molecular markers characteristic for osteoblasts, stem cells, and cell cycle control by immunohistochemistry, reverse transcriptase-PCR, Western blotting and flow cytometry. OS1 have aberrant G-banded karyotypes, possibly reflecting chromosomal abnormalities related to p53 deficiency. OS1 had ossification profiles similar to human fetal osteoblasts rather than SAOS-2 which ossifies ab initio (P < 0.05). Absence of p53 correlates with increased Runx2 expression, while the slow proliferation of OS1 cells is perhaps attenuated by pRB retention. OS1 express mesenchymal stem cell markers (CD44, CD105) and differ in relative expression of CD29, CD63, and CD71 to SAOS-2. (P < 0.05). Cell cycle synchronization with nocodazole did not affect Runx2 and CDK1 levels but decreased cyclin-E and increased cyclin-A (P < 0.05). Xenotransplantion of OS1 in SCID mice yields spontaneous tumors that were larger and grew faster than SAOS-2 transplants. Hence, OS1 is a new osteosarcoma cell culture model derived from a pre-chemotherapeutic ethnic Chinese patient, for mechanistic studies and development of therapeutic strategies to counteract metastasis and deregulation of mesenchymal development.
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Diferenciación Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Osteoblastos/patología , Osteosarcoma/metabolismo , Osteosarcoma/patología , Proteína de Retinoblastoma/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Adolescente , Animales , Antígenos CD/metabolismo , Pueblo Asiatico , Calcificación Fisiológica/fisiología , Ciclo Celular/efectos de los fármacos , Desdiferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Niño , Aberraciones Cromosómicas , Colágeno Tipo I/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Femenino , Expresión Génica/genética , Humanos , Antígeno Ki-67/metabolismo , Ratones , Ratones SCID , Nocodazol/farmacología , Osteocalcina/metabolismo , Osteosarcoma/diagnóstico , Osteosarcoma/genética , Proteína de Retinoblastoma/genética , Proteína p53 Supresora de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In this study, poly (epsilon-caprolactone) [PCL] and its collagen composite blend (PCL/Col) were fabricated to scaffolds using electrospinning method. Incorporated collagen was present on the surface of the fibers, and it modulated the attachment and proliferation of pig bone marrow mesenchymal cells (pBMMCs). Osteogenic differentiation markers were more pronounced when these cells were cultured on PCL/Col fibrous meshes, as determined by immunohistochemistry for collagen type I, osteopontin, and osteocalcin. Matrix mineralization was observed only on osteogenically induced PCL/Col constructs. Long bone analogs were created by wrapping osteogenic cell sheets around the PCL/Col meshes to form hollow cylindrical cell-scaffold constructs. Culturing these constructs under dynamic conditions enhanced bone-like tissue formation and mechanical strength. We conclude that electrospun PCL/Col mesh is a promising material for bone engineering applications. Its combination with osteogenic cell sheets offers a novel and promising strategy for engineering of tubular bone analogs.
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Trasplante Óseo , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Huesos/citología , Huesos/efectos de los fármacos , Huesos/fisiología , Caproatos/farmacología , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Colágeno Tipo I/farmacología , Inmunohistoquímica , Lactonas/farmacología , Mesodermo/citología , Microscopía Electrónica de Rastreo , Sus scrofaRESUMEN
To understand the molecular etiology of osteosarcoma, we isolated and characterized a human osteosarcoma cell line (OS1). OS1 cells have high osteogenic potential in differentiation induction media. Molecular analysis reveals OS1 cells express the pocket protein pRB and the runt-related transcription factor Runx2. Strikingly, Runx2 is expressed at higher levels in OS1 cells than in human fetal osteoblasts. Both pRB and Runx2 have growth suppressive potential in osteoblasts and are key factors controlling competency for osteoblast differentiation. The high levels of Runx2 clearly suggest osteosarcomas may form from committed osteoblasts that have bypassed growth restrictions normally imposed by Runx2. Interestingly, OS1 cells do not exhibit p53 expression and thus lack a functional p53/p21 DNA damage response pathway as has been observed for other osteosarcoma cell types. Absence of this pathway predicts genomic instability and/or vulnerability to secondary mutations that may counteract the anti-proliferative activity of Runx2 that is normally observed in osteoblasts. We conclude OS1 cells provide a valuable cell culture model to examine molecular events that are responsible for the pathologic conversion of phenotypically normal osteoblast precursors into osteosarcoma cells.
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Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Osteosarcoma/genética , Proteína de Retinoblastoma/genética , Línea Celular Transformada , Línea Celular Tumoral , Proliferación Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Ciclina D , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Ciclinas/metabolismo , Humanos , Microscopía Fluorescente , Osteoblastos/metabolismo , Osteoblastos/ultraestructura , Osteosarcoma/metabolismo , Proteína de Retinoblastoma/metabolismo , Transducción de Señal , Estadísticas no Paramétricas , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: Human periodontium contains different cell types that have various potential roles in hard and soft tissue regeneration. However, there is limited knowledge about how these diverse cell populations contribute to the regenerative process. In this study, we investigated the surface marker difference between different periodontal cells (alveolar osteoblasts [AOs], periodontal ligament fibroblasts [PDLFs], and gingival fibroblasts [GFs]) and their differentiation potential toward osteogenic and adipogenic phenotypes. METHODS: Periodontal cells (AOs, PDLFs, and GFs) from 14 subjects were isolated. The surface antigen expression pattern of cells was analyzed by cell flow cytometry, and the molecular and histologic characterizations under osteogenic and adipogenic inductions were monitored by reverse transcription-polymerase chain reaction, Western blot, and immunocytohistology. RESULTS: The cell phenotypes of AOs were verified by the high expressions of CD29 and CD49a, whereas PDLFs showed distinctively low levels of CD63 and CD73. Under adipogenic induction, limited AOs formed cube-shaped adipose-like cells, whereas PDLFs formed spindle-shaped adipose-like cells. All three cell types expressed baseline osteo-related genes. AOs demonstrated the highest osteogenic ability followed by PDLFs and GFs. CONCLUSIONS: Cells in alveolar bone and periodontal ligament contain osteogenic and adipogenic progenitors. These observations indicate a possible application for periodontium cells in hard or soft tissue regeneration.
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Adipogénesis , Células Madre Adultas/fisiología , Proceso Alveolar/citología , Osteogénesis , Ligamento Periodontal/citología , Adipocitos/citología , Adipogénesis/genética , Adulto , Proceso Alveolar/fisiología , Antígenos de Superficie/análisis , Calcificación Fisiológica , Diferenciación Celular , Forma de la Célula , Células Cultivadas , Femenino , Fibroblastos/fisiología , Expresión Génica , Humanos , Masculino , Células Madre Multipotentes/fisiología , Osteoblastos/fisiología , Osteogénesis/genética , Ligamento Periodontal/fisiologíaRESUMEN
Cell-sheet techniques have been proven effective in various soft tissue engineering applications. In this experiment, we investigated the feasibility of bone tissue engineering using a hybrid of mesenchymal stem cell (MSC) sheets and PLGA meshes. Porcine MSCs were cultured to a thin layer of cell sheets via osteogenic induction. Tube-like long bones were constructed by wrapping the cell sheet on to PLGA meshes resulting in constructs which could be cultured in spinner flasks, prior to implantation in nude rats. Our results showed that the sheets were composed of viable cells and dense matrix with a thickness of about 80-120 microm, mineral deposition was also observed in the sheet. In vitro cultures demonstrated calcified cartilage-like tissue formation and most PLGA meshes were absorbed during the 8-week culture period. In vivo experiments revealed that dense mineralized tissue was formed in subcutaneous sites and the 8-week plants shared similar micro-CT characteristics with native bone. The neo tissue demonstrated histological markers for both bone and cartilage, indicating that the bone formation pathway in constructs was akin to endochondral ossification, with the residues of PLGA having an effect on the neo tissue organization and formation. These results indicate that cell-sheet approaches in combination with custom-shaped scaffolds have potential in producing bone tissue.
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Desarrollo Óseo/fisiología , Células Madre Mesenquimatosas/fisiología , Ingeniería de Tejidos/métodos , Animales , Técnicas de Cultivo de Célula , Células Madre Mesenquimatosas/citología , Ratas , Ratas Desnudas , Porcinos , Andamios del TejidoRESUMEN
In this study, cell sheets comprising multilayered porcine bone marrow stromal cells (BMSC) were assembled with fully interconnected scaffolds made from medical-grade polycaprolactone-calcium phosphate (mPCL-CaP), for the engineering of structural and functional bone grafts. The BMSC sheets were harvested from culture flasks and wrapped around pre-seeded composite scaffolds. The layered cell sheets integrated well with the scaffold/cell construct and remained viable, with mineralized nodules visible both inside and outside the scaffold for up to 8 weeks culture. Cells within the constructs underwent classical in vitro osteogenic differentiation with the associated elevation of alkaline phosphatase activity and bone-related protein expression. In vivo, two sets of cell-sheet-scaffold/cell constructs were transplanted under the skin of nude rats. The first set of constructs (5 x 5 x 4mm(3)) were assembled with BMSC sheets and cultured for 8 weeks before implantation. The second set of constructs (10 x 10 x 4mm(3)) was implanted immediately after assembly with BMSC sheets, with no further in vitro culture. For both groups, neo cortical and well-vascularised cancellous bone were formed within the constructs with up to 40% bone volume. Histological and immunohistochemical examination revealed that neo bone tissue formed from the pool of seeded BMSC and the bone formation followed predominantly an endochondral pathway, with woven bone matrix subsequently maturing into fully mineralized compact bone; exhibiting the histological markers of native bone. These findings demonstrate that large bone tissues similar to native bone can be regenerated utilizing BMSC sheet techniques in conjunction with composite scaffolds whose structures are optimized from a mechanical, nutrient transport and vascularization perspective.