RESUMO
Human amniotic transplantation has been proposed to improve the therapeutic efficacy of intrauterine adhesions (IUAs). Human amniotic mesenchymal stem stromal cells (hAMSCs) can differentiate into multiple tissue types. This study aimed to investigate the mechanism by which hAMSCs transplantation promotes endometrial regeneration. The rat models with IUA were established through mechanical and infective methods, and PKH26-labeled hAMSCs were transplanted through the tail vein (combined with/without estrogen). Under three different conditions, hAMSCs differentiated into endometrium-like cells. HE and Mason staining assays, and immunohistochemistry were used to compare the changes in rat models treated with hAMSCs and/or estrogen transplantation. To define the induction of hAMSCs to endometrium-like cells in vitro, an induction medium (cytokines, estrogen) was used to investigate the differentiation of hAMSCs into endometrium-like cells. qRT-polymerase chain reaction (PCR) and western blotting were performed to detect the differentiation of hAMSCs into endometrium-like cells. A greater number of glands, fewer endometrial fibrotic areas, and stronger expression of vascular endothelial growth factor and cytokeratin in the combined group (hAMSCs transplantation combined with estrogen) than in the other treatment groups were observed. hAMSCs could be induced into endometrium-like cells by cytokine treatment (TGF-ß1, EGF, and PDGF-BB). Transplantation of hAMSCs is an effective alternative for endometrial regeneration after injury in rats. The differentiation protocol for hAMSCs will be useful for further studies on human endometrial regeneration.
Assuntos
Endométrio , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Regeneração , Doenças Uterinas , Animais , Feminino , Humanos , Ratos , Endométrio/fisiologia , Estrogênios/metabolismo , Células-Tronco Mesenquimais/fisiologia , Aderências Teciduais/cirurgia , Aderências Teciduais/terapia , Doenças Uterinas/cirurgia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Reconstruction of bone defects is one of the most substantial and difficult clinical challenges in orthopedics. Transforming growth factor beta 1 (TGFß1) might play an important role in stimulating osteogenic differentiation of bone morphogenetic protein 9 (BMP9)-induced C3H10T1/2 mesenchymal stem cells. In our current study, we examined the potential synergy between TGFß1 and BMP9 in promoting the osteogenesis of C3H10T1/2 cells, and whether such effects could contribute to bone formation in vivo. Our experiment data indicated that TGFß1 could increase the expression of osteogenic markers and the formation of mineralized calcium nodules in, while suppressing the proliferation of, BMP9-induced C3H10T1/2 cells. Furthermore, mice intramuscularly injected with BMP9/TGFß1-transduced C3H10T1/2 cells into the gastrocnemius muscle on their tibiae developed ectopic bone masses with more mature osteoid structures, compared to those grafted with cells expressing BMP9/RFP. Subsequent mechanistic studies found that TGFß1-induced enhancement of osteogenesis in BMP9-overexpressing C3H10T1/2 cells was accompanied by augmented expression of heat shock protein 47 (HSP47), a collagen-specific molecular chaperone essential for collagen biosynthesis, and can be attenuated by pirfenidone, a known anti-fibrotic inhibitor. Interestingly, protein microarray analysis suggested that TGFß1/BMP9-dependent osteogenesis of C3H10T1/2 cells seemed to involve several non-canonical signaling pathways such as Janus kinase-signal transducer and activator of transcription, phosphoinositide-3-kinase-protein kinase B, and mitogen-activated protein kinase. These results provided further evidence that TGFß1 could promote bone formation from BMP9-induced C3H10T1/2 cells and shed important light on the underlying molecular mechanisms.
Assuntos
Calcificação Fisiológica/genética , Fator 2 de Diferenciação de Crescimento/genética , Proteínas de Choque Térmico HSP47/genética , Osteogênese/genética , Fator de Crescimento Transformador beta1/genética , Animais , Calcificação Fisiológica/fisiologia , Diferenciação Celular/genética , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Fosforilação/genética , Transdução de Sinais/genéticaRESUMO
The cranial suture complex is a heterogeneous tissue consisting of osteogenic progenitor cells and mesenchymal stem cells (MSCs) from bone marrow and suture mesenchyme. The fusion of cranial sutures is a highly coordinated and tightly regulated process during development. Craniosynostosis is a congenital malformation caused by premature fusion of cranial sutures. While the progenitor cells derived from the cranial suture complex should prove valuable for studying the molecular mechanisms underlying suture development and pathogenic premature suture fusion, primary human cranial suture progenitors (SuPs) have limited life span and gradually lose osteoblastic ability over passages. To overcome technical challenges in maintaining sufficient and long-term culture of SuPs for suture biology studies, we establish and characterize the reversibly immortalized human cranial suture progenitors (iSuPs). Using a reversible immortalization system expressing SV40 T flanked with FRT sites, we demonstrate that primary human suture progenitor cells derived from the patent sutures of craniosynostosis patients can be efficiently immortalized. The iSuPs maintain long-term proliferative activity, express most of the consensus MSC markers and can differentiate into osteogenic and adipogenic lineages upon BMP9 stimulation in vitro and in vivo. The removal of SV40 T antigen by FLP recombinase results in a decrease in cell proliferation and an increase in the endogenous osteogenic and adipogenic capability in the iSuPs. Therefore, the iSuPs should be a valuable resource to study suture development, intramembranous ossification and the pathogenesis of craniosynostosis, as well as to explore cranial bone tissue engineering.
Assuntos
Suturas Cranianas/metabolismo , Craniossinostoses/genética , Efeito Fundador , Fatores de Diferenciação de Crescimento/genética , Células-Tronco Mesenquimais/metabolismo , Osteogênese/genética , Adipócitos/citologia , Adipócitos/metabolismo , Diferenciação Celular , Linhagem Celular Transformada , Proliferação de Células , Suturas Cranianas/patologia , Craniossinostoses/metabolismo , Craniossinostoses/patologia , Expressão Gênica , Fator 2 de Diferenciação de Crescimento , Fatores de Diferenciação de Crescimento/metabolismo , Humanos , Lactente , Masculino , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Osteoblastos/metabolismo , Vírus 40 dos Símios/genética , Vírus 40 dos Símios/metabolismo , Transformação GenéticaRESUMO
BACKGROUND: BMP9 induces both osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs). Nell1 is a secretory glycoprotein with osteoinductive and anti-adipogenic activities. We investigated the role of Nell1 in BMP9-induced osteogenesis and adipogenesis in MSCs. METHODS: Previously characterized MSCs iMEFs were used. Overexpression of BMP9 and NELL1 or silencing of mouse Nell1 was mediated by adenoviral vectors. Early and late osteogenic and adipogenic markers were assessed by staining techniques and qPCR analysis. In vivo activity was assessed in an ectopic bone formation model of athymic mice. RESULTS: We demonstrate that Nell1 expression was up-regulated by BMP9. Exogenous Nell1 potentiated BMP9-induced late stage osteogenic differentiation while inhibiting the early osteogenic marker. Forced Nell1 expression enhanced BMP9-induced osteogenic regulators/markers and inhibited BMP9-upregulated expression of adipogenic regulators/markers in MSCs. In vivo ectopic bone formation assay showed that exogenous Nell1 expression enhanced mineralization and maturity of BMP9-induced bone formation, while inhibiting BMP9-induced adipogenesis. Conversely, silencing Nell1 expression in BMP9-stimulated MSCs led to forming immature chondroid-like matrix. CONCLUSION: Our findings indicate that Nell1 can be up-regulated by BMP9, which in turn accelerates and augments BMP9-induced osteogenesis. Exogenous Nell1 may be exploited to enhance BMP9-induced bone formation while overcoming BMP9-induced adipogenesis in regenerative medicine.
Assuntos
Adipogenia , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular , Glicoproteínas/metabolismo , Fator 2 de Diferenciação de Crescimento/metabolismo , Osteogênese , Adipogenia/efeitos dos fármacos , Animais , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/genética , Fator 2 de Diferenciação de Crescimento/genética , Células HEK293 , Humanos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Nus , Osteogênese/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transplante HomólogoRESUMO
BACKGROUND/AIMS: While recombinant adenoviruses are among the most widely-used gene delivery vectors and usually propagated in HEK-293 cells, generating recombinant adenoviruses remains time-consuming and labor-intense. We sought to develop a rapid adenovirus production and amplification (RAPA) line by assessing human Ad5 genes (E1A, E1B19K/55K, pTP, DBP, and DNA Pol) and OCT1 for their contributions to adenovirus production. METHODS: Stable transgene expression in 293T cells was accomplished by using piggyBac system. Transgene expression was determined by qPCR. Adenoviral production was assessed with titering, fluorescent markers and/or luciferase activity. Osteogenic activity was assessed by measuring alkaline phosphatase activity. RESULTS: Overexpression of both E1A and pTP led to a significant increase in adenovirus amplification, whereas other transgene combinations did not significantly affect adenovirus amplification. When E1A and pTP were stably expressed in 293T cells, the resultant RAPA line showed high efficiency in adenovirus amplification and production. The produced AdBMP9 infected mesenchymal stem cells with highest efficiency and induced most effective osteogenic differentiation. Furthermore, adenovirus production efficiency in RAPA cells was dependent on the amount of transfected DNA. Under optimal transfection conditions high-titer adenoviruses were obtained within 5 days of transfection. CONCLUSION: The RAPA cells are highly efficient for adenovirus production and amplification.
Assuntos
Adenoviridae/fisiologia , Biotecnologia/métodos , Engenharia Genética , Vetores Genéticos/metabolismo , Adenoviridae/genética , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Diferenciação Celular , Linhagem Celular , Citometria de Fluxo , Vetores Genéticos/genética , Fator 2 de Diferenciação de Crescimento/genética , Fator 2 de Diferenciação de Crescimento/metabolismo , Células HEK293 , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação ViralRESUMO
BACKGROUND/AIMS: Mesenchymal stem cells (MSCs) are multipotent progenitors that can differentiate into several lineages including bone. Successful bone formation requires osteogenesis and angiogenesis coupling of MSCs. Here, we investigate if simultaneous activation of BMP9 and Notch signaling yields effective osteogenesis-angiogenesis coupling in MSCs. METHODS: Recently-characterized immortalized mouse adipose-derived progenitors (iMADs) were used as MSC source. Transgenes BMP9, NICD and dnNotch1 were expressed by adenoviral vectors. Gene expression was determined by qPCR and immunohistochem¡stry. Osteogenic activity was assessed by in vitro assays and in vivo ectopic bone formation model. RESULTS: BMP9 upregulated expression of Notch receptors and ligands in iMADs. Constitutively-active form of Notch1 NICD1 enhanced BMP9-induced osteogenic differentiation both in vitro and in vivo, which was effectively inhibited by dominant-negative form of Notch1 dnNotch1. BMP9- and NICD1-transduced MSCs implanted with a biocompatible scaffold yielded highly mature bone with extensive vascularization. NICD1 enhanced BMP9-induced expression of key angiogenic regulators in iMADs and Vegfa in ectopic bone, which was blunted by dnNotch1. CONCLUSION: Notch signaling may play an important role in BMP9-induced osteogenesis and angiogenesis. It's conceivable that simultaneous activation of the BMP9 and Notch pathways should efficiently couple osteogenesis and angiogenesis of MSCs for successful bone tissue engineering.
Assuntos
Fator 2 de Diferenciação de Crescimento/metabolismo , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica , Osteogênese , Receptor Notch1/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Fator 2 de Diferenciação de Crescimento/genética , Células-Tronco Mesenquimais/citologia , Camundongos , Receptor Notch1/genéticaRESUMO
BACKGROUND/AIMS: Ovarian cancer is the most lethal gynecologic malignancy, and there is an unmet clinical need to develop new therapies. Although showing promising anticancer activity, Niclosamide may not be used as a monotherapy. We seek to investigate whether inhibiting IGF signaling potentiates Niclosamide's anticancer efficacy in human ovarian cancer cells. METHODS: Cell proliferation and migration are assessed. Cell cycle progression and apoptosis are analyzed by flow cytometry. Inhibition of IGF signaling is accomplished by adenovirus-mediated expression of siRNAs targeting IGF-1R. Cancer-associated pathways are assessed using pathway-specific reporters. Subcutaneous xenograft model is used to determine anticancer activity. RESULTS: We find that Niclosamide is highly effective on inhibiting cell proliferation, cell migration, and cell cycle progression, and inducing apoptosis in human ovarian cancer cells, possibly by targeting multiple signaling pathways involved in ELK1/SRF, AP-1, MYC/MAX and NFkB. Silencing IGF-1R exert a similar but weaker effect than that of Niclosamide's. However, silencing IGF-1R significantly sensitizes ovarian cancer cells to Niclosamide-induced anti-proliferative and anticancer activities both in vitro and in vivo. CONCLUSION: Niclosamide as a repurposed anticancer agent may be more efficacious when combined with agents that target other signaling pathways such as IGF signaling in the treatment of human cancers including ovarian cancer.
Assuntos
Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Niclosamida/farmacologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/terapia , RNA Interferente Pequeno/genética , Receptor IGF Tipo 1/antagonistas & inibidores , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Antiparasitários/farmacologia , Apoptose/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Reposicionamento de Medicamentos , Feminino , Células HEK293 , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , RNA Interferente Pequeno/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Elk-1 do Domínio ets/genética , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
BACKGROUND: BMPs play important roles in regulating stem cell proliferation and differentiation. Using adenovirus-mediated expression of the 14 types of BMPs we demonstrated that BMP9 is one of the most potent BMPs in inducing osteogenic differentiation of mesenchymal stem cells (MSCs), which was undetected in the early studies using recombinant BMP9 proteins. Endogenous BMPs are expressed as a precursor protein that contains an N-terminal signal peptide, a prodomain and a C-terminal mature peptide. Most commercially available recombinant BMP9 proteins are purified from the cells expressing the mature peptide. It is unclear how effectively these recombinant BMP9 proteins functionally recapitulate endogenous BMP9. METHODS: A stable cell line expressing the full coding region of mouse BMP9 was established in HEK-293 cells by using the piggyBac transposon system. The biological activities and stability of the conditioned medium generated from the stable line were analyzed. RESULTS: The stable HEK-293 line expresses a high level of mouse BMP9. BMP9 conditioned medium (BMP9-cm) was shown to effectively induce osteogenic differentiation of MSCs, to activate BMP-R specific Smad signaling, and to up-regulate downstream target genes in MSCs. The biological activity of BMP9-cm is at least comparable with that induced by AdBMP9 in vitro. Furthermore, BMP9-cm exhibits an excellent stability profile as its biological activity is not affected by long-term storage at -80ºC, repeated thawing cycles, and extended storage at 4ºC. CONCLUSIONS: We have established a producer line that stably expresses a high level of active BMP9 protein. Such producer line should be a valuable resource for generating biologically active BMP9 protein for studying BMP9 signaling mechanism and functions.
Assuntos
Diferenciação Celular/genética , Fator 2 de Diferenciação de Crescimento/biossíntese , Células-Tronco Mesenquimais/citologia , Osteogênese/genética , Animais , Meios de Cultivo Condicionados/metabolismo , Fator 2 de Diferenciação de Crescimento/genética , Células HEK293 , Humanos , Camundongos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
BACKGROUND/AIMS: Although osteosarcoma (OS) is the most common primary malignancy of bone, its molecular pathogenesis remains to be fully understood. We previously found the calcium-binding protein S100A6 was expressed in â¼80% of the analyzed OS primary and/or metastatic tumor samples. Here, we investigate the role of S100A6 in OS growth and progression. METHODS: S100A6 expression was assessed by qPCR and Western blotting. Overexpression or knockdown of S100A6 was carried out to determine S100A6's effect on proliferation, cell cycle, apoptosis, tumor growth, and osteogenic differentiation. RESULTS: S100A6 expression was readily detected in human OS cell lines. Exogenous S100A6 expression promoted cell proliferation in vitro and tumor growth in an orthotopic xenograft model of human OS. S100A6 overexpression reduced the numbers of OS cells in G1 phase and increased viable cells under serum starvation condition. Conversely, silencing S100A6 expression induced the production of cleaved caspase 3, and increased early stage apoptosis. S100A6 knockdown increased osteogenic differentiation activity of mesenchymal stem cells, while S100A6 overexpression inhibited osteogenic differentiation. BMP9-induced bone formation was augmented by S100A6 knockdown. CONCLUSION: Our findings strongly suggest that S100A6 may promote OS cell proliferation and OS tumor growth at least in part by facilitating cell cycle progression, preventing apoptosis, and inhibiting osteogenic differentiation. Thus, it is conceivable that targeting S100A6 may be exploited as a novel anti-OS therapy.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Osteogênese , Osteossarcoma/patologia , Proteínas S100/fisiologia , Animais , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Camundongos , Proteína A6 Ligante de Cálcio S100RESUMO
BACKGROUND: The objective of the current study was to establish a rat model to investigate apoptosis in steroid-induced femoral head osteonecrosis occurring via the Wnt/ß-catenin pathway. METHODS: Male Sprague-Dawley (SD) rats were randomly divided into a control group (group A), model group (group B) and sFRP1 group (group C), each consisting of 24 rats, and the rats were intravenously injected with LPS (10 µg/kg body weight). After 24 h, three injections of MPS (20 mg/kg body weight) were administered intramuscularly at 24-h intervals. The rats in group C were injected intramuscularly with 1 µg/kg sFRP1 protein per day for 30 days, beginning at the time of the first MPS administration. The group A rats were fed and housed under identical conditions but received saline injection. All animals were sacrificed at weeks 2, 4 and 8 from the first MPS injection. Histopathological staining was preformed to evaluated osteonecrosis. Apoptosis was detected via quantitative terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate nick-end labelling (TUNEL) staining, caspase-3 activity assay, and detection of Bcl-2 and Bax protein expression by immunohistochemistry and Western blotting. Wnt/ß-catenin pathway signalling molecules, including activated ß-catenin and c-Myc, were detected by immunohistochemistry and Western blotting. RESULTS: Typical osteonecrosis was observed in groups B and C. Apoptosis gradually increased with increasing time in both groups B and C. More severe osteonecrosis and apoptosis were observed in group C compared with group B. The expression levels of caspase-3 and Bax were higher while that of Bcl-2 was lower in group C compared with group B. The expression levels of activated ß-catenin and c-Myc gradually decreased with increasing time in both groups B and C, and they were lower in group C compared with group B. CONCLUSIONS: The Wnt/ß-catenin pathway is involved in the pathogenesis of early stage SANFH, as we have demonstrated in an SANFH rat model, and it may act through the regulation of c-Myc, which affects the cell cycle and cell apoptosis.
Assuntos
Necrose da Cabeça do Fêmur/metabolismo , Cabeça do Fêmur/metabolismo , Metilprednisolona , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Apoptose , Western Blotting , Caspase 3/metabolismo , Modelos Animais de Doenças , Cabeça do Fêmur/patologia , Necrose da Cabeça do Fêmur/induzido quimicamente , Necrose da Cabeça do Fêmur/patologia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Lipopolissacarídeos , Masculino , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ratos Sprague-Dawley , Fatores de Tempo , Proteína X Associada a bcl-2/metabolismoRESUMO
Brain metastases (BrMs) are the leading cause of death in patients with solid cancers. BrMs exhibit a highly immunosuppressive milieu and poor response to immunotherapies; however, the underlying mechanism remains largely unclear. Here, we show that upregulation of HSP47 in tumor cells drives metastatic colonization and outgrowth in the brain by creating an immunosuppressive microenvironment. HSP47-mediated collagen deposition in the metastatic niche promotes microglial polarization to the M2 phenotype via the α2ß1 integrin/nuclear factor κB pathway, which upregulates the anti-inflammatory cytokines and represses CD8+ T cell anti-tumor responses. Depletion of microglia reverses HSP47-induced inactivation of CD8+ T cells and abolishes BrM. Col003, an inhibitor disrupting HSP47-collagen association restores an anti-tumor immunity and enhances the efficacy of anti-PD-L1 immunotherapy in BrM-bearing mice. Our study supports that HSP47 is a critical determinant of M2 microglial polarization and immunosuppression and that blocking the HSP47-collagen axis represents a promising therapeutic strategy against brain metastatic tumors.
Assuntos
Neoplasias Encefálicas , Linfócitos T CD8-Positivos , Colágeno , Proteínas de Choque Térmico HSP47 , Microglia , Animais , Microglia/metabolismo , Microglia/efeitos dos fármacos , Microglia/imunologia , Neoplasias Encefálicas/secundário , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Colágeno/metabolismo , Camundongos , Proteínas de Choque Térmico HSP47/metabolismo , Proteínas de Choque Térmico HSP47/genética , Linhagem Celular Tumoral , Humanos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Microambiente Tumoral/imunologia , Camundongos Endogâmicos C57BL , Polaridade Celular/efeitos dos fármacos , Feminino , NF-kappa B/metabolismoRESUMO
Clinical epidemiological studies have confirmed that tobacco smoking disrupts bone homeostasis and is an independent risk factor for the development of osteoporosis. The low viability and inferior osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) are important etiologies of osteoporosis. However, few basic studies have elucidated the specific mechanisms that tobacco toxins devastated BMSCs and consequently induced or exacerbated osteoporosis. Herein, our clinical data showed the bone mineral density (BMD) values of femoral neck in smokers were significantly lower than non-smokers, meanwhile cigarette smoke extract (CSE) exposure led to a significant decrease of BMD in rats and dysfunction of rat BMSCs (rBMSCs). Transcriptomic analysis and phenotype experiments suggested that the ferroptosis pathway was significantly activated in CSE-treated rBMSCs. Accumulated intracellular reactive oxygen species activated AMPK signaling, furtherly promoted NCOA4-mediated ferritin-selective autophagic processes, increased labial iron pool and lipid peroxidation deposition, and ultimately led to ferroptosis in rBMSCs. Importantly, in vivo utilization of ferroptosis and ferritinophagy inhibitors significantly alleviated BMD loss in CSE-exposed rats. Our study innovatively reveals the key mechanism of smoking-related osteoporosis, and provides a possible route targeting on the perspective of BMSC ferroptosis for future prevention and treatment of smoking-related bone homeostasis imbalance.
Assuntos
Ferroptose , Osteoporose , Ratos , Animais , Nicotiana/efeitos adversos , Osteogênese , Osteoporose/etiologia , Ferro/metabolismoRESUMO
Osteoarthritis (OA) is a common disease in orthopedics. RNA N6-methyladenosine (m6A) exerts an essential effect in a variety of biological processes in the eukaryotes. In this study, we determined the effect of m6A regulators in the OA along with performing the subtype classification. Differential analysis of OA and normal samples in the database of Gene Expression Omnibus identified 9 significantly differentially expressed m6A regulators. These regulators were monitored by a random forest algorithm so as to evaluate the risk of developing OA disease. On the basis of these 9 moderators, a nomogram was established. The results of decision curve analysis suggested that the patients could benefit from a nomogram model. The OA sample was classified as 2 m6A models through a consensus clustering algorithm in accordance with these 9 regulators. These 2 m6A patterns were then assessed with principal component analysis. We also determined the m6A scores for the 2 m6A patterns and their correlation with immune infiltration. The results indicated that type A had a higher m6A score than type B. Thus, we suggest that the m6A pattern may provide a new approach for diagnose and provide novel ideas for molecular targeted therapy of OA.
Assuntos
Algoritmos , RNA , Humanos , Metilação , Análise por Conglomerados , ConsensoRESUMO
Long non-coding RNAs are important regulators of biological processes, but their roles in the osteogenic differentiation of mesenchymal stem cells (MSCs) remain unclear. Here we investigated the role of murine HOX transcript antisense RNA (mHotair) in BMP9-induced osteogenic differentiation of MSCs using immortalized mouse adipose-derived cells (iMADs). Touchdown quantitative polymerase chain reaction analysis found increased mHotair expression in bones in comparison with most other tissues. Moreover, the level of mHotair in femurs peaked at the age of week-4, a period of fast skeleton development. BMP9 could induce earlier peak expression of mHotair during in vitro iMAD osteogenesis. Silencing mHotair diminished BMP9-induced ALP activity, matrix mineralization, and expression of osteogenic, chondrogenic and adipogenic markers. Cell implantation experiments further confirmed that knockdown of mHotair attenuated BMP9-induced ectopic bone formation and mineralization of iMADs, leading to more undifferentiated cells. Crystal violet staining and cell cycle analysis revealed that silencing of mHotair promoted the proliferation of iMAD cells regardless of BMP9 induction. Moreover, ectopic bone masses developed from mHotair-knockdown iMAD cells exhibited higher expression of PCNA than the control group. Taken together, our results demonstrated that murine mHotair is an important regulator of BMP9-induced MSC osteogenesis by targeting cell cycle and proliferation.
Assuntos
Diferenciação Celular/genética , Proliferação de Células/genética , Células-Tronco Mesenquimais , Ossificação Heterotópica/genética , Osteogênese/genética , RNA Longo não Codificante/genética , Adipogenia/genética , Fosfatase Alcalina/metabolismo , Animais , Ciclo Celular/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Condrogênese/genética , Técnicas de Silenciamento de Genes , Fator 2 de Diferenciação de Crescimento/farmacologia , Camundongos , Ossificação Heterotópica/metabolismo , Osteogênese/efeitos dos fármacos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Microtomografia por Raio-XRESUMO
Adenovirus (Ad) is a non-enveloped linear double-stranded DNA virus with >50 serotypes in humans. Ad vectors have been used as gene delivery vehicles to express transgenes, small interfering RNAs (siRNAs) for gene silencing, or CRISPR/Cas and designer nucleases for genome editing. Although several methods are used to generate Ad vectors, the Ad-making process remains technically challenging and time consuming. Moreover, the Ad-making techniques have not been improved for the past two decades. Gibson DNA Assembly (GDA) technology allows one-step isothermal DNA assembly of multiple overlapping fragments. Here, we developed a one-step construction of Ad (OSCA) system using GDA technology. Specifically, we first engineered several adenoviral recipient vectors that contain the ccdB suicide gene flanked with two 20-bp unique sequences, which serve as universal sites for GDA reactions in the Ad genome ΔE1 region. In two proof-of-principle experiments, we demonstrated that the GDA reactions were highly efficient and that the resulting Ad plasmids could be effectively packaged into Ads. Ad-mediated expression of mouse BMP9 in mesenchymal stem cells was shown to effectively induce osteogenic differentiation both in vitro and in vivo. Collectively, our results demonstrate that the OSCA system drastically streamlines the Ad-making process and should facilitate Ad-based applications in basic, translational, and clinical research.
RESUMO
Shuttle effect and the low utilization of dissolved lithium polysulfides (LiPSs) are two prevailing concerns in Li-S battery (LSB) research. Energy efficiency on the other hand is often overlooked but vital to the commercial deployment of battery technology. In this work, a composite of hyperbranched poly(amidoamine)-modified multiwalled carbon nanotubes (PAMAM-CNTs) is successfully prepared by chemical grafting and employed as an interlayer material in LSBs. The high content and highly dispersed polar functional groups of PAMAM can efficiently adsorb and enhance the redox reaction of LiPSs. The CNTs function as a scaffold and current collector that reduces the internal polarization. The assembled LSB displays a high energy efficiency of 86% and a low capacity fading rate of 0.037% per cycle over 1200 cycles at 2 C. The cell also shows excellent cycle performance, high sulfur utilization, and improved stability at a high areal capacity of 9 mAh cm-2 (achieved at a sulfur loading of 8.7 mg cm-2) and low electrolyte/sulfur ratio of 6.1 mL g-1. This thin (12 µm) and lightweight (0.34 mg cm-2) interlayer has a negligible impact on the overall cell energy density.
RESUMO
Bone morphogenetic protein 9 (BMP9) (or GDF2) was originally identified from fetal mouse liver cDNA libraries. Emerging evidence indicates BMP9 exerts diverse and pleiotropic functions during postnatal development and in maintaining tissue homeostasis. However, the expression landscape of BMP9 signaling during development and/or in adult tissues remains to be analyzed. Here, we conducted a comprehensive analysis of the expression landscape of BMP9 and its signaling mediators in postnatal mice. By analyzing mouse ENCODE transcriptome datasets we found Bmp9 was highly expressed in the liver and detectable in embryonic brain, adult lungs and adult placenta. We next conducted a comprehensive qPCR analysis of RNAs isolated from major mouse tissues/organs at various ages. We found that Bmp9 was highly expressed in the liver and lung tissues of young adult mice, but decreased in older mice. Interestingly, Bmp9 was only expressed at low to modest levels in developing bones. BMP9-associated TGFß/BMPR type I receptor Alk1 was highly expressed in the adult lungs. Furthermore, the feedback inhibitor Smads Smad6 and Smad7 were widely expressed in mouse postnatal tissues. However, the BMP signaling antagonist noggin was highly expressed in fat and heart in the older age groups, as well as in kidney, liver and lungs in a biphasic fashion. Thus, our findings indicate that the circulating BMP9 produced in liver and lungs may account for its pleiotropic effects on postnatal tissues/organs although possible roles of BMP9 signaling in liver and lungs remain to be fully understood.
RESUMO
Graphene-based materials are used in many fields but have found only limited applications in biomedicine, including bone tissue engineering. Here, we demonstrate that novel hybrid materials consisting of gelatin-derived graphene and silicate nanosheets of Laponite (GL) are biocompatible and promote osteogenic differentiation of mesenchymal stem cells (MSCs). Homogeneous cell attachment, long-term proliferation, and osteogenic differentiation of MSCs on a GL-scaffold were confirmed using optical microscopy and scanning electron microscopy. GL-powders made by pulverizing the GL-scaffold were shown to promote bone morphogenetic protein (BMP9)-induced osteogenic differentiation. GL-powders increased the alkaline phosphatase (ALP) activity in immortalized mouse embryonic fibroblasts but decreased the ALP activity in more-differentiated immortalized mouse adipose-derived cells. Note, however, that GL-powders promoted BMP9-induced calcium mineral deposits in both MSC lines, as assessed using qualitative and quantitative alizarin red assays. Furthermore, the expression of chondro-osteogenic regulator markers such as Runx2, Sox9, osteopontin, and osteocalcin was upregulated by the GL-powder, independent of BMP9 stimulation; although the powder synergistically upregulated the BMP9-induced Osterix expression, the adipogenic marker PPARγ was unaffected. Furthermore, in vivo stem cell implantation experiments demonstrated that GL-powder could significantly enhance the BMP9-induced ectopic bone formation from MSCs. Collectively, our results strongly suggest that the GL hybrid materials promote BMP9-induced osteogenic differentiation of MSCs and hold promise for the development of bone tissue engineering platforms.
Assuntos
Células-Tronco Mesenquimais , Animais , Diferenciação Celular , Gelatina , Grafite , Fator 2 de Diferenciação de Crescimento , Camundongos , Osteogênese , SilicatosRESUMO
The liver provides vital metabolic, exocrine and endocrine functions in the body as such pathological conditions of the liver lead to high morbidity and mortality. The liver is highly regenerative and contains facultative stem cells that become activated during injury to replicate to fully recover mass and function. Canonical Wnt/ß-catenin signaling plays an important role in regulating the proliferation and differentiation of liver progenitor cells during liver regeneration. However, possible roles of noncanonical Wnts in liver development and regeneration remain undefined. We previously established a reversibly-immortalized hepatic progenitor cell line (iHPx), which retains hepatic differentiation potential. Here, we analyze the expression pattern of the essential components of both canonical and noncanonical Wnt signaling pathways at different postnatal stages of mouse liver tissues and iHPx cells. We find that noncanonical Wnt4, Wnt5a, Wnt9b, Wnt10a and Wnt10b, are highly expressed concordantly with the high levels of canonical Wnts in late stages of liver tissues. Wnt5a, Wnt9b, Wnt10a and Wnt10b are able to antagonize Wnt3a-induced ß-catenin/TCF activity, reduce the stemness of iHPx cells, and promote hepatic differentiation of liver progenitors. Stem cell implantation assay demonstrates that Wnt5a, Wnt9b, Wnt10a and Wnt10b can inhibit cell proliferation and promote hepatic differentiation of the iHPx progenitor cells. Our results strongly suggest that noncanonical Wnts may play an important role in fine-tuning Wnt/ß-catenin functions during liver development and liver regeneration. Thus, understanding regulatory mechanisms governing proliferation and differentiation of liver progenitor cells may hold great promise to facilitate liver regeneration and/or progenitor cell-based therapies for liver diseases.
Assuntos
Diferenciação Celular , Autorrenovação Celular , Fígado/citologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Animais , Biomarcadores , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Autorrenovação Celular/genética , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Humanos , Fígado/fisiologia , Regeneração Hepática/genética , Masculino , Camundongos , Transplante de Células-Tronco , TranscriptomaRESUMO
Head and neck squamous cell carcinoma (HNSCC) is one of the most common and aggressive types of human cancers worldwide. Nearly a half of HNSCC patients experience recurrence within five years of treatment and develop resistance to chemotherapy. Thus, there is an urgent clinical need to develop safe and novel anticancer therapies for HNSCC. Here, we investigate the possibility of repurposing the anthelmintic drug mebendazole (MBZ) as an anti-HNSCC agent. Using the two commonly-used human HNSCC lines CAL27 and SCC15, we demonstrate MBZ exerts more potent anti-proliferation activity than cisplatin in human HNSCC cells. MBZ effectively inhibits cell proliferation, cell cycle progression and cell migration, and induces apoptosis of HNSCC cells. Mechanistically, MBZ can modulate the cancer-associated pathways including ELK1/SRF, AP1, STAT1/2, MYC/MAX, although the regulatory outcomes are context-dependent. MBZ also synergizes with cisplatin in suppressing cell proliferation and inducing apoptosis of human HNSCC cells. Furthermore, MBZ is shown to promote the terminal differentiation of CAL27 cells and keratinization of CAL27-derived xenograft tumors. Our results are the first to demonstrate that MBZ may exert its anticancer activity by inhibiting proliferation while promoting differentiation of certain HNSCC cancer cells. It's conceivable the anthelmintic drug MBZ can be repurposed as a safe and effective agent used in combination with other frontline chemotherapy drugs such as cisplatin in HNSCC treatment.