RESUMO
Neuronal growth regulator 1 (NEGR1) is a newly identified raft-associated protein, which has recently been spotlighted as a new locus related to human obesity. Niemann-Pick disease Type C2 (NPC2) protein functions as a key player in the intracellular cholesterol trafficking, and its defect is linked to a fatal human neurodegenerative disease, NPC. In this study, we identified that NEGR1 interacts with NPC2 and increases its protein stability. Ectopically expressed NEGR1 proteins relieved an abnormal cholesterol accumulation in endosomal compartments. Importantly, NEGR1-defective mouse embryonic fibroblast cells exhibit increased cholesterol levels and triglyceride contents. These findings provide the first insight into the role of NEGR1 in intracellular cholesterol homeostasis, possibly explaining the missing link between NEGR1 with human obesity.
Assuntos
Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Colesterol/química , Doença de Niemann-Pick Tipo C/metabolismo , Animais , Endossomos/metabolismo , Fibroblastos/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Microdomínios da Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Doenças Neurodegenerativas/metabolismo , Obesidade/metabolismo , Fases de Leitura Aberta , Triglicerídeos/químicaRESUMO
BACKGROUND: Atopic dermatitis (AD), a chronic inflammatory skin disease with T cell activation as a key feature, in which Th2 cell-mediated responses play a pivotal role. Regulatory T cells (Treg) are central immune cells that restrict autoimmunity and inflammation in the body. Patients with immune dysregulation, polyendocrinopathy, or enteropathy X-linked syndrome, an immune disease characterized by a deficiency in Treg, develop skin inflammation and allergic disorders, indicating that Treg play a crucial role in the development of allergic skin inflammation. OBJECTIVE: we investigated the underlying mechanisms by which Treg control cutaneous allergic inflammation. METHODS: An allergic skin inflammation mouse model was constructed using MC903, and Treg-depleted mouse model was constructed using diphtheria toxin. Neutralization of IFN-γ was constructed using anti-mouse-IFN-γ mouse antibody. Neutrophil infiltration was analyzed by flow cytometry and immunohistochemistry. Neutrophil extracellular traps (NETs), a process called NETosis, were detected using immunofluorescence. In vitro neutrophil stimulation and immunocytochemistry was conducted to demonstrate the effect of IFN-γ on NETosis. RESULTS: The depletion of Foxp3+ Treg led to significantly exacerbated AD-like skin inflammation, including increased recruitment of neutrophils and expression of Th1 cytokine IFN-γ. Neutrophil infiltrating in skin of Treg-depleted mice released more NETs than wild type. Neutralization of IFN-γ abolished neutrophil infiltration and NETosis in Treg-depleted mice. Neutrophils stimulated with IFN-γ were more prone to release NETs in vitro. Finally, Foxp3+ Treg control cutaneous allergic inflammation by regulating IFN-γ-driven neutrophilic infiltration and NETosis. CONCLUSION: Our results highlight the previously underestimated Treg-IFN-γ-neutrophil inflammatory axis.
Assuntos
Dermatite Atópica , Modelos Animais de Doenças , Armadilhas Extracelulares , Fatores de Transcrição Forkhead , Interferon gama , Infiltração de Neutrófilos , Neutrófilos , Pele , Linfócitos T Reguladores , Animais , Linfócitos T Reguladores/imunologia , Interferon gama/metabolismo , Interferon gama/imunologia , Fatores de Transcrição Forkhead/metabolismo , Camundongos , Armadilhas Extracelulares/imunologia , Dermatite Atópica/imunologia , Dermatite Atópica/patologia , Dermatite Atópica/induzido quimicamente , Pele/imunologia , Pele/patologia , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Humanos , Camundongos Endogâmicos C57BL , FemininoRESUMO
Dendritic cells (DCs) are readily generated from the culture of mouse bone marrow (BM) treated with either granulocyte macrophage-colony stimulating factor (GM-CSF) or FMS-like tyrosine kinase 3 ligand (FLT3L). CD11c+MHCII+ or CD11c+MHCIIhi cells are routinely isolated from those BM cultures and generally used as in vitro-generated DCs for a variety of experiments and therapies. Here, we examined CD11c+ cells in the BM culture with GM-CSF or FLT3L by staining with a monoclonal antibody 2A1 that is known to recognize mature or activated DCs. Most of the cells within the CD11c+MHCIIhi DC gate were 2A1+ in the BM culture with GM-CSF (GM-BM culture). In the BM culture with FLT3L (FL-BM culture), almost of all the CD11c+MHCIIhi cells were within the classical DC2 (cDC2) gate. The analysis of FL-BM culture revealed that a majority of cDC2-gated CD11c+MHCIIhi cells exhibited a 2A1-CD83-CD115+CX3CR1+ phenotype, and the others consisted of 2A1+CD83+CD115-CX3CR1- and 2A1-CD83-CD115-CX3CR1- cells. According to the antigen uptake and presentation, morphologies, and gene expression profiles, 2A1-CD83-CD115-CX3CR1- cells were immature cDC2s and 2A1+CD83+CD115-CX3CR1- cells were mature cDC2s. Unexpectedly, however, 2A1-CD83-CD115+CX3CR1+ cells, the most abundant cDC2-gated MHCIIhi cell subset in FL-BM culture, were non-DCs. Adoptive cell transfer experiments in the FL-BM culture confirmed that the cDC2-gated MHCIIhi non-DCs were precursors to cDC2s, i.e., MHCIIhi pre-cDC2s. MHCIIhi pre-cDC2s also expressed the higher level of DC-specific transcription factor Zbtb46 as similarly as immature cDC2s. Besides, MHCIIhi pre-cDC2s were generated only from pre-cDCs and common DC progenitor (CDP) cells but not from monocytes and common monocyte progenitor (cMoP) cells, verifying that MHCIIhi pre-cDC2s are close lineage to cDCs. All in all, our study identified and characterized a new cDC precursor, exhibiting a CD11c+MHCIIhiCD115+CX3CR1+ phenotype, in FL-BM culture.
Assuntos
Medula Óssea , Antígenos de Histocompatibilidade Classe II , Camundongos , Animais , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Receptor 1 de Quimiocina CX3C/metabolismo , Células da Medula Óssea , Células Dendríticas , Diferenciação Celular , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
CENP-W was originally identified as a putative oncogene, cancer-upregulated gene 2 (CUG2) that was commonly up-regulated in many cancer tissues. Recently, CENP-W has also been identified as a new centromeric component that interacts with CENP-T. As a complex with CENP-T, CENP-W plays crucial roles in assembly of the functional kinetochore complex. In this study, the subnuclear localization of CENP-W was extensively analyzed using various approaches. We found that ectopically expressed CENP-W primarily accumulated in the nucleolus and remained substantially associated with the nucleolus in stable cells. The following fractionation study also showed that CENP-W is associated with RNA as well as DNA. Moreover, a considerable amount of CENP-W was found in the nuclear mesh-like structure, nuclear matrix, possibly indicating that CENP-W participates in diverse subnuclear activities. Finally, biochemical affinity binding analysis revealed that CENP-W specifically interacts with the nucleolar phosphoprotein, nucleophosmin (B23). Depletion of cellular B23 by siRNA treatment induced a dramatic decrease of CENP-W stability and severe mislocalization during prophase. Our data proposed that B23 may function in the assembly of the kinetochore complex by interacting with CENP-W during interphase.
Assuntos
Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , RNA/química , Linhagem Celular Tumoral , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Células HeLa , Humanos , Cinetocoros/metabolismo , Microscopia de Fluorescência/métodos , Proteínas Nucleares/metabolismo , Nucleofosmina , Estrutura Terciária de Proteína , RNA/metabolismo , RNA Interferente Pequeno/metabolismo , Frações Subcelulares/metabolismoRESUMO
Neuronal growth regulator 1 (NEGR1) is a GPI-anchored membrane protein that is involved in neural cell adhesion and communication. Multiple genome wide association studies have found that NEGR1 is a generic risk factor for multiple human diseases, including obesity, autism, and depression. Recently, we reported that Negr1-/- mice showed a highly increased fat mass and affective behavior. In the present study, we identified Na/K-ATPase, beta1-subunit (ATP1B1) as an NEGR1 binding partner by yeast two-hybrid screening. NEGR1 and ATP1B1 were found to form a relatively stable complex in cells, at least partially co-localizing in membrane lipid rafts. We found that NEGR1 binds with ATP1B1 at its C-terminus, away from the binding site for the alpha subunit, and may contribute to intercellular interactions. Collectively, we report ATP1B1 as a novel NEGR1-interacting protein, which may help deciphering molecular networks underlying NEGR1-associated human diseases. [BMB Reports 2021; 54(3): 164-169].
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Comunicação Celular , Células Cultivadas , Proteínas Ligadas por GPI/metabolismo , HumanosRESUMO
The skin is the outermost barrier organ in the body, which contains several types of dendritic cells (DCs), a group of professional antigen-presenting cells. When the skin encounters invading pathogens, different cutaneous DCs initiate a distinct T cell immune response to protect the body. Among the invading pathogens, fungal infection specifically drives a protective interleukin-17-producing Th17 immune response. A protocol was developed to efficiently differentiate Th17 cells by intradermal Candida albicans infection to investigate a subset of cutaneous DCs responsible for inducing Th17 immunity. Flow cytometry and gene expression analyses revealed a prominent induction of Th17 immune response in skin-draining lymph nodes and infected skin. Using diphtheria toxin-induced DC subset-depleting mouse strains, CD301b+ dermal DCs were found to be responsible for mounting optimal Th17 differentiation in this model. Thus, this protocol provides a valuable method to study in vivo function of differential subsets of cutaneous DCs to determine Th17 immunity against deep skin fungal infection.
Assuntos
Candida albicans , Células Th17 , Animais , Células Dendríticas , Células de Langerhans , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Cancer-upregulated gene 2 (CUG2), which was named since it was originally identified as a putative oncogene up-regulated in various human cancers, was recently renamed CENP-W based on the new findings that it is a component of the centromeric complex playing a crucial role in the assembly of functional kinetochore complex during mitosis. To understand the transcriptional regulation of CENP-W, we analyzed its TATA-less promoter and identified a GC-rich putative Sp1 binding site located at -46 to -36 that was critical in CENP-W expression. Competitive electrophoretic gel mobility shift assay using mutated oligos and supershift assays with Sp1 and Sp3 antibodies demonstrated that both proteins specifically bound to this promoter region. Moreover, we found that CENP-W was highly induced by serum stimulation followed by serum deprivation, with Sp1 and Sp3 transcription factors involved in this transactivation. Taken together, our results suggest that Sp1 together with Sp3 may function as the main regulator of the basal and serum-induced transcription of CENP-W.
Assuntos
Proteínas Cromossômicas não Histona/genética , Regulação da Expressão Gênica , Soro/metabolismo , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/metabolismo , Sequência de Bases , Sítios de Ligação , Proteínas Cromossômicas não Histona/metabolismo , Clonagem Molecular , Células HeLa , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas/genética , Ligação ProteicaRESUMO
PURPOSE: Malignant gliomas are aggressive spinal cord tumors. In this study, we hypothesized that combination therapy using an anti-angiogenic agent, bevacizumab, and hypoxia-inducible glioblastoma-specific suicide gene could reduce tumor growth. MATERIALS AND METHODS: In the present study, we evaluated the effect of combination therapy using bevacizumab and pEpo-NI2-SV-TK in reducing the proliferation of C6 cells and tumor growth in the spinal cord. Spinal cord tumor was generated by the injection of C6 cells into the T5 level of the spinal cord. Complexes of branched polyethylenimine (bPEI)/pEpo-NI2-SV-TK were injected into the spinal cord tumor. Bevacizumab was then administered by an intraperitoneal injection at a dose of 7 mg/kg. The anti-cancer effects of combination therapy were analyzed by histological analyses and magnetic resonance imaging (MRI). The Basso, Beattie and Bresnahan scale scores for all of the treatment groups were recorded every other day for 15 days to assess the rat hind-limb strength. RESULTS: The complexes of bPEI/pEpo-NI2-SV-TK inhibited the viability of C6 cells in the hypoxia condition at 5 days after treatment with ganciclovir. Bevacizumab was decreased in the cell viability of human umbilical vein endothelial cells. Combination therapy reduced the tumor size by histological analyses and MRI. The combination therapy group showed improved hind-limb function compared to the other groups that were administered pEpo-NI2-SV-TK alone or bevacizumab alone. CONCLUSION: This study suggests that combination therapy using bevacizumab with the pEpo-NI2-SV-TK therapeutic gene could be useful for increasing its therapeutic benefits for intramedullary spinal cord tumors.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Bevacizumab/uso terapêutico , Glioma/patologia , Neoplasias da Medula Espinal/terapia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Terapia Combinada , Células Endoteliais/patologia , Elementos Facilitadores Genéticos , Eritropoetina/genética , Genes Reporter , Genes Transgênicos Suicidas , Glioblastoma/patologia , Membro Posterior/diagnóstico por imagem , Injeções Intraperitoneais , Imageamento por Ressonância Magnética , Ratos , Neoplasias da Medula Espinal/patologiaRESUMO
Glioma is the most malignant type of primary central nervous system tumors, and has an extremely poor prognosis. One potential therapeutic approach is to induce the terminal differentiation of glioma through the forced expression of pro-neural factors. Our goal is to show the proof of concept of the neuronal conversion of C6 glioma through the combined action of small molecules. We investigated the various changes in gene expression, cell-specific marker expression, signaling pathways, physiological characteristics, and morphology in glioma after combination treatment with two small molecules (CHIR99021, a glycogen synthase kinase 3 [GSK3] inhibitor and forskolin, a cyclic adenosine monophosphate [cAMP] activator). Here, we show that the combined action of CHIR99021 and forskolin converted malignant glioma into fully differentiated neurons with no malignant characteristics; inhibited the proliferation of malignant glioma; and significantly down-regulated gene ontology and gene expression profiles related to cell division, gliogenesis, and angiogenesis in small molecule-induced neurons. In vivo, the combined action of CHIR99021 and forskolin markedly delayed neurological deficits and significantly reduced the tumor volume. We suggest that reprogramming technology may be a potential treatment strategy replacing the therapeutic paradigm of traditional treatment of malignant glioma, and a combination molecule comprising a GSK3 inhibitor and a cAMP inducer could be the next generation of anticancer drugs.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Glioma/tratamento farmacológico , Glioma/genética , Quinase 3 da Glicogênio Sintase/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colforsina/administração & dosagem , AMP Cíclico/antagonistas & inibidores , AMP Cíclico/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/patologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Humanos , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Imagem Óptica , Piridinas/administração & dosagem , Pirimidinas/administração & dosagem , Ratos , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Vascular endothelial growth factor (VEGF) is an angiogenic cytokine that stimulates the differentiation and function of vascular endothelial cells. VEGF has been implicated in improving nervous system function after injury. However, uncontrolled overexpression of VEGF increases the risk of tumor formation at the site of gene delivery. For this reason, VEGF expression needs to be strictly controlled. The goal of the present study was to understand the effects of hypoxia-induced gene expression system to control VEGF gene expression in neural stem cells (NSCs) on the regeneration of neural tissue after sciatic nerve injury. In this study, we used the erythropoietin (Epo) enhancer-SV40 promoter system (EpoSV-VEGF-NSCs) for hypoxia-specific VEGF expression. We used three types of NSCs: DsRed-NSCs as controls, SV-VEGF-NSCs as uncontrolled VEGF overexpressing NSCs, and EpoSV-VEGF-NSCs. For comparison of VEGF expression at normoxia and hypoxia, we measured the amount of VEGF secreted. VEGF expression decreased at normoxia and increased at hypoxia for EpoSV-VEGF-NSCs; thus, EpoSV-VEGF-NSCs controlled VEGF expression, dependent upon oxygenation condition. To demonstrate the therapeutic effect of EpoSV-VEGF-NSCs, we transplanted each cell line in a neuropathic pain sciatic nerve injury rat model. The transplanted EpoSV-VEGF-NSCs improved sciatic nerve functional index (SFI), mechanical allodynia, and re-myelination similar to the SV-VEGF-NSCs. Additionally, the number of blood vessels increased to a level similar to that of the SV-VEGF-NSCs. However, we did not observe tumor generation in the EpoSV-VEGF-NSC animals that were unlikely to have tumor formation in the SV-VEGF-NSCs. From our results, we determined that EpoSV-VEGF-NSCs safely regulate VEGF gene expression which is dependent upon oxygenation status. In addition, we found that they are therapeutically appropriate for treating sciatic nerve injury.
Assuntos
Células-Tronco Neurais/transplante , Neuralgia/terapia , Nervo Isquiático/lesões , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Hipóxia Celular , Linhagem Celular , Eritropoetina/genética , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Masculino , Camundongos , Células-Tronco Neurais/metabolismo , Neuralgia/genética , Neuralgia/patologia , Neuralgia/fisiopatologia , Plasmídeos/genética , Regiões Promotoras Genéticas , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/metabolismo , Nervo Isquiático/patologia , Nervo Isquiático/fisiopatologiaRESUMO
Electrospun silk fibroin (SF) scaffolds have drawn much attention because of their resemblance to natural tissue architecture such as extracellular matrix, and the biocompatibility of SF as a candidate material to replace collagen. However, electrospun scaffolds lack the physical integrity of bone tissue scaffolds, which require resistance to mechanical loadings. In this work, we propose membrane-reinforced electrospun SF scaffolds by a serial process of electrospinning and freeze-drying of SF solutions in two different solvents: formic acid and water, respectively. After wet electrospinning followed by replacement of methanol with water, SF nanofibers dispersed in water were mixed with aqueous SF solution. Freeze-drying of the mixed solution resulted in 3D membrane-connected SF nanofibrous scaffolds (SF scaffolds) with a thickness of a few centimeters. We demonstrated that the SF concentration of aqueous SF solution controlled the degree of membrane reinforcement between nanofibers. It was also shown that both increase in degree of membrane reinforcement and inclusion of hydroxyapatite (HAP) nanoparticles resulted in higher resistance to compressive loadings of the SF scaffolds. Culture of human osteoblasts on collagen, SF, and SF-HAP scaffolds showed that both SF and SF-HAP scaffolds had biocompatibility and cell proliferation superior to that of the collagen scaffolds. SF-HAP scaffolds with and without BMP-2 were used for in vivo studies for 4 and 8 weeks, and they showed enhanced bone tissue formation in rat calvarial defect models.
Assuntos
Substitutos Ósseos/química , Fibroínas/química , Alicerces Teciduais/química , Animais , Fenômenos Biomecânicos , Regeneração Óssea , Adesão Celular , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Força Compressiva , Durapatita/química , Consolidação da Fratura , Humanos , Masculino , Teste de Materiais , Microscopia Eletrônica de Varredura , Nanofibras/química , Osteoblastos/citologia , Osteogênese , Porosidade , Ratos , Ratos Sprague-Dawley , Crânio/diagnóstico por imagem , Crânio/lesões , Crânio/patologia , Engenharia Tecidual , Microtomografia por Raio-XRESUMO
Previously, we determined that vascular endothelial growth factor (VEGF) improves the survival of neural stem cells (NSCs) transplanted into an ischemic environment and effectively enhances angiogenesis. Here, we applied NSCs expressing VEGF (SV-VEGF-NSCs) to treat neuropathic pain. In this study, our goal was to verify the therapeutic effect of SV-VEGF-NSCs by transplanting the cells in a sciatic nerve injury model. We compared the amount of VEGF secreted from DsRed-NSCs (control) or SV-VEGF-NSCs and observed that SV-VEGF-NSCs have a much higher expression level of VEGF. We next investigated whether transplantation with SV-VEGF-NSCs aids functional recovery and pain reduction. We confirmed that transplantation with SV-VEGF-NSCs enhances functional recovery, pain reduction, and remyelination as well as the number of blood vessels compared with the control groups. Our results show that VEGF aids functional recovery and pain reduction in a sciatic nerve injury model.
Assuntos
Células-Tronco Neurais/metabolismo , Neuralgia/terapia , Transplante de Células-Tronco/métodos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/fisiologia , Modelos Animais de Doenças , Hiperalgesia/patologia , Hiperalgesia/fisiopatologia , Hiperalgesia/terapia , Masculino , Bainha de Mielina/patologia , Bainha de Mielina/fisiologia , Neuralgia/patologia , Neuralgia/fisiopatologia , Medição da Dor , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/fisiologia , Nervo Isquiático/lesões , Tato , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
STUDY DESIGN: Neuronal cell-specific gene expression system and neural stem cells (NSCs) were combined for treatment of spinal cord injury (SCI). OBJECTIVE: To verify the reproducibility of the neuronal cell-specific therapeutic gene overexpression system, we develop a neuronal cell-specific granulocyte-macrophage colony-stimulating factor expression system (NSE-GMCSF), and then examine the characteristics of GMCSF overexpression and protective effect on neural cells in vitro and vivo. SUMMARY OF BACKGROUND DATA: The stem cell transplantation is considered a promising therapy for SCI. However, stem cell monotherapy strategy is insufficient for complete recovery after SCI. Therefore, combined treatment method based on stem cells with other therapeutic system may be effective for improving the therapeutic efficacy. In this study, we established the gene and stem cell therapy platform based on NSCs and neuronal cell-specific gene expression system. METHODS: To examine the GMCSF expression pattern, we compared the amount of secreted GMCSF from the neuronal cell-specific GMCSF expressing NSCs with control GMCSF-expressing NSCs (respectively, NSE-GMCSF-NSCs vs. SV-GMCSF-NSCs) by ELISA in vitro and in vivo, and then verified the neuronal protective effect of these cells in vitro and vivo. RESULTS: The results showed that NSE-GMCSF-NSCs secreted more GMCSF compared with SV-GMCSF-NSCs in normoxia, hypoxia and cytotoxic conditions. The cell viability of NSE-GMCSF-NSCs was increased depending on the amount of secreted GMCSF in cytotoxic condition. In addition, the amount of secreted GMCSF by NSE-GMCSF-NSCs transplanted into injured spinal cord was significantly higher than SV-GMCSF-NSCs. Higher amount of secreted GMCSF decreased the expression of proapoptotic protein, Bax. CONCLUSION: In this study, we demonstrated that the neuronal cell-specific gene expression system induced overexpression of GMCSF in NSCs. These combined NSCs & gene therapy treatment protocol would be an effective therapeutic system for SCI. LEVEL OF EVIDENCE: N/A.
Assuntos
Apoptose/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Células-Tronco Neurais/metabolismo , Substâncias Protetoras/metabolismo , Traumatismos da Medula Espinal/metabolismo , Animais , Linhagem Celular , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Modelos Biológicos , Substâncias Protetoras/farmacologia , TransfecçãoRESUMO
Electrospun silk fibroin (SF) scaffolds provide large surface area, high porosity, and interconnection for cell adhesion and proliferation and they may replace collagen for many tissue engineering applications. Despite such advantages, electrospun SF scaffolds are still limited as bone tissue replacement due to their low mechanical strengths. While enhancement of mechanical strengths by incorporating inorganic ceramics into polymers has been demonstrated, electrospinning of a mixture of SF and inorganic ceramics such as hydroxyapatite is challenging and less studied due to the aggregation of ceramic particles within SF. In this study, we aimed to enhance the mechanical properties of electrospun SF scaffolds by uniformly dispersing hydroxyapatite (HAp) nanoparticles within SF nanofibers. HAp nanoaprticles were modified by γ-glycidoxypropyltrimethoxysilane (GPTMS) for uniform dispersion and enhanced interfacial bonding between HAp and SF fibers. Optimal conditions for electrospinning of SF and GPTMS-modified HAp nanoparticles were identified to achieve beadless nanofibers without any aggregation of HAp nanoparticles. The MTT and SEM analysis of the osteoblasts-cultured scaffolds confirmed the biocompatibility of the composite scaffolds. The mechanical properties of the composite scaffolds were analyzed by tensile tests for the scaffolds with varying contents of HAp within SF fibers. The mechanical testing showed the peak strengths at the HAp content of 20 wt.%. The increase of HAp content up to 20 wt.% increased the mechanical properties of the composite scaffolds, while further increase above 20 wt.% disrupted the polymer chain networks within SF nanofibers and weakened the mechanical strengths.