Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 566
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Cell Biochem Biophys ; 78(1): 23-30, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31875277

RESUMO

The CRISPR/Cas9 system is an effective tool for gene editing. However, this conventional expression system cannot control the timing of gene editing and does not utilize resistance screening markers. Therefore, carrying out CRISPR/Cas9 experiments is extremely inconvenient. Our aim is to develop an inducible lentiviral vector-based gene-editing system for C-X-C chemokine receptor 4 (CXCR4) by CRISPR/Cas9, and to demonstrate its function in MKN-45 cell. The DNA fragments of Blasticidin and T2A-GFP were produced using the lenti-Cas9-BLAST and PX458 plasmids as templates. The PCR products were harvested and cloned into the lenti-guide-puro plasmid to yield the lenti-guide-BLAST-GFP plasmid. Three double-stranded guide RNA (gRNA) sequences targeting the exon 2 of CXCR4 gene were designed online (http://crispr.mit.edu), synthesized, and recombined into the lenti-guide-BLAST-GFP plasmid, to yield the lenti-guide-BLAST-GFP-gRNA plasmid. The pCW-Cas9 and lenti-guide-BLAST-GFP-gRNA plasmids were packaged with lentiviral vectors, which were then transfected into MKN-45 cells, to identify the CXCR4 gene-editing effects using the T7 endonuclease 1 (T7E1) and Western blot assays. The lenti-guide-BLAST-GFP and lenti-guide-BLAST-GFP-gRNA plasmids were successfully constructed and packaged, to yield lentiviral particles. Transfection of the pCW-Cas9 and lenti-guide-BLAST-GFP-gRNA viral vectors could decrease the expression of CXCR4 protein, and lead to gene editing in MKN-45 cells. The efficiencies of gRNA-1, gRNA-2, and gRNA-3 were 45.6%, 53.6%, and 56.7%, respectively. Furthermore, the chemotactic efficiency of the dual viral vector-infected MKN-45 cells was significantly decreased in response to SDF-1. The numbers of migratory cells in the lower chamber of the transwell system were 30.0 ± 0.23, 29.7 ± 1.55, 28.2 ± 1.11 and 36.1 ± 2.00 cells per field (400×) for gRNA-1, gRNA-2, gRNA-3 and the control, respectively (P < 0.05). We constructed an inducible CXCR4 gene-editing, dual-vector CRISPR/Cas9 system, which could induce CXCR4 gene editing in MKN-45 cells in a doxycycline-dependent manner and thus reduce the migration of MKN-45 cells.


Assuntos
Sistemas CRISPR-Cas/genética , Receptores CXCR4/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12/farmacologia , Doxiciclina/farmacologia , Éxons , Edição de Genes , Expressão Gênica/efeitos dos fármacos , Humanos , Lentivirus/genética , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , Receptores CXCR4/metabolismo
2.
ACS Appl Mater Interfaces ; 11(38): 34621-34633, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31483598

RESUMO

In situ tissue repair holds great potential as a cell-free regenerative strategy. A critical aspect of this approach is the selection of cell instructive materials that can efficiently regulate the defect microenvironment via the release of chemoattractant factors to mobilize and recruit endogenous stem cells toward the site of implantation. Here we report the design of a DNA-based hydrogel as a drug delivery platform for the sustained release of a promising chemoattractant, SDF-1α. The hydrogel is composed of chemically cross-linked DNA strands, which are bridged via silicate nanodisks (nSi). Silicate nanodisks electrostatically interact with the negatively charged DNA backbone resulting in the formation of a dual cross-linked nanocomposite hydrogel with a combination of chemical and physical cross-link points. The formulated nanocomposites display enhanced elasticity and mechanical toughness as compared to their nonsilicate containing counterparts. Moreover, the electrostatic interaction between nSi and SDF-1α leads to sustained release of the chemokine from the hydrogels. The in vitro bioactivity assays confirm the retention of chemotactic properties of the protein after its release. Overall, the dual cross-linked DNA-based hydrogel platform could be potentially used as a cell-instructive material for the recruitment of host stem cells to guide the process of in situ tissue repair.


Assuntos
Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12 , DNA/química , Hidrogéis/química , Nanoestruturas/química , Silicatos/química , Células-Tronco/metabolismo , Animais , Quimiocina CXCL12/química , Quimiocina CXCL12/farmacocinética , Quimiocina CXCL12/farmacologia , Humanos , Camundongos , Células RAW 264.7 , Células-Tronco/citologia
3.
Colloids Surf B Biointerfaces ; 181: 963-972, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31382347

RESUMO

There is an urgent clinical demand to develop a functional small diameter vascular graft with long-term patency, which mainly relies on the anticoagulation and endothelialization of the vascular graft. In addition, improved degradation of vascular graft provides more space for cell infiltration and facilitates remodeling of blood vessel. In this study, an elastic and biomimetic nanofibrous vascular scaffold with improved degradability was prepared by adding poly(lactic-co-glycolic acid) (PLGA) into the poly(L-lactic acid) (PLLA)/poly(L-lactide-co-ε-caprolactone) (PLCL) blend using thermally induced phase separation (TIPS) technique. The incorporation of PLGA also improved the hydrophilicity of the composite scaffold. Then the vascular scaffold was surface modified by combination of heparin and stromal cell-derived factor-1 alpha (SDF-1α). The results of whole blood clotting kinetics and plasma recalcification profiles indicated that heparinized modification significantly enhanced the anticoagulation of vascular scaffold. In vitro cell culture assays demonstrated the immobilized SDF-1α facilitated recruitment of endothelial progenitor cells (EPCs), migration and proliferation of mature endothelial cells, human umbilical vein endothelial cells (HUVECs), and expression of VE-cadherin/CD144 and endothelial nitric oxide synthase (eNOS) genes in HUVECs, thereby accelerating the endothelialization of the modified vascular scaffold. Besides, the nanofibrous vascular scaffold modified with heparin and SDF-1α inhibited the proliferation of human vascular smooth muscle cells (HVSMCs). Therefore, the phase separated nanofibrous vascular scaffold modified with heparin and SDF-1α shows the promising in vitro performance as a functional small diameter vascular graft.


Assuntos
Anticoagulantes/farmacologia , Quimiocina CXCL12/farmacologia , Heparina/farmacologia , Nanofibras/química , Polímeros/farmacologia , Anticoagulantes/química , Coagulação Sanguínea/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiocina CXCL12/química , Células Endoteliais/efeitos dos fármacos , Heparina/química , Humanos , Tamanho da Partícula , Polímeros/química , Propriedades de Superfície , Engenharia Tecidual
4.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(5): 393-398, 2019 May.
Artigo em Chinês | MEDLINE | ID: mdl-31223107

RESUMO

Objective To explore the effects of over-expression of C-X-C motif chemokine receptor 4 (CXCR4) on the homing capacity of bone marrow mesenchymal stem cells (BMMSCs) in vivo and in vitro, and the proliferation activity of BMMSCs. Methods The CXCR4-BMMSCs were constructed by the lentiviral vector-mediated BMMSC over-expressing the CXCR4 gene. The effect of chemokine SDF-1 on the migration ability of BMMSCs was detected by TranswellTM migration assay; and MTT assay was used to detect cell proliferation. The BALB/c mice were randomly divided into two groups after receiving total body irradiation (TBI), 10 mice in each group. BMMSCs control group: mice were infused with BMMSCs (5×105) transducted by EGFP via tail vein after TBI; CXCR4-BMMSC group: mice were infused with BMMSCs (5×105) simultaneously transducted by GFP and CXCR4 gene via tail vein after TBI. The thymus of mice was frozen and sectioned, and fluorescence microscopy was used to observe the homing of BMMSCs into thymus tissue. Furthermore, flow cytometry was performed to detect the homing efficiency of BMMSCs to thymus in recipient mice. Results Transwell assay showed that over-expresion of CXCR4 could promote the migration and recruitment of BMMSCs to chemokine SDF-1; the proliferation activity of CXCR4-BMMSCs increased significantly compared with BMMSC control group. Compared with the control group, the frozen section showed that over-expression of CXCR4 could significantly improve the efficiency of BMMSC homing to thymus tissue. Flow cytometry suggested that CXCR4-BMMSCs homing to thymus were more than those in the control group. Conclusion Over-expression of CXCR4 gene mediated by lentivirus vector can enhance the migration and recruitment of BMMSCs by SDF-1, and promote BMMSCs homing to damaged thymus in vivo. In addition, CXCR4 also promotes the proliferation of BMMSCs.


Assuntos
Movimento Celular , Proliferação de Células , Células-Tronco Mesenquimais/citologia , Receptores CXCR4/genética , Animais , Células da Medula Óssea , Quimiocina CXCL12/farmacologia , Lentivirus , Camundongos , Camundongos Endogâmicos BALB C , Transdução de Sinais
5.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(4): 320-326, 2019 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-31167691

RESUMO

Objective To investigating the effect of bone marrow mesenchymal stem cells (BMSCs) pretreated with stromal cell-derived factor-1 (SDF-1) on carotid stenosis in rats. Methods The plasmid carrying enhanced green fluorescent protein (EGFP) was transfected into BMSCs and then intravenously injected into rats. The rats were divided into carotid artery injury model control group, BMSCs transplantation group, and BMSCs pretreated with SDF-1 group. Two weeks after the cell transplantation, the injured vascular tissues were collected and EGFP expression was detected by immunofluorescence histochemistry to determine the homing of BMSCs. Four weeks after cell transplantation, the endothelialization of injured intima was observed by Evans blue staining, and CD31 expression in injured vessels was detected by the immunofluorescence technique. The neointimal hyperplasia of injured carotid arteries was observed by HE staining. The expression of proliferating cell nuclear antigen (PCNA) in injured vessels was detected by immunohistochemical staining. The protein level of vascular endothelial growth factor (VEGF) was tested by Western blot analysis. Results The transplantation of BMSCs pretreated with SDF-1 could effectively promote the homing of BMSCs to injured blood vessels, and promote the re-endothelialization of injured vessels. The neointimal area and neointimal area/medial area in the two BMSC-transplantation groups were both lower than those in the model control group, which were more significantly different from the BMSCs pretreated with SDF-1 group. The transplantation of BMSCs pretreated with SDF-1 could significantly increase the expression of CD31 and VEGF in injured intima and inhibit the expression of PCNA. Conclusion The transplantation of BMSCs pretreated with SDF-1 can inhibit the proliferation of smooth muscle cells in the media and reduce arterial stenosis by promoting the migration of BMSCs to the injured site and inducing the differentiation of BMSCs.


Assuntos
Artérias Carótidas/citologia , Estenose das Carótidas/terapia , Quimiocina CXCL12/farmacologia , Endotélio/citologia , Transplante de Células-Tronco Mesenquimais , Miócitos de Músculo Liso/citologia , Animais , Células da Medula Óssea/citologia , Artérias Carótidas/patologia , Proliferação de Células , Ratos , Fator A de Crescimento do Endotélio Vascular/metabolismo
6.
Cell Tissue Res ; 378(2): 207-220, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31152245

RESUMO

Pulp-dentin regeneration in the apical region of immature permanent teeth represents a significant clinical challenge. Tissue engineering approaches using bioactive molecules and scaffolds may have the potential to regenerate the natural apical structure of these teeth, representing a superior alternative to existing treatment regimens. The aims of this study are (i) to evaluate the VitroGel 3D system, an animal origin-free polysaccharide hydrogel, as a possible injectable scaffold for pulp-dentin regeneration and (ii) to investigate the effects of stromal cell-derived factor-1α (SDF-1α) and bone morphogenetic protein-2(BMP-2) cotreatment on odontogenic differentiation of human stem cells from apical papilla (SCAP) cultured in the VitroGel 3D system. The morphology, viability and proliferation of SCAP cultured in the VitroGel 3D system were measured via scanning electron microscopy (SEM), live and dead cell staining and CCK-8 assays. Alkaline phosphatase (ALP) activity, real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR) and Western blot analysis were further used to evaluate the odontogenic differentiation of SCAP cultured in the VitroGel 3D system in vitro. Finally, the odontogenic differentiation was assessed in vivo through ectopic subcutaneous injection. The results showed that SCAP cultured in 3D hydrogel demonstrated favorable viability and proliferation. SDF-1α and BMP-2 cotreatment enhanced odontogenic differentiation-related gene and protein expression in vitro and promoted odontogenic differentiation of SCAP in vivo. In conclusion, the present study demonstrated that the VitroGel 3D system promoted SCAP proliferation and differentiation. Moreover, SDF-1α cotreatment had synergistic effects on BMP-2-induced odontogenic differentiation of human SCAP cultured in the VitroGel 3D system both in vitro and in vivo.


Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Quimiocina CXCL12/farmacologia , Papila Dentária/citologia , Odontogênese/efeitos dos fármacos , Células-Tronco/citologia , Adolescente , Diferenciação Celular , Células Cultivadas , Meios de Cultura , Feminino , Humanos , Hidrogéis , Masculino , Engenharia Tecidual/métodos , Tecidos Suporte
7.
ACS Appl Mater Interfaces ; 11(16): 14608-14618, 2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-30938503

RESUMO

Continuous delivery of growth factors to the injury site is crucial to creating a favorable microenvironment for cartilage injury repair. In the present study, we fabricated a novel sustained-release scaffold, stromal-derived factor-1α (SDF-1α)/transforming growth factor-ß1 (TGF-ß1)-loaded silk fibroin-porous gelatin scaffold (GSTS). GSTS persistently releases SDF-1α and TGF-ß1, which enhance cartilage repair by facilitating cell homing and chondrogenic differentiation. Scanning electron microscopy showed that GSTS is a porous microstructure and the protein release assay demonstrated the sustainable release of SDF-1α and TGF-ß1 from GSTS. Bone marrow-derived mesenchymal stem cells (MSCs) maintain high in vitro cell activity and excellent cell distribution and phenotype after seeding into GSTS. Furthermore, MSCs acquired enhanced chondrogenic differentiation capability in the TGF-ß1-loaded scaffolds (GSTS or GST: loading TGF-ß1 only) and the conditioned medium from SDF-1α-loaded scaffolds (GSTS or GSS: loading SDF-1α only) effectively promoted MSCs migration. GSTS was transplanted into the osteochondral defects in the knee joint of rats, and it could promote cartilage regeneration and repair the cartilage defects at 12 weeks after transplantation. Our study shows that GSTS can facilitate in vitro MSCs homing, migration, chondrogenic differentiation and SDF-1α and TGF-ß1 have a synergistic effect on the promotion of in vivo cartilage forming. This SDF-1α and TGF-ß1 releasing GSTS have promising therapeutic potential in cartilage repair.


Assuntos
Cartilagem , Quimiocina CXCL12 , Condrogênese/efeitos dos fármacos , Fibroínas , Gelatina , Fator de Crescimento Transformador beta1 , Animais , Cartilagem/lesões , Cartilagem/metabolismo , Cartilagem/patologia , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12/química , Quimiocina CXCL12/farmacocinética , Quimiocina CXCL12/farmacologia , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Preparações de Ação Retardada/farmacologia , Fibroínas/química , Fibroínas/farmacocinética , Fibroínas/farmacologia , Gelatina/química , Gelatina/farmacocinética , Gelatina/farmacologia , Masculino , Porosidade , Ratos , Fator de Crescimento Transformador beta1/química , Fator de Crescimento Transformador beta1/farmacocinética , Fator de Crescimento Transformador beta1/farmacologia
8.
Mol Med Rep ; 19(5): 4388-4400, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30942441

RESUMO

Osteoarthritis (OA) is an aseptic inflammatory disease which is associated with the stromal cell­derived factor 1/C­X­C chemokine receptor type 4 (SDF­1/CXCR4) axis. Accumulating studies have identified numbers of microRNAs (miRNAs) that serve important roles in the pathogenesis of OA. However, whether and how the inhibition of the SDF­1/CXCR4 axis induces alterations in miRNA expression remains largely unclear. miRNA profiling was performed in OA chondrocytes stimulated with SDF­1 alone, or SDF­1 with the CXCR4 antagonist TN14003 by miRNA microarray. Candidate miRNAs were verified by reverse transcription quantitative polymerase chain reaction. Bioinformatic analyses including target prediction, gene ontology (GO) and pathway analysis were performed to explore the potential functions of candidate miRNAs. Notably, 7 miRNAs (miR­146a­5p, miR­221­3p, miR­126­3p, miR­185­5p, miR­155­5p, miR­124­3p and miR­130a­3p) were significantly differentially expressed. GO analysis indicated that miR­146a­5p and its associated genes were enriched in receptor regulatory activity, nuclear factor­kappa­light­chain­enhancer of activated B cells (NF­κB)­inducing kinase activity, cellular response to interleukin­1, cytokine­cytokine receptor interaction, NF­κB signaling pathway and osteoclast differentiation pathways. CXCR4 was predicted to be a target of miR­146a­5p with high importance. The mRNA and protein levels of key factors involved in cartilage degeneration were measured following manipulation of the expression levels of miR­146a­5p in OA chondrocytes. CXCR4 and MMP­3 levels were negatively associated with miR­146a­5p expression, while the levels of type II collagen and aggrecan were positively associated. These data reveal that TN14003 upregulates miR­146a­5p expression, and also pinpoints a novel role of miR­146a­5p in inhibiting cartilage degeneration by directly targeting the SDF­1/CXCR4 axis.


Assuntos
Quimiocina CXCL12/farmacologia , MicroRNAs/metabolismo , Osteoartrite/patologia , Peptídeos/farmacologia , Regulação para Cima/efeitos dos fármacos , Idoso , Antagomirs/metabolismo , Biomarcadores/metabolismo , Cartilagem/metabolismo , Cartilagem/patologia , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Feminino , Humanos , Masculino , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Osteoartrite/diagnóstico , Osteoartrite/metabolismo , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transdução de Sinais/efeitos dos fármacos
9.
Neurochem Int ; 126: 59-63, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30858017

RESUMO

We investigated the impact of the prolonged exposure of rat hippocampal synaptosomes to CXCL12 (3 nM) on the NMDA-mediated release of [3H]D-aspartate ([3H]D-Asp) or [3H]noradrenaline ([3H]NA). Synaptosomes were stimulated twice with NMDA/CXCL12 and the amount of the NMDA-evoked tritium release (S1 and S2) quantified to calculate the S2/S1 ratio. The S2/S1 ratio for both transmitters was drastically decreased by 3 nM CXCL12 between the two stimuli (CXCL12-treated synaptosomes) in a AMD3100-sensitive manner. The phosphorylation of the GluN1 subunit in Ser 896 was reduced in CXCL12-treated synaptosomes, while the overall amount of GluN1 and GluN2B proteins as well as the GluN2B insertion in synaptosomal plasmamembranes were unchanged. We conclude that the CXCR4/NMDA cross-talk is dynamically regulated by the time of activation of the CXCR4s. Our results unveil a functional cross-talk that might account for the severe impairments of central transmission that develop in pathological conditions characterized by CXCL12 overproduction.


Assuntos
Hipocampo/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores CXCR4/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinaptossomos/metabolismo , Animais , Quimiocina CXCL12/farmacologia , Hipocampo/efeitos dos fármacos , N-Metilaspartato/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores CXCR4/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Sinaptossomos/efeitos dos fármacos
10.
Biol Reprod ; 100(1): 61-70, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30084961

RESUMO

Successful implantation and pregnancy is dependent on sufficient endometrial growth during each reproductive cycle. Here, we report the therapeutic effect of either bone marrow-derived cells (BMDCs) or the stem cell chemo-attractant C-X-C motif chemokine 12 (CXCL12) on endometrial receptivity in a murine ethanol induced thin endometrium model. Endometrial epithelial area was significantly increased in mice treated with BMDCs, CXCL12, or by co-treatment with both compared with PBS-treated controls. Ki-67 and CD31 immunoreactivity was significantly higher in mice treated with either BMDCs, CXCL12, or both. The mRNA expression levels of endometrial receptivity markers leukemia inhibitory factor, interleukin-1ß, and integrin beta-3 were increased in mice treated with either BMDCs, CXCL12, or both. The mRNA levels of matrix metalloproteinase-2 and -9 were significantly decreased by BMDCs but not by CXCL12. Pregnancy rates and litter size were increased after either treatment. Both BMDCs and CXCL12 displayed a comparable efficacy on endometrial regeneration in mice with thin endometrium. Our findings indicate the potential therapeutic effects of BMDCs and CXCL12 on infertility related to thin endometrium. Bone marrow-derived cells and CXCL12 displayed a comparable efficacy on endometrial regeneration in mice with thin endometrium.


Assuntos
Células da Medula Óssea/fisiologia , Transplante de Medula Óssea , Quimiocina CXCL12/farmacologia , Endométrio/efeitos dos fármacos , Infertilidade Feminina , Doenças Uterinas , Animais , Modelos Animais de Doenças , Implantação do Embrião/efeitos dos fármacos , Implantação do Embrião/fisiologia , Endométrio/patologia , Endométrio/fisiologia , Feminino , Infertilidade Feminina/etiologia , Infertilidade Feminina/patologia , Infertilidade Feminina/terapia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Doenças Uterinas/complicações , Doenças Uterinas/patologia , Doenças Uterinas/terapia
11.
Int J Biol Macromol ; 124: 460-468, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30391592

RESUMO

Facial nerve injury is a clinically common disease accompanied by demyelination of damaged nerves. The remyelination of damaged nerves and the unsatisfactory function recovery are problems that have been plaguing people for a long time. The role that CXCL12 plays after facial nerve injury remains unknown. Our experiments found that the expression of CXCL12 was up-regulated in the early stage of facial nerve injury and decreased after two weeks. Further research found that CXCL12 had no effect on Schwann cells proliferation, apoptosis and cell cycle, while significantly promoted Schwann cells migration. Treatment with CXCL12 decreased the phosphorylation of PI3K, AKT and mTOR, but increased autophagy marker LC3II/I. The CXCL12-induced Schwann cells migration was significantly attenuated by inhibition of autophagy and activation of PI3K pathway through pretreatment with 3-MA and IGF-1 respectively, and this effect was enhanced by PI3K pathway inhibitor LY294002. Animal experiment also confirmed that CXCL12 could improve facial nerve function and myelin regeneration. The findings of this study indicate that CXCL12 can promote the migration of Schwann cells and potentially become a key molecule in the repair of facial nerve injury.


Assuntos
Autofagia/efeitos dos fármacos , Quimiocina CXCL12/farmacologia , Traumatismos do Nervo Facial/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Serina-Treonina Quinases TOR/genética , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cromonas/farmacologia , Nervos Cranianos/efeitos dos fármacos , Nervos Cranianos/metabolismo , Nervos Cranianos/patologia , Modelos Animais de Doenças , Nervo Facial/efeitos dos fármacos , Nervo Facial/metabolismo , Nervo Facial/patologia , Traumatismos do Nervo Facial/genética , Traumatismos do Nervo Facial/metabolismo , Traumatismos do Nervo Facial/patologia , Regulação da Expressão Gênica , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Masculino , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Cultura Primária de Células , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Células de Schwann/patologia , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
12.
Oncogene ; 38(1): 73-87, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30082913

RESUMO

Primary glioblastoma is the most frequent human brain tumor in adults and is generally fatal due to tumor recurrence. We previously demonstrated that glioblastoma-initiating cells invade the subventricular zones and promote their radio-resistance in response to the local release of the CXCL12 chemokine. In this work, we show that the mitotic Aurora A kinase (AurA) is activated through the CXCL12-CXCR4 pathway in an ERK1/2-dependent manner. Moreover, the CXCL12-ERK1/2 signaling induces the expression of Ajuba, the main cofactor of AurA, which allows the auto-phosphorylation of AurA.We show that AurA contributes to glioblastoma cell survival, radio-resistance, self-renewal, and proliferation regardless of the exogenous stimulation with CXCL12. On the other hand, AurA triggers the CXCL12-mediated migration of glioblastoma cells in vitro as well as the invasion of the subventricular zone in xenograft experiments. Moreover, AurA regulates cytoskeletal proteins (i.e., Actin and Vimentin) and favors the pro-migratory activity of the Rho-GTPase CDC42 in response to CXCL12. Altogether, these results show that AurA, a well-known kinase of the mitotic machinery, may play alternative roles in human glioblastoma according to the CXCL12 concentration.


Assuntos
Aurora Quinase A/fisiologia , Neoplasias Encefálicas/enzimologia , Quimiocina CXCL12/fisiologia , Glioblastoma/enzimologia , Proteínas de Neoplasias/fisiologia , Animais , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular , Quimiocina CXCL12/farmacologia , Ativação Enzimática , Glioblastoma/patologia , Xenoenxertos , Humanos , Proteínas com Domínio LIM/biossíntese , Proteínas com Domínio LIM/genética , Ventrículos Laterais/patologia , Sistema de Sinalização das MAP Quinases , Camundongos , Invasividade Neoplásica , Fosforilação , Processamento de Proteína Pós-Traducional , Receptores CXCR4/fisiologia , Transdução de Sinais
13.
Int J Nanomedicine ; 13: 7395-7408, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30519022

RESUMO

Background: Stromal cell-derived factor 1 (SDF-1) is an important chemokine for stem cell mobilization, and plays a critical role in mobilization of mesenchymal stem cells (MSCs). Bone morphogenetic protein 2 (BMP-2) plays a critical role in osteogenesis of MSCs. However, the use of SDF-1 and BMP-2 in bone tissue engineering is limited by their short half-lives and rapid degradation in vitro and in vivo. Methods: The chitosan oligosaccharide/heparin nanoparticles (CSO/H NPs) were first prepared via self-assembly. Chitosan-agarose-gelatin (CAG) Scaffolds were then synthesized via gelation technology using cross-linked chitosan, agarose, and gelatin, and were modified by CSO/H NPs. The encapsulation efficiency and release kinetics of SDF-1 and BMP-2 were quantified using an enzyme-linked immunosorbent assay. A CCK-8 assays were used to evaluate biocompatibility of NP-modified scaffolds. The biological activity of the loaded SDF-1 and BMP-2 was evaluated using the transwell migration assay and osteogenic induction assay. An animal MSC recruitment model was used to study the ability of SDF-1 released from NP-modified scaffolds to induce migration of MSCs. Results: In this study, we developed a novel nanoparticle-modified CAG scaffold for the delivery of SDF-1 and BMP-2. CCK-8 assays demonstrated excellent biocompatibility of NP-modified scaffolds. In addition, we investigated the release of SDF-1 and BMP-2 from NP-modified scaffolds, and evaluated the effect of released SDF-1 on MSC migration. The effect of released BMP-2 on MSC osteogenesis was also examined. In vitro cell migration assays showed that SDF-1 released from NP-modified scaffolds retained its migration activity; osteogenesis studies demonstrated that released BMP-2 exhibited a strong ability to induce differentiation towards osteoblasts. Our in vivo recruitment assays showed continuous chemotactic response of MSCs to SDF-1 released from the NP-modified scaffold. Conclusion: The simplicity of synthesizing CSO/H NP-modified CAG scaffolds, combined with its high cytokine loading capacity and sustained release effect, renders NP-modified CAG scaffold an attractive candidate for sustained release of SDF-1 and BMP-2 to promote bone repair and regeneration.


Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Quimiocina CXCL12/farmacologia , Quitosana/química , Gelatina/química , Nanopartículas/química , Sefarose/química , Tecidos Suporte/química , Fosfatase Alcalina/metabolismo , Animais , Movimento Celular , Preparações de Ação Retardada/farmacologia , Liberação Controlada de Fármacos , Feminino , Humanos , Cinética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/enzimologia , Camundongos Endogâmicos C57BL , Nanopartículas/ultraestrutura , Osteoblastos/citologia , Osteogênese/efeitos dos fármacos , Ratos
14.
Cytotherapy ; 20(12): 1427-1436, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30377040

RESUMO

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) enhance islet function both in vitro and in vivo, at least in part by secreting ligands that activate islet G-protein coupled receptors (GPCRs). We assessed whether pre-treatment with a defined "cocktail" of MSC-secreted GPCR ligands enhances islet functional survival in vitro and improves the outcomes of islet transplantation in an experimental model of diabetes. METHODS: Isolated islets were cultured for 48 h with ANXA1, SDF-1 or C3a, alone or in combination. Glucose-stimulated insulin secretion (GSIS) and cytokine-induced apoptosis were measured immediately after the 48 h culture period and at 24 h or 72 h following removal of the ligands from the culture media. Islets were syngeneically transplanted underneath the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice and blood glucose levels monitored for 28 days. RESULTS: Pre-culturing islets with a cocktail of ANXA1/SDF-1/C3a potentiated GSIS and protected islet cells from cytokine-induced apoptosis in vitro. These effects were maintained for up to 72 h after the removal of the factors from the culture medium, suggesting a sustained protection of islet graft functional survival during the immediate post-transplantation period. Islets pre-treated with the cocktail of MSC secretory factors were more effective in reducing blood glucose in diabetic mice, consistent with their improved functional survival in vivo. DISCUSSION: Pre-culturing islets with a cocktail of MSC secretory products offers a well-defined, cell-free approach to improve clinical islet transplantation outcomes while avoiding many of the safety, regulatory and logistical hurdles of incorporating MSCs into transplantation protocols.


Assuntos
Quimiocina CXCL12/farmacologia , Complemento C3a/farmacologia , Transplante das Ilhotas Pancreáticas/métodos , Células-Tronco Mesenquimais/metabolismo , Animais , Anexina A1/genética , Anexina A1/metabolismo , Anexina A1/farmacologia , Apoptose/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Complemento C3a/genética , Complemento C3a/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/terapia , Glucose/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Receptores Acoplados a Proteínas-G/metabolismo
15.
Front Immunol ; 9: 2118, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30283452

RESUMO

Chemokine synergy-inducing molecules are emerging as regulating factors in cell migration. The alarmin HMGB1, in its reduced form, can complex with CXCL12 enhancing its activity on monocytes via the chemokine receptor CXCR4, while the form containing a disulfide bond, by binding to TLR2 or TLR4, initiates a cascade of events leading to production of cytokines and chemokines. So far, the possibility that the CXCL12/HMGB1 heterocomplex could be maintained in chronic inflammation was debated, due to the release of reactive oxygen species. Therefore, we have assessed if the heterocomplex could remain active in Rheumatoid Arthritis (RA) and its relevance in the disease assessment. Monocytes from RA patients with active disease require a low concentration of HMGB1 to enhance CXCL12-induced migration, in comparison to monocytes from patients in clinical remission or healthy donors. The activity of the heterocomplex depends on disease activity, on the COX2 and JAK/STAT pathways, and is determined by the redox potential of the microenvironment. In RA, the presence of an active thioredoxin system correlates with the enhanced cell migration, and with the presence of the heterocomplex in the synovial fluid. The present study highlights how, in an unbalanced microenvironment, the activity of the thioredoxin system plays a crucial role in sustaining inflammation. Prostaglandin E2 stimulation of monocytes from healthy donors is sufficient to recapitulate the response observed in patients with active RA. The activation of mechanisms counteracting the oxidative stress in the extracellular compartment preserves HMGB1 in its reduced form, and contributes to fuel the influx of inflammatory cells. Targeting the heterocomplex formation and its activity could thus be an additional tool for dampening the inflammation sustained by cell recruitment, for those patients with chronic inflammatory conditions who poorly respond to current therapies.


Assuntos
Artrite Reumatoide/metabolismo , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12/farmacologia , Proteína HMGB1/farmacologia , Monócitos/efeitos dos fármacos , Adulto , Idoso , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Movimento Celular/imunologia , Células Cultivadas , Dinoprostona/farmacologia , Sinergismo Farmacológico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/metabolismo , Oxirredução , Ligação Proteica/efeitos dos fármacos , Receptores CXCR4/imunologia , Receptores CXCR4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia
16.
Sci Rep ; 8(1): 14501, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30266921

RESUMO

Skeletal osteoblasts are important regulators of B-lymphopoiesis, serving as a rich source of factors such as CXCL12 and IL-7 which are crucial for B-cell development. Recent studies from our laboratory and others have shown that deletion of Rptor, a unique component of the mTORC1 nutrient-sensing complex, early in the osteoblast lineage development results in defective bone development in mice. In this study, we now demonstrate that mTORC1 signalling in pre-osteoblasts is required for normal B-lymphocyte development in mice. Targeted deletion of Rptor in osterix-expressing pre-osteoblasts (Rptorob-/-) leads to a significant reduction in the number of B-cells in the bone marrow, peripheral blood and spleen at 4 and 12 weeks of age. Rptorob-/- mice also exhibit a significant reduction in pre-B and immature B-cells in the BM, indicative of a block in B-cell development from the pro-B to pre-B cell stage. Circulating levels of IL-7 and CXCL12 are also significantly reduced in Rptorob-/- mice. Importantly, whilst Rptor-deficient osteoblasts are unable to support HSC differentiation to B-cells in co-culture, this can be rescued by the addition of exogenous IL-7 and CXCL12. Collectively, these findings demonstrate that mTORC1 plays an important role in extrinsic osteoblastic regulation of B-cell development.


Assuntos
Linfócitos B/citologia , Linfopoese/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/fisiologia , Osteoblastos/metabolismo , Animais , Linfócitos B/metabolismo , Quimiocina CXCL12/biossíntese , Quimiocina CXCL12/sangue , Quimiocina CXCL12/farmacologia , Técnicas de Cocultura , Regulação para Baixo , Genes Reporter , Interleucina-7/sangue , Interleucina-7/farmacologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , RNA Mensageiro/biossíntese , Proteína Regulatória Associada a mTOR/deficiência , Proteína Regulatória Associada a mTOR/genética , Proteína Regulatória Associada a mTOR/fisiologia , Fator de Transcrição Sp7/metabolismo
17.
J Biosci ; 43(4): 649-659, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30207311

RESUMO

Glucose-induced oxidative stress in the diabetic pancreas directly affects viability and the consequent therapeutic outcome of transplanted stem cells. Pretreatment of stem cells with growth factors induces tolerance in them against various stresses (hypoxia, thermal or hyperglycaemic). This study investigated the effect of pretreatment on insulin-producing cells (IPCs) differentiated from adipose-derived mesenchymal stem cells (ADMSCs), with a combination of stromal cell-derived factor 1 alpha (SDF1 α) and basic fibroblast growth factor (bFGF) against hyperglycaemic stress (17 or 33 mM glucose). The results showed that IPCs pretreated with a combination of SDF1α and bFGF exhibited maximally alleviated apoptosis, senescence and cell damage with a concomitantly increased release of insulin, enhanced cell proliferation and greater upregulation of Insulin 1, Insulin 2, Ngn3, Pdx1 and Nkx6.2 when stressed with 33 mM glucose. These findings may offer an improved therapeutic outcome for the treatment of diabetes.


Assuntos
Quimiocina CXCL12/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Fator 2 de Crescimento de Fibroblastos/farmacologia , Insulina/biossíntese , Células-Tronco Mesenquimais/efeitos dos fármacos , Tecido Adiposo/citologia , Tecido Adiposo/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quimiocina CXCL12/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Glucose/administração & dosagem , Glucose/toxicidade , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Células-Tronco Mesenquimais/citologia , Estresse Oxidativo/efeitos dos fármacos , Pâncreas/metabolismo , Pâncreas/patologia , Ratos
18.
PLoS Biol ; 16(7): e2005315, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30052626

RESUMO

Over half of individuals infected with human immunodeficiency virus (HIV) suffer from HIV-associated neurocognitive disorders (HANDs), yet the molecular mechanisms leading to neuronal dysfunction are poorly understood. Feline immunodeficiency virus (FIV) naturally infects cats and shares its structure, cell tropism, and pathology with HIV, including wide-ranging neurological deficits. We employ FIV as a model to elucidate the molecular pathways underlying HIV-induced neuronal dysfunction, in particular, synaptic alteration. Among HIV-induced neuron-damaging products, HIV envelope glycoprotein gp120 triggers elevation of intracellular Ca2+ activity in neurons, stimulating various pathways to damage synaptic functions. We quantify neuronal Ca2+ activity using intracellular Ca2+ imaging in cultured hippocampal neurons and confirm that FIV envelope glycoprotein gp95 also elevates neuronal Ca2+ activity. In addition, we reveal that gp95 interacts with the chemokine receptor, CXCR4, and facilitates the release of intracellular Ca2+ by the activation of the endoplasmic reticulum (ER)-associated Ca2+ channels, inositol triphosphate receptors (IP3Rs), and synaptic NMDA receptors (NMDARs), similar to HIV gp120. This suggests that HIV gp120 and FIV gp95 share a core pathological process in neurons. Significantly, gp95's stimulation of NMDARs activates cGMP-dependent protein kinase II (cGKII) through the activation of the neuronal nitric oxide synthase (nNOS)-cGMP pathway, which increases Ca2+ release from the ER and promotes surface expression of AMPA receptors, leading to an increase in synaptic activity. Moreover, we culture feline hippocampal neurons and confirm that gp95-induced neuronal Ca2+ overactivation is mediated by CXCR4 and cGKII. Finally, cGKII activation is also required for HIV gp120-induced Ca2+ hyperactivation. These results thus provide a novel neurobiological mechanism of cGKII-mediated synaptic hyperexcitation in HAND.


Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo II/metabolismo , Síndrome de Imunodeficiência Adquirida Felina/virologia , HIV-1/fisiologia , Vírus da Imunodeficiência Felina/fisiologia , Sinapses/metabolismo , Animais , Cálcio/metabolismo , Gatos , Quimiocina CXCL12/farmacologia , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Proteína gp120 do Envelope de HIV/metabolismo , Hipocampo/patologia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , Modelos Biológicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Subunidades Proteicas/metabolismo , Receptores de AMPA/metabolismo , Proteínas Virais/metabolismo
19.
Domest Anim Endocrinol ; 65: 38-48, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29890304

RESUMO

The strategies for improving the in vitro maturation (IVM) of domestic animal oocytes focus on promoting nuclear and cytoplasmic maturation. The identification of paracrine factors and their supplementation in the culture medium represent effective approaches for oocyte maturation and embryo development. This study investigated the effects of paracrine factor supplementation including connective tissue growth factor (CTGF), nerve growth factor (NGF), hepatocyte growth factor (HGF), and stromal derived factor 1 (SDF1) on ovine oocytes and early parthenogenetic embryos using an in vitro culture system. First, we identified the optimal concentrations of CTGF (30 ng/mL), SDF1 (10 ng/mL), NGF (3 ng/mL), and HGF (100 ng/mL) for promoting oocyte maturation, which combined, induced nuclear maturation in 94.19% of oocytes. This combination also promoted cumulus cell expansion and inhibited oocyte/cumulus apoptosis, while enabling a larger proportion (33.04%) of embryos to develop into blastocysts than in the controls and prevented embryo apoptosis. These novel findings demonstrate that the paracrine factors CTGF, SDF1, NGF, and HGF facilitate ovine oocyte and early parthenogenetic embryo development in vitro. Thus, supplementation with these factors may help optimize the IVM of ovine oocytes and early parthenogenetic embryo development strategies.


Assuntos
Quimiocina CXCL12/farmacologia , Fator de Crescimento do Tecido Conjuntivo/farmacologia , Fator de Crescimento de Hepatócito/farmacologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Fator de Crescimento Neural/farmacologia , Ovinos , Animais , Apoptose/efeitos dos fármacos , Blastocisto/efeitos dos fármacos , Blastocisto/fisiologia , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/fisiologia , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Partenogênese
20.
Macromol Biosci ; 18(7): e1800004, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29870589

RESUMO

Dual electrospinning can be used to make multifunctional scaffolds for regenerative medicine applications. Here, two supramolecular polymers with different material properties are electrospun simultaneously to create a multifibrous mesh. Bisurea (BU)-based polycaprolactone, an elastomer providing strength to the mesh, and ureido-pyrimidinone (UPy) modified poly(ethylene glycol) (PEG), a hydrogelator, introducing the capacity to deliver compounds upon swelling. The dual spun scaffolds are modularly tuned by mixing UPyPEG hydrogelators with different polymer lengths, to control swelling of the hydrogel fiber, while maintaining the mechanical properties of the scaffold. Stromal cell derived factor 1 alpha (SDF1α) peptides are embedded in the UPyPEG fibers. The swelling and erosion of UPyPEG increase void spaces and released the SDF1α peptide. The functionalized scaffolds demonstrate preferential lymphocyte recruitment proposed to be created by a gradient formed by the released SDF1α peptide. This delivery approach offers the potential to develop multifibrous scaffolds with various functions.


Assuntos
Quimiocina CXCL12/química , Hidrogéis/química , Poliésteres/química , Polietilenoglicóis/química , Engenharia Tecidual/métodos , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12/farmacologia , Elasticidade , Técnicas Eletroquímicas , Humanos , Hidrogéis/farmacologia , Hidrogéis/efeitos da radiação , Leucócitos Mononucleares , Peptídeos/química , Peptídeos/farmacologia , Poliésteres/farmacologia , Polietilenoglicóis/farmacologia , Porosidade , Cultura Primária de Células , Pirimidinonas/química , Tecidos Suporte , Raios Ultravioleta , Ureia/análogos & derivados
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA