RESUMO
OBJECTIVES: To investigate the long-term outcome of patients with acute ST-segment elevation myocardial infarction (STEMI) and a chronic total occlusion (CTO) in a non-infarct-related artery (IRA) and the risk factors for mortality. METHODS: The enrolled cohort comprised 323 patients with STEMI and multivessel diseases (MVD) that received a primary percutaneous coronary intervention between January 2008 and November 2013. The patients were divided into two groups: the CTO group (n = 97) and the non-CTO group (n = 236). The long-term major adverse cardiovascular and cerebrovascular events (MACCE) experienced by each group were compared. RESULTS: The rates of all-cause mortality and MACCE were significantly higher in the CTO group than they were in the non-CTO group. Cox regression analysis showed that an age ≥ 65 years (OR = 3.94, 95% CI: 1.47-10.56, P = 0.01), a CTO in a non-IRA(OR = 5.09, 95% CI: 1.79 ~ 14.54, P < 0.01), an in-hospital Killip class ≥ 3 (OR = 4.32, 95% CI: 1.71 ~ 10.95, P < 0.01), and the presence of renal insufficiency (OR = 5.32, 95% CI: 1.49 ~ 19.01, P = 0.01), stress ulcer with gastraintestinal bleeding (SUB) (OR = 6.36, 95% CI: (1.45 ~ 28.01, P = 0.01) were significantly related the 10-year mortality of patients with STEMI and MVD; an in-hospital Killip class ≥ 3 (OR = 2.97,95% CI:1.46 ~ 6.03, P < 0.01) and the presence of renal insufficiency (OR = 5.61, 95% CI: 1.19 ~ 26.39, P = 0.03) were significantly related to the 10-year mortality of patients with STEMI and a CTO. CONCLUSIONS: The presence of a CTO in a non-IRA, an age ≥ 65 years, an in-hospital Killip class ≥ 3, and the presence of renal insufficiency, and SUB were independent risk predictors for the long-term mortality of patients with STEMI and MVD; an in-hospital Killip class ≥ 3 and renal insufficiency were independent risk predictors for the long-term mortality of patients with STEMI and a CTO.
Assuntos
Oclusão Coronária/fisiopatologia , Intervenção Coronária Percutânea , Infarto do Miocárdio com Supradesnível do Segmento ST/terapia , Fatores Etários , Idoso , Doença Crônica , Oclusão Coronária/diagnóstico por imagem , Oclusão Coronária/mortalidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Úlcera Péptica Hemorrágica/mortalidade , Intervenção Coronária Percutânea/efeitos adversos , Intervenção Coronária Percutânea/mortalidade , Insuficiência Renal/mortalidade , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Infarto do Miocárdio com Supradesnível do Segmento ST/diagnóstico por imagem , Infarto do Miocárdio com Supradesnível do Segmento ST/mortalidade , Infarto do Miocárdio com Supradesnível do Segmento ST/fisiopatologia , Fatores de Tempo , Resultado do TratamentoRESUMO
Myocardial infarction is one of the common types of coronary heart disease in the clinic. Its morbidity, lethality and disability are high, and it has become a serious threat to human health. At present, it is shown that in the early stage of acute myocardial infarction, myocardial cells are mainly apoptotic, suggesting that effectively blocking myocardial apoptosis in the early stage of myocardial infarction is of great significance for reducing tissue necrosis in the infarcted area. Recent studies have shown that NG nano-protein complexes have a better therapeutic effect on acute myocardial infarction and can inhibit left ventricular remodeling in patients with acute myocardial infarction. However, there are few studies on the effect of NG nano-protein complexes on myocardial cell apoptosis after ischemia. This study used a rat model of acute myocardial infarction to analyze its effect on apoptotic proteins of myocardial cells in rats with acute myocardial infarction in order to provide a certain theoretical basis for its clinical application. In this study, 45 SD rats were randomly divided into a sham operation group, a myocardial infarction group, and a NG nano-protein complex group, with 15 in each group. The sham operation group only underwent thoracotomy, and received normal saline gavage postoperatively; the myocardial infarction group and the NG nano-protein complex group were ligated to the left anterior descending coronary artery of the rat to establish an acute myocardial infarction model, and were performed separately treatment with saline and NG nanoprotein complexes. Finally, we conclude that this nano-protein complex can significantly reduce the expression level of myocardial apoptosis-related proteins in rats with acute myocardial infarction, and is of great significance in inhibiting the apoptosis of acute myocardial infarction cells.
Assuntos
Infarto do Miocárdio , Animais , Apoptose , Humanos , Infarto do Miocárdio/tratamento farmacológico , Miocárdio , Miócitos Cardíacos , Ratos , Ratos Sprague-DawleyRESUMO
PURPOSE: Effective therapy for visual loss caused by optic nerve injury or diseases has not been achieved even though the optic nerve has the regeneration potential after injury. This study was designed to modify amniotic epithelial cells (AECs) with basic fibroblast growth factor (bFGF) gene, preliminarily investigating its effect on transected optic nerve. METHODS: A human bFGF gene segment was delivered into rat AECs (AECs/hbFGF) by lentiviral vector, and the gene expression was examined by RT-PCR and ELISA. The AECs/hbFGF and untransfected rat AECs were transplanted into the transected site of the rat optic nerve. At 28 days post transplantation, the survival and migration of the transplanted cells was observed by tracking labeled cells; meanwhile retinal ganglion cells (RGCs) were observed and counted by employing biotin dextran amine (BDA) and Nissl staining. Furthermore, the expression of growth associated protein 43 (GAP-43) within the injury site was examined with immunohistochemical staining. RESULTS: The AECs/hbFGF was proven to express bFGF gene and secrete bFGF peptide. Both AECs/hbFGF and AECs could survive and migrate after transplantation. RGCs counting implicated that RGCs numbers of the cell transplantation groups were significantly higher than that of the control group, and the AECs/hbFGF group was significantly higher than that of the AECs group. Moreover GAP-43 integral optical density value in the control group was significantly lower than that of the cell transplantation groups, and the value in the AECs/hbFGF group was significantly higher than that of the AECs group. CONCLUSIONS: AECs modified with bFGF could reduce RGCs loss and promote expression of GAP-43 in the rat optic nerve transected model, facilitating the process of neural restoration following injury.
Assuntos
Células Epiteliais/transplante , Fator 2 de Crescimento de Fibroblastos/genética , Regeneração Nervosa/fisiologia , Traumatismos do Nervo Óptico/terapia , Nervo Óptico/metabolismo , Âmnio/citologia , Animais , Biomarcadores/metabolismo , Contagem de Células , Diferenciação Celular , Sobrevivência Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Proteína GAP-43/genética , Proteína GAP-43/metabolismo , Expressão Gênica , Vetores Genéticos , Humanos , Lentivirus/genética , Masculino , Nervo Óptico/patologia , Nervo Óptico/cirurgia , Traumatismos do Nervo Óptico/metabolismo , Traumatismos do Nervo Óptico/patologia , Ratos , Ratos Sprague-Dawley , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , Transdução Genética , TransgenesRESUMO
Despite advances in antibiotic therapy and intensive care, the mortality caused by systemic inflammatory response syndrome and severe sepsis remains high. The use of anti-inflammatory agents to attenuate inflammatory response during acute systemic inflammatory reactions may improve survival rates. Here we show that a newly synthesized 2-pyridone compound (FJU-C4) can suppress the expression of late inflammatory mediators such as iNOS and COX-2 in murine macrophages. The pro-inflammatory cytokines, including TNFα, IL-1ß, and IL-6, were dose-dependently suppressed by FJU-C4 both in mRNA and protein levels. In addition, the expression of TNFα was inhibited from as early as 2 hours after exposure to LPS stimulation. The production of mature pro-inflammatory cytokines was also suppressed by pretreatment with FJU-C4 in either cell culture medium or mice serum when stimulated by LPS. FJU-C4 prolongs mouse survival and prevents mouse death from LPS-induced systemic inflammation when the dose of FJU-C4 is over 5 mg/kg. The activities of ERK, JNK, and p38MAPK were induced by LPS stimulation on murine macrophage cell line, but only p38MAPK signaling was dramatically suppressed by pretreatment with the FJU-C4 compound in a dose-dependent manner. NF-κB activation also was suppressed by FJU-C4 compound. These findings suggest that the FJU-C4 compound may act as a promising therapeutic agent against inflammatory diseases by inhibiting the p38MAPK and NF-κB signaling pathway.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , NF-kappa B/genética , Piridonas/farmacologia , Síndrome de Resposta Inflamatória Sistêmica/tratamento farmacológico , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Animais , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Feminino , Regulação da Expressão Gênica , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/antagonistas & inibidores , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolissacarídeos , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Transdução de Sinais , Síndrome de Resposta Inflamatória Sistêmica/induzido quimicamente , Síndrome de Resposta Inflamatória Sistêmica/genética , Síndrome de Resposta Inflamatória Sistêmica/patologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: Evidence indicates that early reperfusion therapy in patients with ST-elevation myocardial infarction (STEMI) reduces complications. This study was undertaken to compare the in-hospital delay to primary percutaneous coronary intervention (PPCI) for patients with STEMI between specialized hospitals and non-specialized hospitals in Beijing, China. METHODS: Two specialized hospitals and fifteen non-specialized hospitals capable of performing PPCI were selected to participate in this study. A total of 308 patients, within 12 hours of the onset of symptoms and undergoing PPCI between November 1, 2005 and December 31, 2006 were enrolled. Data were collected by structured interview and review of medical records. RESULTS: The median in-hospital delay was 98 (interquartile range 105 to 180) minutes, and 16.9% of the patients were treated within 90 minutes. Total in-hospital delay and ECG-to-treatment decision-making time were longer in the non-specialized hospitals than in the cardiac specialized hospitals (147 minutes vs. 120 minutes, P < 0.001; 55 minutes vs. 45 minutes, P = 0.035). After controlling the confounding factors, the non-specialized hospitals were independently associated with an increased risk of being in the upper median of in-hospital delays. CONCLUSIONS: There were substantial in-hospital delays between arrival at the hospital and the administration of PPCI for patients with STEMI in Beijing. Patients admitted to the cardiac specialized hospitals had a shorter in-hospital delay than those to the non-specialized hospitals because of a shorter time of ECG-to-treatment decision-making.
Assuntos
Angioplastia Coronária com Balão , Hospitais/estatística & dados numéricos , Infarto do Miocárdio/terapia , China , Tomada de Decisões , Eletrocardiografia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Qualidade da Assistência à Saúde , Fatores de Tempo , Resultado do TratamentoRESUMO
Sciatic nerves in adult male rats were transected and reunited via a silicone chamber. This was followed by a focal injection of recombinant ciliary neurotrophic factor (CNTF). To evaluate the effect of this therapeutic approach and to explore its possible mechanisms, nerve regeneration was traced by horseradish peroxidase retrograde labeling. Functional recovery was evaluated by functional assessment of the hind feet and the expression of a number of proteins was detected using immunohistochemistry. The results showed that a single administration of CNTF could promote regeneration of motor axons, with improved functional recovery in adult rats. Growth associated protein (GAP)-43, S100, CD68 and major histocompatibility complex class II immunoreactivity in the regenerative and distal nerves suggested that CNTF could promote axon regeneration, Schwann cell migration, monocyte infiltration and activation. CNTF might also indirectly promote axonal regeneration by further activating the JAK-STAT3 pathway and subsequently upregulating phosphotyrosine, GAP-43 and S100 expression to enhance proliferation, growth and migration of Schwann cells. CNTF has suggested important targets for pharmacological intervention in peripheral nerve disease and injury.
Assuntos
Fator Neurotrófico Ciliar/farmacologia , Expressão Gênica/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Implantação de Prótese/métodos , Neuropatia Ciática/tratamento farmacológico , Neuropatia Ciática/cirurgia , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Modelos Animais de Doenças , Proteína GAP-43/metabolismo , Expressão Gênica/fisiologia , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/metabolismo , Cones de Crescimento/patologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Imuno-Histoquímica , Janus Quinase 1/metabolismo , Masculino , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Regeneração Nervosa/fisiologia , Próteses e Implantes , Ratos , Ratos Sprague-Dawley , Proteínas S100/metabolismo , Fator de Transcrição STAT3/metabolismo , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Neuropatia Ciática/metabolismo , Silicones/uso terapêuticoRESUMO
OBJECTIVE: To evaluate the effects of socioeconomic status on the distribution of cardiovascular risk factors and clinical treatments of patients with acute myocardial infarction in Beijing. METHODS: In Beijing, a prospective, multi-center, registration study was carried out which including 800 patients who were consecutively hospitalized for ST-segment elevation acute myocardial infarction within 24 hours after event attack in 19 different hospitals in Beijing between November, 2005 and December, 2006. Indicators of socioeconomic status included self-reported personal income (< 500, 500-2000, > 2000 RMB/ month), educational attainment (< or = 12 and > 12 years) and status of medical insurance (yes/no). According to categories of education, patients were categorized into two groups of lower socioeconomic status and higher socioeconomic status. Differences of cardiovascular risk factors and clinical treatments were compared across the two groups respectively. RESULTS: Proportion of diabetes and hyperlipidemia in patients with higher socioeconomic status was much higher than that of patients with lower socioeconomic status (P < 0.05, P < 0.01 respectively). Patients with lower socioeconomic status were more likely to be smokers (P < 0.05). The rates of receiving coronary angiography and PTCA were much lower in patients with lower socioeconomic status. Medical insurance and income were the most important two socioeconomic factors determining the use of PTCA. CONCLUSION: Compared to patients with lower socioeconomic status, patients with higher socioeconomic status had higher rates of hyperlipidemia and diabetes but lower smoking rate among cardiovascular risk factors. The rates of receiving interventional therapies were much lower in patients with lower socioeconomic status.
Assuntos
Infarto do Miocárdio/economia , Infarto do Miocárdio/terapia , Classe Social , Idade de Início , Idoso , Angioplastia Coronária com Balão/economia , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/terapia , China/epidemiologia , Escolaridade , Feminino , Humanos , Renda , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores de RiscoRESUMO
OBJECTIVE: To explore the effect of low-dose folate on plasma homocysteinemia (Hcy) and chemokine levels in patients with hyperhomocysteinemia (HHcy). METHODS: Forty HHcy patients were treated with 0.8 mg/d folate for 6 months. Plasma levels of Hcy, monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), malondialdehyde (MDA), and superoxide dismutase (SOD) were measured before and after folate treatment. RESULTS: Plasma level of Hcy significantly decreased after folate treatment [(57.1 +/- 18.0) micromol/L vs (25.8 +/- 12.0) micromol/L, P <0.05]. However, the plasma levels of MCP-1, IL-8, SOD, and MDA were not changed after folate treatment. CONCLUSION: Folate treatment can decrease the plasma Hcy level in HHcy patients; however, it has no obvious effects on the chemokine levels.
Assuntos
Quimiocinas/sangue , Ácido Fólico/uso terapêutico , Homocisteína/sangue , Hiper-Homocisteinemia/sangue , Hiper-Homocisteinemia/tratamento farmacológico , Idoso , Feminino , Ácido Fólico/administração & dosagem , Humanos , Masculino , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
AIM: G protein-coupled inwardly rectifying potassium channels (GIRK) are important for neuronal signaling and membrane excitability. In the present study, we intend to find whether GIRK channels express functionally in adult rat dorsal root ganglion (DRG) neurons. METHODS: We used RT-PCR to detect mRNA for 4 subunits of GIRK in the adult DRG. The whole-cell patch clamp recording was used to confirm GIRK channels functionally expressed. RESULTS: The mRNA for the 4 subunits of GIRK were detected in the adult DRG. GTPgammaS enhanced inwardly rectifying potassium (K+) currents of the DRG neurons, while Ba2+ inhibited such currents. Furthermore, the GIRK channels were shown to be coupled to the GABA(B) receptor, a member of the G protein-coupled receptor family, as baclofen increased the inwardly rectifying K+ currents. CONCLUSION: GIRK channels are expressed and functionally coupled with GABA(B) receptors in adult rat DRG neurons.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/fisiologia , Gânglios Espinais/metabolismo , Animais , Compostos de Bário/farmacologia , Cloretos/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/química , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/efeitos dos fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de GABA-B/análiseRESUMO
OBJECTIVE: To evaluate the effectiveness and safety of subcutaneous low molecular weight heparin (LMWH) used in acute management of patients with non-ST segment elevation acute coronary syndrome (ACS). METHODS: A total of 102 patients with non-ST segment elevation ACS were treated for at least 48 hours ( > or =5 times) with subcutaneous nadroparin (1 mg/kg each 12 hours). All 102 patients underwent coronary angiographies (CAG) within 8 hours after LMWH injection, followed by immediate percutaneous coronary intervention (PCI). RESULTS: Anti-Xa activity at the time of catheterization was (0.62 +/- 0.18) IU/ml, and 90% of the patients had anti-Xa activity > 0.5 IU/ml. No death, myocardial infarction relapse or emergent revascularization occurred after PCI. Thrombosis and/or embolism occurred in 2 patients (3.5%) during PCI. Mild hemorrhage was observed in 4 patients (3.9%) of PCI group and in 2 patients (4.4%) in CAG group. No major hemorrhage occurred. CONCLUSION: PCI within 8-12 hours of the last dose after > or =48 hours nadroparin subcutaneous injection seems to be effective and safe.
Assuntos
Síndrome Coronariana Aguda/terapia , Anticoagulantes/uso terapêutico , Nadroparina/uso terapêutico , Síndrome Coronariana Aguda/sangue , Angioplastia com Balão , Anticoagulantes/efeitos adversos , Inibidores do Fator Xa , Humanos , Nadroparina/efeitos adversosRESUMO
RET receptor signalling is essential for glial-cell-line-derived neurotrophic factor (GDNF)-induced survival and differentiation of various neurons such as mesencephalic neurons. To identify proteins that mediate RET-dependent signaling, yeast two-hybrid screening was performed with the intracellular domain of RET as bait. We identified a new interaction between RET and the adapter protein SH2-Bbeta. Upon GDNF stimulation of PC12-GFRalpha1-RET cells (that stably overexpress GDNF receptor alpha1 and RET), wild-type SH2-Bbeta co-immunoprecipitated with RET, whereas the dominant-negative SH2-Bbeta mutant R555E did not. RET interacted with endogenous SH2-Bbeta both in PC12-GFRalpha1-RET cells and in rat tissues. Mutagenesis analysis revealed that Tyr981 within the intracellular domain of RET was crucial for the interaction with SH2-Bbeta. Morphological evidence showed that SH2-Bbeta and RET colocalized in mesencephalic neurons. Furthermore, functional analysis indicated that overexpression of SH2-Bbeta facilitated GDNF-induced neurite outgrowth in both PC12-GFRalpha1-RET cells and cultured mesencephalic neurons, whereas the mutant R555E inhibited the effect. Moreover, inhibition of SH2-Bbeta expression by RNA interference caused a significant decrease of GDNF-induced neuronal differentiation in PC12-GFRalpha1-RET cells. Taken together, our results suggest that SH2-Bbeta is a new signaling molecule involved in GDNF-induced neurite outgrowth.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/fisiologia , Neuritos/fisiologia , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Diferenciação Celular , Crescimento Celular , Linhagem Celular , Células Cultivadas , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Dados de Sequência Molecular , Células PC12 , Ratos , Transdução de Sinais , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Olfactory ensheathing cell (OEC) transplantation has emerged as a very promising therapy for spinal cord repair. In this study, we tested the ability of genetically modified OECs to secrete high levels of glial cell line-derived neurotrophic factor (GDNF) to promote spinal cord repair. The GDNF gene was transduced into OECs using a retroviral-based system. The engineered OECs were first characterized by their ability to express and secrete biologically active GDNF in vitro. After implantation into the spinal cord of adult rats with complete spinal cord transection, OEC survival and GDNF production were examined. The locomotor functions of animals were assessed and axon regeneration was evaluated at the morphological level. To our knowledge, we report for the first time that the genetically modified OECs are capable of producing GDNF in vivo to significantly improve recovery after spinal cord injury (SCI). This work combined the outgrowth-promoting property of OECs with the neuroprotective effects of the additionally overexpressed neurotrophic factors and opens new avenues for the treatment of SCI.
Assuntos
Transplante de Células/métodos , Terapia Genética/métodos , Bainha de Mielina/transplante , Fatores de Crescimento Neural/genética , Bulbo Olfatório/citologia , Traumatismos da Medula Espinal/terapia , Animais , Axônios/fisiologia , Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Feminino , Expressão Gênica , Vetores Genéticos/administração & dosagem , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Imuno-Histoquímica/métodos , Bainha de Mielina/metabolismo , Fatores de Crescimento Neural/metabolismo , Regeneração Nervosa , Ratos , Ratos Sprague-Dawley , Retroviridae/genética , Transdução Genética/métodosRESUMO
Glial cell line-derived neurotrophic factor (GDNF) plays a critical role in neurodevelopment and survival of midbrain dopaminergic and spinal motor neurons in vitro and in vivo. The biological actions of GDNF are mediated by a two-receptor complex consisting of a glycosylphosphatidylinositol-linked cell surface molecule, the GDNF family receptor alpha1 (GFRalpha1), and receptor protein tyrosine kinase Ret. Although structural analysis of GDNF has been extensively examined, less is known about the structural basis of GFRalpha1 function. In this study, based on evolutionary trace method and relative solvent accessibility prediction of residues, a set of trace residues that are solvent-accessible was selected for site-directed mutagenesis. A series of GFRalpha1 mutations was made, and PC12 cell lines stably expressing different GFRalpha1 mutants were generated. According to the survival and differentiation responses of these stable PC12 cells upon GDNF stimulation and the GDNF-GFRalpha1-Ret interaction assay, residues 152NN153, Arg259, and 316SNS318 in the GFRalpha1 central region were found to be critical for GFRalpha1 binding to GDNF and eliciting downstream signal transduction. The single mutation R259A in the GFRalpha1 molecule simultaneously lost its binding ability to GDNF and Ret. However N152A/N153A or S316A/N317A/S318A mutation in the GFRalpha1 molecule still retained the ability to bind with Ret. These findings suggest that distinct structural elements in GFRalpha1 may be involved in binding to GDNF and Ret.
Assuntos
Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/metabolismo , Substituição de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Dicroísmo Circular , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/metabolismo , Células PC12 , Fosforilação , Desnaturação Proteica , Estrutura Secundária de Proteína , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-ret , Ratos , Receptores Proteína Tirosina Quinases/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeamento por RestriçãoRESUMO
To observe the influence of tumor necrosis factor-alpha (TNF-alpha) on differentiation of rat mesencephalic neural stem cells (NSCs), the numbers of neurons, astrocytes and oligodendrocytes generated from NSCs were analyzed after differentiation for 3 days by using immunocytochemistry technique. The results show that: (1) TNF-alpha enhanced the proportions of neurons and oligodendrocytes in progeny of NSCs; and (2) TNF-alpha induced the proliferation of oligodendrocytes derived from NSCs, but the proliferation of astrocytes was not influenced by TNF-alpha. We conclude that the TNF-alpha could influence the application of NSCs.
Assuntos
Diferenciação Celular/fisiologia , Mesencéfalo/citologia , Células-Tronco Neurais/citologia , Oligodendroglia/citologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Proliferação de Células , Neurônios/citologia , Ratos , Ratos Sprague-DawleyRESUMO
The GFRalpha1 cDNA was amplified by RT-PCR from fetal rat hippocampus. The soluble recombinant GFRalpha1 and its mutants were obtained from an Escherichia coli expression system. The biological activity of soluble GFRalpha1 and its mutants were evaluated in PC12 cells. The results suggest that the central domain of GFRalpha1 is a crucial determinant for ligand binding. This established a solid basis for further study to find the key amino acid mediating the binding of GDNF and GFRalpha1.
Assuntos
Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Primers do DNA/genética , Escherichia coli/metabolismo , Deleção de Genes , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Hipocampo/química , Humanos , Modelos Moleculares , Células PC12 , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína/genética , Proteínas Proto-Oncogênicas/farmacologia , Proteínas Proto-Oncogênicas c-ret , Ratos , Receptores Proteína Tirosina Quinases/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Relação Estrutura-AtividadeRESUMO
There are numerous parallels between the heamatolymphopoietic and nervous systems in terms of the mechanisms regulating their development. We proposed that neural stem cells (NSCs) may respond to the microenvironmental signals provided by bone marrow stromal cells (BMSCs) which regulate the differentiation and maturation of hematolymphopoietic stem cells. First, we isolated and proliferated BMSCs from the femur and tibia, and NSCs from the midbrain of Sprague-Dawley (SD) rats, and then investigated the effects of BMSCs on the differentiation of NSCs into neurons, astrocytes and oligodendrocytes by directly plating neurospheres on BMSC monolayers in serum-free conditions. The results confirmed that BMSCs induced NSCs to differentiate selectively into neurons. The percentage of neurons significantly increased in 7 days in vitro co-cultures of NSCs and BMSCs as compared to NSCs cultures alone. When the duration of the cultures was extended to 12 days in vitro, BMSCs enhanced the survival of neurons derived from these NSCs; our investigation then focused on the underlying mechanism for this effect of BMSCs. NSCs were cultured with BMSC conditioned-medium and co-cultured with membrane fragments of live BMSCs or paraformaldehyde fixed BMSCs, the inducing activity of BMSCs was solely detectable in BMSC conditioned-medium, indicating that soluble factors secreted by BMSCs were responsible for its effect on the neuronal differentiation of NSCs. Therefore, BMSCs may provide a powerful tool for therapeutic neurological applications.
Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular/fisiologia , Mesencéfalo/citologia , Proteínas do Tecido Nervoso , Neurônios/citologia , Células-Tronco/citologia , Células Estromais/fisiologia , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/metabolismo , Animais , Animais Recém-Nascidos , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Imunoquímica , Proteínas de Filamentos Intermediários/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Nestina , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Ratos , Ratos Sprague-Dawley/crescimento & desenvolvimento , Ratos Sprague-Dawley/metabolismo , Receptores da Transferrina , Células-Tronco/fisiologia , Células Estromais/metabolismo , Fatores de TempoRESUMO
AIM: To investigate the effect of ciliary neurotrophic factor (CNTF) on the nuclear translocation of protein kinase C (PKC) following NMDA administration in the primary cultured hippocampal neurons. METHODS: (1) PKCGAMMA or PKCepsilon- immunocytochemistry staining method was used after treating neurons with NMDA or CNTF. (2) The gray of the nucleus of the PKC-positive neurons were measured under the image pattern analysis system. RESULTS: (1) After NMDA administration of different concentration and time, Nucleus appear PKCgamma and PKCepsilon activities, especially the 100 micromol/L NMDA 30 min group. (2) The gray of nucleus in CNTF + 500 micromol/L NMDA group is similar to control group. CONCLUSION: NMDA can induce nuclear translocation of PKC in the primary cultured hippocampal neurons, and CNTF can inhibit the translocation. It suggests that the inhibition of PKC translocation induced by NMDA is one of the important reasons for the neuro-protective effects of CNTF.
Assuntos
Fator Neurotrófico Ciliar/farmacologia , Hipocampo/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteína Quinase C/metabolismo , Animais , Células Cultivadas , N-Metilaspartato/farmacologia , Transporte Proteico , Ratos , Ratos Sprague-DawleyRESUMO
Objective. To examine the effect of electromagnetic fields (EMF) (20 Hz, 8 mT; 5 Hz, 8 mT) on the neuron-orientated differentiation of neural stem cells (NSCs) from midbrains of new-bom rats. Method. Differentiated NSCs were exposed to EMF for 2 x 15 min per day lasting for I d, 5 d, or 10 d. The sham-exposure controls were correspondingly established. Cells were fixed and processed for immunofluorescent staining using the antibody against neuron-specific marker MAP2, then the percentage of MAP2+ cells was calculated. Result. The two EMFs promoted the differentiation of a neuronal fate in different ways. Both of them came into effect even after 1 d exposure. When cells exposed to the 20 Hz EMF, the percentage of neuron-orientated cells gradually increased with longer-term exposure and the most significant effect appeared in 10 d group while that happened in 5 d group under the condition of 5 Hz EMF. The effect contrasted horizontally, significant differences between the two EMFs were observed only at 10 d groups, 20 Hz EMF having more favorable effect than 5 Hz EMF. Conclusion. 20 Hz and 5 Hz EMF could promote the differentiation of midbrain NSCs to a neuronal phenotype in different ways, suggesting that the physical induction might be another strategy to manipulate the differentiation of NSCs.
Assuntos
Diferenciação Celular/efeitos da radiação , Campos Eletromagnéticos , Mesencéfalo/efeitos da radiação , Neurônios/efeitos da radiação , Células-Tronco/efeitos da radiação , Animais , Animais Recém-Nascidos , Diferenciação Celular/fisiologia , Células Cultivadas/efeitos da radiação , Mesencéfalo/citologia , Neurônios/fisiologia , Ratos , Células-Tronco/fisiologia , Fatores de TempoRESUMO
It is widely accepted that monoamine transmitters are playing an important role in the progress of depression. The functions of monoamine transmitters and their receptors change profoundly during depression and its treatment. This paper is attempted to review the new progress of investigation into serotonin receptors, adrenoceptors and dopamine receptors related with depression mechanism respectively.