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1.
Acta Histochem ; 121(2): 253-259, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30611528

RESUMO

Understanding the mechanisms of adipogenic differentiation may lead to the discovery of novel therapeutic targets for obesity. The natural plant polyphenol compound curcumin can improve obesity-associated inflammation and diabetes in obese mice. The role of curcumin in adipogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs) is still unclear. We used hMSCs to investigate the details of the mechanism underlying the adipogenic effects of curcumin. At different time points (i.e., 5 days and 10 days) of hMSC adipocyte differentiation, an accumulation of large lipid droplets was analyzed in Oil Red O-stained cultured cells in two curcumin (5 µM and 10 µM) groups and the control group. The cells were also harvested for the detection of mRNA and protein expressions by quantitative real-time polymerase chain reaction and Western blot analysis. The results showed that curcumin can suppresses adipocyte differentiation in a dose-dependent manner and inhibited the expression of PPARγ, C/EBPα, and FABP4. Importantly, curcumin can also suppress the expression of Kruppel-like factor 15, which may bind to the PPARγ promoter, resulting in downregulation of PPARγ expression to inhibit the adipogenic differentiation of hMSCs.


Assuntos
Adipócitos/citologia , Adipogenia/fisiologia , Células da Medula Óssea/citologia , Diferenciação Celular/fisiologia , Células-Tronco Mesenquimais/citologia , Regulação para Baixo/fisiologia , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição/metabolismo
2.
Eur J Med Chem ; 157: 1395-1405, 2018 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-30196062

RESUMO

Chalcone, a natural structure, demonstrates many pharmacological activities including anticancer, and one promising mechanism is to modulate the generation of ROS. It has been known that pyroptosis is associated with anticancer effects, whereas there is fewer researches about ROS-mediated pyroptosis triggered by chemotherapy drugs. Moreover, incorporation of a α,ß-unsaturated ketone unit into chalcone may be an effective strategy for development of chemotherapy drugs. Hence, a number of chalcone analogues bearing a α,ß-unsaturated ketone were synthesized from chalcone analogues 1 with modest anticancer activities as the lead compound. Structure-activity relationship (SAR) studies confirmed the function of α,ß-unsaturated ketone to improve anticancer activity. Notably, compound 8, bearing a α,ß-unsaturated ketone, is the most potent inhibitor of cancer, with IC50 values on NCI-H460, A549 and H1975 cells of 2.3 ±â€¯0.3, 3.2 ±â€¯0.0 and 5.7 ±â€¯1.4 µM, respectively. Besides, 8 showed antiproliferative ability against NCI-H460 cells in a time- and concentration-dependent manner through modulating ROS to induce caspase-3-mediated pyroptosis, and displayed a better safety profile in vivo. Overall, these results demonstrated that compound 8 is a candidate agent and a potential lead compound for development of chemotherapy drugs, and can be used as a probe to further examine the mechanism of ROS-dependent pyroptosis.


Assuntos
Antineoplásicos/farmacologia , Chalcona/farmacologia , Desenho de Drogas , Cetonas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Piroptose/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Chalcona/síntese química , Chalcona/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Cetonas/química , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Estrutura Molecular , Relação Estrutura-Atividade , Células Tumorais Cultivadas
3.
Eur J Med Chem ; 144: 493-503, 2018 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-29288946

RESUMO

Molecular hybridization is considered as an effective tactic to develop drugs for the treatment of cancer. A series of novel hybrid compounds of isatin and Michael acceptor were designed and synthesized on the basis of association principle. These hybrid compounds were tested for cytotoxic potential against human cancer cell lines namely, BGC-823, SGC-7901 and NCI-H460 by MTT assay. Most compounds showed good anti-growth activities in all tested human cancer cells. SAR and QSAR analysis may provide vital information for the future development of novel anti-cancer inhibitors. Notably, compound 6a showed potent growth inhibition on BGC-823, SGC-7901 and NCI-H460 with the IC50 values of 3.6 ±â€¯0.6, 5.7 ±â€¯1.2, 3.2 ±â€¯0.7 µM, respectively. Besides, colony formation assays, wound healing assays and flow cytometry analysis indicated 6a exhibited a potent anti-growth and anti-migration ability in a concentration-dependence manner through arrested cells in the G2/M phase of cell cycle. Moreover, 6a significantly repressed tumor growth in a NCI-H460 xenograft mouse model. Overall, our findings suggested isatin analogues inspired Michael acceptor may provide promising lead compounds for the development of cancer chemotherapeutics.


Assuntos
Antineoplásicos/farmacologia , Desenho de Drogas , Isatina/farmacologia , Relação Quantitativa Estrutura-Atividade , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Isatina/síntese química , Isatina/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Células Tumorais Cultivadas
4.
Mol Med Rep ; 15(4): 1571-1576, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28260060

RESUMO

The present study aimed to screen several differentially expressed genes (DEGs) and differentially expressed microRNAs (miRNAs) for two types of mesenchymal stem cell (MSC) differentiation. Bone morphogenetic protein 6 (BMP­6) and dexamethasone were used to induce MSCs towards osteoblastic differentiation or adipocytic differentiation. The t­test in the Bioconductor bioinformatics software tool was used to screen DEGs and differentially expressed miRNAs in the two samples. Subsequent gene ontology (GO) and pathway analyses on the DEGs were performed using the GO and Kyoto Encyclopedia of Genes and Genomes databases, respectively; potential target genes for the screened miRNAs were predicted using the TargetScan database. In addition, an interaction network between the DEGs and miRNAs was constructed. Numerous DEGs and miRNAs were screened during osteoblastic and adipocytic differentiation of MSCs. Important pathways, such as glutathione metabolism, pathogenic Escherichia coli infection and Parkinson's disease, and GO terms, including cytoskeletal protein binding and phospholipase inhibitor activity, were enriched in the screened DEGs from MSCs undergoing osteogenic differentiation and adipocytic differentiation. miRNAs, including miRNA (miR)­382 and miR­203, and DEGs, including neuronal growth regulator 1 (NEGR1), phosphatidic acid phosphatase 2B (PPAP2B), platelet­derived growth factor receptor alpha (PDGFRA), interleukin 6 signal transducer (IL6ST) and sortilin 1 (SORT1), were demonstrated to be involved in osteoblastic differentiation. In addition, the downregulated miRNAs (including miR­495, miR­376a and miR­543), the upregulated miR­106a, the upregulated DEGs, including enabled homolog (ENAH), polypeptide N­acetylgalactosaminyltransferase 1 and acyl­CoA synthetase long­chain family member 1, and the downregulated repulsive guidance molecule family member B and semaphorin SEMA7A were demonstrated to be involved in adipocytic differentiation. The results of the present study suggested that miRNAs (miR­203 and miR­382) and DEGs (NEGR1, PPAP2B, PDGFRA, IL6ST and SORT1) may serve pivotal functions in the osteoblastic differentiation of MSCs, whereas miR­495, which is also involved in osteoblast differentiation and had four targets, including NEGR1, miR­376a, miR­543 and ENAH may have crucial roles in adipocytic differentiation of MSCs.


Assuntos
Adipócitos/citologia , Diferenciação Celular , Biologia Computacional/métodos , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Adipócitos/metabolismo , Diferenciação Celular/genética , Perfilação da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , Humanos , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Osteoblastos/metabolismo , Mapas de Interação de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/genética
5.
Exp Hematol ; 44(11): 1013-1019, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27473568

RESUMO

Adoptive transfer of T cells can be an effective anticancer treatment. However, uncontrolled or unpredictable immediate or persistent toxic effects are a source of concern. The ability to conditionally eliminate aberrant cells in vivo is therefore becoming a critical step for the successful translation of this approach to the clinic. We review the evolution of safety systems, focusing on a suicide switch that can be expressed stably and efficiently in human T cells without impairing phenotype, function, or antigen specificity. This system is based on the fusion of human caspase-9 to a modified human FK-binding protein, allowing conditional dimerization in the presence of an otherwise bio-inert small molecule drug. When exposed to the synthetic dimerizing drug, the inducible caspase-9 becomes activated, resulting in the rapid apoptosis of cells expressing this construct. We have illustrated the clinical feasibility and efficacy of this approach after haploidentical hematopoietic stem cell transplant. Here we review the benefits and limitations of the approach.


Assuntos
Caspase 9/genética , Engenharia Celular , Terapia Baseada em Transplante de Células e Tecidos , Regulação da Expressão Gênica , Engenharia Genética , Terapia Genética , Linfócitos T/metabolismo , Animais , Anticorpos Monoclonais , Caspase 9/química , Caspase 9/metabolismo , Transplante de Células/efeitos adversos , Transplante de Células/métodos , Terapia Baseada em Transplante de Células e Tecidos/efeitos adversos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Genes Transgênicos Suicidas , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Doença Enxerto-Hospedeiro/etiologia , Humanos , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Depleção Linfocítica , Pró-Fármacos/farmacologia , Multimerização Proteica , Linfócitos T/imunologia , Linfócitos T/transplante , Doadores de Tecidos
6.
Mol Ther ; 24(4): 736-45, 2016 04.
Artigo em Inglês | MEDLINE | ID: mdl-26639404

RESUMO

Safety switches are becoming relevant for the clinical translation of T-cell-based immunotherapies. In patients receiving an allogeneic hematopoietic stem cell transplant, the inducible caspase-9 gene (iC9) safety switch expressed by donor-derived T lymphocytes efficiently controls acute graft versus host disease (GvHD). However, in vivo elimination of iC9-T cells by the chemical inducer of dimerization (CID) that activates the iC9 protein is incomplete. To study this effect, we characterized the clonal diversity and dynamics of vector insertion sites (VIS) in iC9-T cells pre- and post-CID administration in four patients who developed GvHD. We identified 3,203 VIS among four patients and followed their in vivo clonal dynamics up to 161 days post-CID. VIS were categorized by their proximity to host genome elements, gene associations, and cis-modulatory relationship to mapped promoters. We found that VIS are preferentially located near open chromatin and promoter regions; furthermore, there was no evidence for selection bias among VIS surviving the CID treatment. The majority of iC9-T cells with high normalized VIS copy number at the time of GvHD onset were eliminated by CID, while iC9-T cells detectable post-CID generally have low normalized VIS copy number. We propose that suboptimal iC9 transgene expression is responsible for the incomplete elimination of iC9-T cells and illustrate here by simple model how cis-modulatory influences of local genome context and T-cell receptor activation status at time of CID treatment contribute to stochastic sparing of iC9-T cells.


Assuntos
Caspase 9/metabolismo , Doença Enxerto-Hospedeiro/tratamento farmacológico , Linfócitos T/metabolismo , Integração Viral , Caspase 9/genética , Cromatina/genética , Genoma Humano , Doença Enxerto-Hospedeiro/genética , Transplante de Células-Tronco Hematopoéticas , Humanos , Imunoterapia , Compostos Orgânicos/administração & dosagem , Regiões Promotoras Genéticas , Linfócitos T/transplante , Transgenes , Transplante Homólogo
7.
Mol Ther ; 24(4): 823-31, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26708005

RESUMO

Activation of the inducible caspase 9 (iC9) safety gene by a dimerizing drug (chemical inducer of dimerization (CID) AP1903) effectively resolves the symptoms and signs of graft-versus-host disease (GvHD) in haploidentical stem cell transplant (HSCT) recipients. However, after CID treatment, 1% of iC9-T cells remain and can regrow over time; although these resurgent T cells do not cause recurrent GvHD, it remains unclear whether repeat CID treatments are a safe and feasible way to further deplete residual gene-modified T cells should any other adverse effects associated with them occur. Here, we report a patient who received an infusion of haploidentical iC9-T cells after HSCT and subsequently received three treatments with AP1903. There was a mild (grade 2) and transient pancytopenia following each AP1903 administration but no non-hematological toxicity. Ninety five percent of circulating iC9-T cells (CD3(+)CD19(+)) were eliminated after the first AP1903 treatment. Three months later, the residual cells had expanded more than eightfold and had a lower level of iC9 expression. Each repeated AP1903 administration eliminated a diminishing percentage of the residual repopulating cells, but elimination could be enhanced by T-cell activation. These data support the safety and efficiency of repeated CID treatments for persistent or recurring toxicity from T-cell therapies.


Assuntos
Caspase 9/genética , Doença Enxerto-Hospedeiro/prevenção & controle , Linfócitos T/efeitos dos fármacos , Linfócitos T/transplante , Criança , Relação Dose-Resposta a Droga , Esquema de Medicação , Genes Transgênicos Suicidas , Doença Enxerto-Hospedeiro/imunologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Ativação Linfocitária , Masculino , Compostos Orgânicos/administração & dosagem , Compostos Orgânicos/farmacologia , Transplante de Células-Tronco , Resultado do Tratamento
8.
Nan Fang Yi Ke Da Xue Xue Bao ; 37(2): 199-203, 2016 Feb 20.
Artigo em Chinês | MEDLINE | ID: mdl-28219863

RESUMO

OBJECTIVE: To screen the differentially expressed miRNAs and their target genes in adipogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs) to better understand the mechanism for regulating the balance between osteoblast and adipocyte differentiation. METHODS: Cultured hMSCs were induced for adipogenic differentiation, and at 0, 7, 14, and 21 days of induction, the cells were examined for miRNA and mRNA expression profiles using miRNA chip and transcriptome sequencing (RNA-seq) techniques. Correlation analysis was carried out for the miRNAs and mRNAs of potential interest. The databases including TargetScan, PicTar and miRanda were used to predict the target genes of the differentially expressed miRNA. RESULTS: The expression of miR-140-5p was down-regulated and leukemia inhibitory factor receptor (LIFR) expression increased progressively during adipogenic differentiation of hMSCs, showing a negative correlation between them. Target gene prediction using the 3 databases identified LIFR as the target gene of miR-140-5p. CONCLUSION: miRNA-140-5p may play an important role by regulating its target gene LIFR during adipogenic differentiation of hMSCs.


Assuntos
Adipogenia , Diferenciação Celular , Células-Tronco Mesenquimais/citologia , MicroRNAs/genética , Adipócitos/citologia , Células Cultivadas , Regulação para Baixo , Humanos , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Osteoblastos/citologia , RNA Mensageiro , Transcriptoma
9.
Methods Mol Biol ; 1317: 87-105, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26072403

RESUMO

Although cellular therapies may be effective in cancer treatment, their potential for expansion, damage of normal organs, and malignant transformation is a source of concern. The ability to conditionally eliminate aberrant cells in vivo would ameliorate these concerns and broaden the application of cellular therapy. We devised an inducible T-cell safety switch that can be stably and efficiently expressed in human T cells without impairing phenotype, function, or antigen specificity. This system is based on the fusion of human caspase 9 to a modified human FK-binding protein, allowing conditional dimerization using a small-molecule drug. When exposed to a synthetic dimerizing drug, the inducible caspase 9 (iC9) becomes activated and leads to the rapid apoptosis of cells expressing this construct. We have demonstrated the clinical feasibility and efficacy of this approach after haploidentical hematopoietic stem cell transplant (haplo-HSCT). A single dose of a small-molecule drug (AP1903) eliminated more than 90 % of the modified T cells within 30 min after administration and symptoms resolved without recurrence. This system has the potential to broaden the clinical applications of cellular therapy.


Assuntos
Caspase 9/metabolismo , Genes Transgênicos Suicidas , Complexo CD3/metabolismo , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Fibronectinas/farmacologia , Citometria de Fluxo , Humanos , Imunotoxinas/metabolismo , Interleucina-2/farmacologia , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Depleção Linfocítica , Muromonab-CD3/metabolismo , Proteínas Recombinantes/farmacologia , Retroviridae/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Doadores de Tecidos , Transdução Genética , Transgenes
10.
Pharmaceuticals (Basel) ; 8(2): 230-49, 2015 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-26110321

RESUMO

T-cells genetically redirected with a chimeric antigen receptor (CAR) to recognize tumor antigens and kill tumor cells have been infused in several phase 1 clinical trials with success. Due to safety concerns related to on-target/off-tumor effects or cytokine release syndrome, however, strategies to prevent or abate serious adverse events are required. Pharmacologic therapies; suicide genes; or novel strategies to limit the cytotoxic effect only to malignant cells are under active investigations. In this review, we summarize results and toxicities of investigations employing CAR redirected T-cells, with a focus on published strategies to grant safety of this promising cellular application.

11.
Blood ; 125(26): 4103-13, 2015 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-25977584

RESUMO

To test the feasibility of a single T-cell manipulation to eliminate alloreactivity while sparing antiviral and antitumor T cells, we infused 12 haploidentical hematopoietic stem cell transplant patients with increasing numbers of alloreplete haploidentical T cells expressing the inducible caspase 9 suicide gene (iC9-T cells). We determined whether the iC9-T cells produced immune reconstitution and if any resultant graft-versus-host disease (GVHD) could be controlled by administration of a chemical inducer of dimerization (CID; AP1903/Rimiducid). All patients receiving >10(4) alloreplete iC9-T lymphocytes per kilogram achieved rapid reconstitution of immune responses toward 5 major pathogenic viruses and concomitant control of active infections. Four patients received a single AP1903 dose. CID infusion eliminated 85% to 95% of circulating CD3(+)CD19(+) T cells within 30 minutes, with no recurrence of GVHD within 90 days. In one patient, symptoms and signs of GVHD-associated cytokine release syndrome (CRS-hyperpyrexia, high levels of proinflammatory cytokines, and rash) resolved within 2 hours of AP1903 infusion. One patient with varicella zoster virus meningitis and acute GVHD had iC9-T cells present in the cerebrospinal fluid, which were reduced by >90% after CID. Notably, virus-specific T cells recovered even after AP1903 administration and continued to protect against infection. Hence, alloreplete iC9-T cells can reconstitute immunity posttransplant and administration of CID can eliminate them from both peripheral blood and the central nervous system (CNS), leading to rapid resolution of GVHD and CRS. The approach may therefore be useful for the rapid and effective treatment of toxicities associated with infusion of engineered T lymphocytes. This trial was registered at www.clinicaltrials.gov as #NCT01494103.


Assuntos
Caspase 9/genética , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Linfócitos T/transplante , Adolescente , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Genes Transgênicos Suicidas , Haplótipos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Transtornos Linfoproliferativos/cirurgia , Masculino , Pessoa de Meia-Idade , Compostos Orgânicos/uso terapêutico , Reação em Cadeia da Polimerase em Tempo Real , Adulto Jovem
12.
Oncol Rep ; 33(3): 1505-11, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25607216

RESUMO

The human ubiquitin-specific processing enzyme 22 (USP22) plays a crucial role in regulating cell cycle processes and its overexpression has been linked to tumor progression. However, the mechanisms leading to USP22 transcriptional activation in human cancer cells are still unclear. Previously, we characterized the 5'-flanking sequence of the human USP22 gene and found a potential CREB/ATF binding site within the basic promoter region. The present study found that this site was required for constitutive USP22 transcriptional activity in HeLa and HepG2 cells. Chromatin immunoprecipitation assay confirmed that CREB interacted with this site. siRNA knockdown of CREB decreased USP22 transcriptional activation and endogenous expression, whereas CREB overexpression did not affect transcriptional levels. Furthermore, USP22 promoter activity and expression were decreased by inhibiting PKA with H-89, but were not responsive to forskolin induction. All of these results demonstrate that PKA/CREB is involved in the regulation of constitutive promoter activity of the USP22 gene.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Neoplasias/genética , Regiões Promotoras Genéticas/genética , Tioléster Hidrolases/genética , Ativação Transcricional/genética , Sítios de Ligação/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Colforsina/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Células HeLa , Células Hep G2 , Humanos , Isoquinolinas/farmacologia , Mutação/genética , Neoplasias/patologia , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , RNA Interferente Pequeno , Sulfonamidas/farmacologia
13.
Zhong Nan Da Xue Xue Bao Yi Xue Ban ; 39(8): 764-8, 2014 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-25202959

RESUMO

OBJECTIVE: To clone 5' untranslated region of human IPO8 gene and determine its transcription activity. METHODS: We used 5' rapid amplification of cDNA ends (RACE) analysis to identify the IPO8 transcription start site (TSS), and amplified series truncated 5' UTR fragment containing transcription start site. The PCR productions were inserted into luciferase report vector pGL3- Basic. After confirmation by restriction enzyme digestion, the recombinant plasmids were cotransfected into Saos-2 cells with plasmid pRL-TK. The luciferase activities were measured by dual luciferase reporter system. RESULTS: The IPO8 gene transcription start site was established. The electrophoresis analysis of restriction enzyme digestion and DNA sequencing verified the fragments were successfully amplified and inserted into pGL3-Basic. After the recombinant plasmids transfected, the highexpressions of luciferase were detected in Saos-2 cells. CONCLUSION: The recombinant vector containing IPO8 promoter is constructed successfully, which provides a foundation for determining expressional regulation of IPO8 in the further study.


Assuntos
Regiões Promotoras Genéticas , beta Carioferinas/genética , Clonagem Molecular , DNA Complementar , Vetores Genéticos , Humanos , Luciferases , Plasmídeos , Transfecção
14.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 30(7): 717-20, 2014 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-25001936

RESUMO

OBJECTIVE: To verify whether insulin promotes the differentiation of skeletal myoblasts into myocytes and whether wortmannin and U0126 inhibit the promoting effect with an attempt to explore the molecular mechanisms of inducing the differentiation of muscular cells in rats. METHODS: Primary skeletal myoblasts were separated and cultured from rats, and then were treated in DMEM containing various concentrations of insulin. The morphology of cells was monitored under a phase-contrast microscope. And the expression of myogenin was detected by immunocytochemistry and Western blotting. The change in the effect of insulin on the differentiation of myoblasts was observed after the intervention of a phosphatidylinositide 3-kinase (PI3K) inhibitor wortmannin and a specific MEK inhibitor U0126. RESULTS: Insulin markedly promoted myotube formation of myoblasts. Two days after insulin treatment, myotubes started to form; later, more and more myotubes appeared, and to the peak at 7 days. Insulin increased the expression of myogenin in a concentration-dependent manner. However, wortmannin and U0126 inhibited the effect of insulin on the differentiation of skeletal myoblasts in rats. CONCLUSION: Wortmannin and U0126 can suppress the promoting effect of insulin on the differentiation of skeletal myoblasts into myocytes in rats and decrease the formation of myotubes and the expression of myogenin.


Assuntos
Androstadienos/farmacologia , Butadienos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Insulina/farmacologia , Mioblastos Esqueléticos/efeitos dos fármacos , Nitrilos/farmacologia , Animais , Western Blotting , Células Cultivadas , Relação Dose-Resposta a Droga , Imuno-Histoquímica , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , Microscopia de Contraste de Fase , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/metabolismo , Miogenina/metabolismo , Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Wortmanina
15.
Blood ; 123(25): 3895-905, 2014 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-24753538

RESUMO

Adoptive transfer of donor-derived T lymphocytes expressing a safety switch may promote immune reconstitution in patients undergoing haploidentical hematopoietic stem cell transplant (haplo-HSCT) without the risk for uncontrolled graft versus host disease (GvHD). Thus, patients who develop GvHD after infusion of allodepleted donor-derived T cells expressing an inducible human caspase 9 (iC9) had their disease effectively controlled by a single administration of a small-molecule drug (AP1903) that dimerizes and activates the iC9 transgene. We now report the long-term follow-up of 10 patients infused with such safety switch-modified T cells. We find long-term persistence of iC9-modified (iC9-T) T cells in vivo in the absence of emerging oligoclonality and a robust immunologic benefit, mediated initially by the infused cells themselves and subsequently by an apparently accelerated reconstitution of endogenous naive T lymphocytes. As a consequence, these patients have immediate and sustained protection from major pathogens, including cytomegalovirus, adenovirus, BK virus, and Epstein-Barr virus in the absence of acute or chronic GvHD, supporting the beneficial effects of this approach to immune reconstitution after haplo-HSCT. This study was registered at www.clinicaltrials.gov as #NCT00710892.


Assuntos
Caspase 9/genética , Transplante de Células-Tronco Hematopoéticas/métodos , Linfócitos T/transplante , Transgenes/genética , Adolescente , Aspergilose/imunologia , Aspergilose/microbiologia , Aspergilose/prevenção & controle , Aspergillus fumigatus/imunologia , Caspase 9/biossíntese , Criança , Pré-Escolar , Indução Enzimática/efeitos dos fármacos , Feminino , Seguimentos , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Imunoterapia Adotiva/métodos , Linfoma Anaplásico de Células Grandes/imunologia , Linfoma Anaplásico de Células Grandes/terapia , Masculino , Síndromes Mielodisplásicas/imunologia , Síndromes Mielodisplásicas/terapia , Compostos Orgânicos/administração & dosagem , Compostos Orgânicos/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Tempo , Transplante Homólogo , Resultado do Tratamento , Viroses/imunologia , Viroses/prevenção & controle , Viroses/virologia
16.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 29(4): 350-3, 2013 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-23643163

RESUMO

OBJECTIVE: To construct and identify the recombinant adenovirus of muramidase-released protein (MRP) gene fragment from Streptococcus suis type 2 (SS2). METHODS: The specific primers were designed based on the sequence of MRP gene fragment. The MRP gene fragment (467-1351 bp) was amplified by PCR method with genomic DNA of SS2 as a template. PCR products were cloned in pMD18-T vector. Then MRP gene fragment was linked into the adenovirus shuttle plasmid (pShuttle-CMV) to construct recombinant shuttle plasmid (pShuttle-CMV-MRP). After PmeI digestion, it was transformed into BJ5183-AD-1 competent cells containing adenoviral backbone plasmid pAdEasy-1 to construct homogeneous recombinant adenovirus plasmid (pAdeno-CMV-MRP). Then the recombinant adenovirus plasmid was linearized by PmeI and then transfected into AD-293 cells for viral packaging. Finally, the virus liquid was tested by PCR and Western blotting. RESULTS: Cytopathic effect (CPE) was observed at 8 d after transfection of linear pAdeno-CMV-MRP in AD-293 cells. MRP gene fragment and protein expression were also detected in the virus liquid. CONCLUSION: The recombinant adenovirus of MRP gene fragment (rAdeno-MRP) from SS2 was constructed successfully.


Assuntos
Adenoviridae/genética , Adenoviridae/metabolismo , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Streptococcus suis/genética , Streptococcus suis/metabolismo , Linhagem Celular , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção/métodos
17.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 23(3): 324-6, 329, 2011 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-22164506

RESUMO

OBJECTIVE: To study the published papers and its citation of Chinese Journal of Schistosomiasis Control, so as to provide evidence for the improvement of its quality. METHODS: The published papers in Chinese Journal of Schistosomiasis Control from 2008 to 2010 were searched, biblimetrics analysis was employed, and the number of published papers, the proportion of fund articles, the cooperation rate of scientific research, the distribution of authors, the citations and their types, the half-life periods and the Price's index were analysed. RESULTS: From 2008 to 2010, the number of papers published were 217, 203, 210, respectively, and the proportions of fund articles were 32.72%, 38.92% and 49.52%, respectively. The cooperation rates of scientific research were 87.56%, 95.07%, 94.76%, respectively. The average amounts of citation were 5.49, 10.14, 13.33 per paper, respectively, and the citations were mainly from books and journals. The Price's indexes were 47.23%, 50.12% and 51.48%, respectively. CONCLUSIONS: The quality and academic level of papers published in Chinese Journal of Schistosomiasis Control is improving year by year, the journal can satisfy its authors and readers with the latest information of scientific research in schistosomiasis and parasitic diseases study and control. But it requires to enlarge the author group and to increase the amount of citation to its further development.


Assuntos
Bibliometria , Publicações Periódicas como Assunto/estatística & dados numéricos , Editoração/estatística & dados numéricos , Esquistossomose , China , Humanos , Linguagem , Publicações Periódicas como Assunto/normas , Editoração/normas , Controle de Qualidade , Esquistossomose/epidemiologia , Esquistossomose/imunologia , Esquistossomose/parasitologia
18.
Mol Ther ; 19(1): 211-7, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20959814

RESUMO

Although the benefits of adoptive T-cell therapy can be increased by prior lymphodepletion of the recipient, this process usually requires chemotherapy or radiation. Vaccination with antigens to which the transferred T cells respond should be a less toxic means of enhancing their activity, but to date such vaccines have not been effective. We, therefore, determined which characteristics an adenoviral vaccine has to fulfill to optimally activate and expand adoptively transferred antigen-specific T cells in vivo. We evaluated (i) antigen, (ii) flagellin, a Toll-like receptor (TLR) 5 ligand, and (iii) an inhibitor of the antigen-presenting attenuator A20. Vaccination of mice before T-cell transfer with a vaccine that contained all three components dramatically enhanced the effector function of ovalbumin (OVA)-specific T cells as judged by the regression of established B16-OVA tumors compared to one- and two-component vaccines. Immunization with the three-component vaccine induced a strong Th1 environment, which was critical for the observed synergy and proved as effective as cytoxan-induced lymphodepletion in enhancing in vivo T-cell expansion. Thus, the combination of our vaccine with T-cell therapy has the potential to enhance and broaden adoptive cellular immunotherapy.


Assuntos
Adenoviridae/imunologia , Vacinas Anticâncer/imunologia , Imunoterapia Adotiva/métodos , Linfócitos T/imunologia , Células Th1/imunologia , Vacinas Virais/imunologia , Animais , Apresentação do Antígeno/imunologia , Antígenos/imunologia , Linhagem Celular , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Flagelina/genética , Imunização Secundária/métodos , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina/imunologia , RNA Interferente Pequeno/genética , Vacinas Sintéticas/genética , Vacinas Sintéticas/metabolismo
19.
Proc Natl Acad Sci U S A ; 107(42): 18109-14, 2010 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-20921400

RESUMO

Mutations in WNK1 and WNK4 lead to familial hyperkalemic hypertension (FHHt). Because FHHt associates net positive Na(+) balance together with K(+) and H(+) renal retention, the identification of WNK1 and WNK4 led to a new paradigm to explain how aldosterone can promote either Na(+) reabsorption or K(+) secretion in a hypovolemic or hyperkalemic state, respectively. WNK1 gives rise to L-WNK1, an ubiquitous kinase, and KS-WNK1, a kinase-defective isoform expressed in the distal convoluted tubule. By inactivating KS-WNK1 in mice, we show here that this isoform is an important regulator of sodium transport. KS-WNK1(-/-) mice display an increased activity of the Na-Cl cotransporter NCC, expressed specifically in the distal convoluted tubule, where it participates in the fine tuning of sodium reabsorption. Moreover, the expression of the ROMK and BKCa potassium channels was modified in KS-WNK1(-/-) mice, indicating that KS-WNK1 is also a regulator of potassium transport in the distal nephron. Finally, we provide an alternative model for FHHt. Previous studies suggested that the activation of NCC plays a central role in the development of hypertension and hyperkalemia. Even though the increase in NCC activity in KS-WNK1(-/-) mice was less pronounced than in mice overexpressing a mutant form of WNK4, our study suggests that the activation of Na-Cl cotransporter is not sufficient by itself to induce a hyperkalemic hypertension and that the deregulation of other channels, such as the Epithelial Na(+) channel (ENaC), is probably required.


Assuntos
Canais Epiteliais de Sódio/metabolismo , Hipertensão/prevenção & controle , Córtex Renal/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Receptores de Droga/metabolismo , Simportadores/metabolismo , Animais , Canais Epiteliais de Sódio/genética , Imunofluorescência , Masculino , Camundongos , Camundongos Knockout , Antígenos de Histocompatibilidade Menor , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Membro 3 da Família 12 de Carreador de Soluto , Proteína Quinase 1 Deficiente de Lisina WNK
20.
J Am Soc Nephrol ; 21(10): 1724-31, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20813867

RESUMO

WNK1 and WNK4 encode two members of the WNK serine-threonine kinase subfamily. Greater WNK1 expression associates with higher BP. A combination of promoters, enhancers, repressors, and insulators regulate WNK1 expression, but whether microRNAs also modulate WNK1 expression is unknown. Here, computational analysis revealed the presence of a target sequence for miR-192 and miR-215 at the same site in the 3' untranslated region of the ubiquitous L- and the kidney-specific KS-WNK1. We functionally validated this target sequence by transient transfection and reporter assays. Although we observed expression of both miRs along the distal nephron, only miR-192 regulated endogenous WNK1 ex vivo. Furthermore, a potassium load, sodium depletion, and aldosterone infusion each significantly reduced miR-192 expression in the kidney. Taken together, these results suggest a miR-driven mechanism of gene regulation by aldosterone and a role for miR-192 in the regulation of sodium and potassium balance in the kidney.


Assuntos
Aldosterona/metabolismo , Túbulos Renais Distais/metabolismo , MicroRNAs/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Linhagem Celular , Cães , Camundongos , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Menor , Potássio/metabolismo , Proteínas Serina-Treonina Quinases/genética , Sódio na Dieta/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNK
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