RESUMO
The effects of Hedgehog signaling inhibitor (cyclopamine) and activator (Shh) on drug resistance of U251-MG human glioma cells and human astrocyte culture to cisplatin, temozolomide, and doxorubicin were studied. Cyclopamine and Shh modified the drug resistance of U251-MG cells but not of human astrocytes. Experiments with cyclopamine, Shh, and chemical drugs can contribute to detection of the mechanisms of signaling effects on the drug resistance processes, while the experimental data can serve as one of the criteria for choosing individual chemotherapy for patients.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/genética , Neuroglia/efeitos dos fármacos , Transdução de Sinais/genética , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Células Cultivadas , Cisplatino/farmacologia , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Doxorrubicina/farmacologia , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Humanos , Neuroglia/metabolismo , Neuroglia/patologia , Peptídeos/farmacologia , Temozolomida , Alcaloides de Veratrum/farmacologiaRESUMO
We studied the effect of an activator (ShTh) and an inhibitor (cyclopamine) of the Hedgehog signaling pathway on proliferation of human glioma cell lines U87-MG and U251-MG and cultured human astrocytes. The Hedgehog signaling pathway is activated in glioma cells, but not in cultured human astrocytes. Experiments with Shh and cyclopamine can serve as an additional criterion for assessing activity of Hedgehog signaling in known cell lines and primary cultured cells.
Assuntos
Proliferação de Células , Glioblastoma/metabolismo , Transdução de Sinais , Astrócitos/fisiologia , Linhagem Celular Tumoral , Glioblastoma/patologia , Proteínas Hedgehog/agonistas , Proteínas Hedgehog/metabolismo , Humanos , Ligantes , Alcaloides de Veratrum/farmacologiaRESUMO
To study demyelination and remyelination processes and their response to different drugs, a protocol for modeling multiple sclerosis using the copper chelator cuprizone was developed. Magnetic resonance imaging confirmed the presence of demyelination lesions on week 4 of 0.6% cuprizone-containing diet. Immunohistochemical staining with polyclonal antibodies to glial fibrillary acidic protein (pAb GFAP) confirmed the increase in the number of reactive astrocytes on week 4 of diet and during remyelination (week 2 after diet). Analysis of neurophysiological functions in mice with cuprizone-induced demyelination revealed motor and behavioral deficits. This model can be used as a tool for preclinical studies of the efficiency of multiple sclerosis diagnostic and therapy.
Assuntos
Cuprizona/farmacologia , Doenças Desmielinizantes/induzido quimicamente , Esclerose Múltipla/induzido quimicamente , Bainha de Mielina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Encéfalo/diagnóstico por imagem , Modelos Animais de Doenças , Feminino , Proteína Glial Fibrilar Ácida , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/terapia , RadiografiaRESUMO
A model of highly metastasizing orthotopic allogeneic breast carcinoma was reproduced and standardized in experiments on BALB/c mice. 4T1 cells characterized by high metastatic activity were transfected with red fluorescent protein (RFP) gene or firefly luciferase (Luc2) gene. Unmodified 4T1 cells and modified 4T1-RFP and 4T1-Luc2 cells were subcutaneously injected to mature female mice into the second mammary fat pads. Quantitative evaluation of the primary node and visceral metastases was performed using magnetic-resonance imaging, X-ray and optical tomography. Modification of 4T1 cells with RFP gene considerably reduced their invasive and metastatic potential and led to spontaneous regression of the primary tumor in 20% cases. Modification of 4T1 cells with Luc2 gene had practically no effect on proliferative, invasive, and metastatic characteristics of the tumor and provided the possibility of quantitative analysis of the primary tumor dynamics by the luminescence intensity. The survival median in mice receiving unmodified 4T1 cells and transfected 4T1-RFP and 4Т1-Luc2 cells was 32, 42, and 38 days, respectively. Neither primary node nor tumor metastases accumulated gadolinium-containing contrast agent and Alasens fluorescent tracer. After implantation of 4T1 and 4Т1-Luc2 cells, multiple metastases were more often detected in the lungs, liver, spleen, spine, and regional lymph nodes and less frequently in the brain, which corresponded to metastasizing profile of human breast cancer. The developed model of orthotopic breast carcinoma 4T1 in BALB/c mice with complex detection of multiple organ metastases using X-ray microCT, optical, and MRI can be recommended for preclinical studies of new antitumor preparations.
Assuntos
Neoplasias da Mama/patologia , Modelos Animais de Doenças , Modelos Biológicos , Metástase Neoplásica/fisiopatologia , Animais , Feminino , Luciferases/farmacologia , Proteínas Luminescentes/metabolismo , Proteínas Luminescentes/farmacologia , Imageamento por Ressonância Magnética/métodos , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica/diagnóstico por imagem , Metástase Neoplásica/ultraestrutura , Análise de Sobrevida , Tomografia Óptica , Microtomografia por Raio-X , Proteína Vermelha FluorescenteRESUMO
cDNA extracellular Ig-like domains I-III of vascular endothelial growth factor receptor 2 (VEGFR2) was cloned in an expressing vector pET_32a. Western blotting showed immunochemical identity of recombinant VEGFR2I-III produced by prokaryotic expression system to the native receptor. BALB/c mice were immunized with VEGFR2I-III for obtaining specific antibodies to VEGFR2. Hybridomas producing monoclonal antibodies were selected by ELISA, Western blotting, and immunocytochemical assay. Thus, we obtained hybridoma producing monoclonal antibodies to VEGFR2 that selectively interact with both recombinant and native extracellular fragment of the receptor.
Assuntos
Anticorpos Monoclonais/imunologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologia , Animais , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
cDNA fragment encoding neuron-specific enolase was amplified from the cDNA library of human brain. Then the fragment was cloned for expression in E. coli using the vector pET28-a. High level of neuron-specific enolase expression was confirmed by SDS-PAAG electrophoresis and immunochemical identity by immunoblot analysis. The constructed producer strain is the cheapest source of neuron-specific enolase suitable for the use in diagnostic applications.
Assuntos
DNA Complementar/genética , Escherichia coli/genética , Fosfopiruvato Hidratase/genética , Clonagem Molecular , HumanosAssuntos
Animais Recém-Nascidos/fisiologia , Barreira Hematoencefálica , Convulsões/fisiopatologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Dexametasona/farmacologia , Feminino , Proteína Glial Fibrilar Ácida/sangue , Glucocorticoides/farmacologia , Hipertermia Induzida , Masculino , Permeabilidade , Ratos , Convulsões/etiologia , Fatores de TempoRESUMO
The dynamic state of hematoencephalic barrier (HEB) was estimated in Russian tick-borne encephalitic (RTBE) and in meningeal syndrome (Lyme's disease, LD). The enzyme immunoassays of neurospecific proteins in blood serum such as alpha 1-BG and enolase (NSE) were performed in the course of disease. The break of HEB at blood-brain direction was proved in patients with RTBE and LD for the proteins mentioned above. The blood serum NSE levels as well as their dynamics confirmed the functional alteration of brain neurons' permeability in LD and pronounced destruction of such neurons in RTBE. The acute increase of blood NSE concentration was observed within some days before the development of clinical signs of paresis. The authors suppose that the enzyme immunoassays of neurospecific proteins which are of rather high value for HEB permeability and neuronal destruction control may be successfully applied in monitoring of both therapy's efficacy and individualization of pharmacotherapy.