RESUMO
Introducción: El carcinoma oral de células escamosas (COCE) es una neoplasia epitelial maligna que se presenta frecuentemente entre la quinta y sexta década de la vida. Su compleja patogénesis incluye el proceso de angiogénesis y la regulación del microambiente tumoral como mecanismos de progresión tumoral. Objetivo: Determinar la relación entre las variables clínicas e histológicas del COCE con la inmunoexpresión de VEGF, FGF-1, FGFR-1, TGFB-1, TGFBR-II y CD105. Material y métodos: Nueve casos de COCE; tres bien (BD), tres moderado (MD) y tres pobremente diferenciados (PD) obtenidos del Departamento de Patología y Medicina Bucal, División de Estudios de Postgrado e Investigación. Se aplicó la técnica de inmunohistoquímica por peroxidasa para identificar la expresión de VEGF, FGF-1, FGFR- 1, TGFB-1, TGFBR-II y CD105. El análisis de inmunoexpresión se realizó con el programa ImageJ. Se aplicó la prueba de Kruskal-Wallis y correlación de Spearman (p < 0.05). Resultados: La inmunoexpresión de VEGF fue mayor en los COCE PD, FGFR-1 fue positivo en los BD, mientras que FGF, TGFB-1 y TGFBR-II fueron negativos. El análisis de microdensidad vascular (MVD) indicó mayor número de vasos CD105 positivos en los carcinomas BD, seguidos de los PD y MD. Conclusión: Considerando los resultados obtenidos podemos concluir que la angiogénesis es un fenómeno constante independiente del grado de diferenciación que durante el proceso de transformación de una neoplasia requerirá la formación de vasos sanguíneos y que este proceso puede ser modulado por factores de crecimiento tales como los analizados en este trabajo (AU)
Introduction: Oral squamous cell carcinoma (OSCC) is a malignant epithelial neoplasm that frequently occurs between the fifth and sixth decade of life. Its complex pathogenesis includes the angiogenesis process and the regulation of the tumor microenvironment as mechanisms of tumor progression. Objective: To determine the relationship between the clinical and histological variables of OSCC with the immunoexpression of VEGF, FGF-1, FGFR-1, TGFB- 1, TGFBR-II and CD105. Material and methods: Nine cases of OSCC; three well (WD), three moderate (MD) and three poorly differentiated (PD) obtained from the Oral Medicine and Pathology Department, Division of Graduate Studies and Research. The peroxidase immunohistochemistry technique was performed to identify the expression of VEGF, FGF-1, FGFR-1, TGFB-1, TGFBR-II and CD105. The immunoexpression analysis was performed with the ImageJ software. The Kruskal-Wallis and Spearman correlation test were performed (p < 0.05). Results: VEGF immunoexpression was higher in PD OSCC, while FGFR-1 was predominantly positive in WD; FGF, TGFB-1 and TGFBR-II were negative. Vascular microdensity analysis (MVD) indicated a greater number of CD105 positive vessels in WD carcinomas, followed by PD and MD. Conclusion: Considering the results obtained, we can conclude that angiogenesis is a constant phenomenon independent of the degree of differentiation; that during the transformation process of a neoplasm it will require the formation of blood vessels and that this process can be modulated by growth factors such as those analyzed in this work (AU)
Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Idoso , Carcinoma de Células Escamosas/imunologia , Fator 1 de Crescimento de Fibroblastos , Fator A de Crescimento do Endotélio Vascular , Vasos Sanguíneos , Imuno-Histoquímica , Técnicas Histológicas , Peptídeos e Proteínas de Sinalização Intercelular , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Endoglina , MéxicoRESUMO
Introdução: Queloides surgem de resposta excessiva à lesão da derme, resultando em proliferação de fibroblastos, produção exagerada de colágeno e comprometimento da pele sadia adjacente. O diagnóstico é clínico e muitos métodos conservadores e cirúrgicos já foram utilizados para tratamento. Porém, dados da eficácia desses tratamentos são limitados e não há consenso na literatura quanto a melhor técnica a ser empregada, permanecendo uma lacuna que necessita ser preenchida, a fim de que seus usos sejam indicados com maior confiabilidade, em um modelo de medicina baseada em evidências. Métodos: Revisão não sistemática da literatura sobre "queloides" nas bases de dados PubMed, Scielo, MEDLINE, UptoDate e livros-texto das áreas de Dermatologia e Cirurgia Dermatológica. Revisão de Literatura: Foram enumeradas e abordadas as principais informações sobre técnicas cirúrgicas e adjuvantes empregadas para essas lesões, que são: excisão, injeções intralesionais, crioterapia, laserterapia, revestimento com gel de silicone, radioterapia e pressoterapia. Torna-se relevante o levantamento dessas informações, tendo em vista que, além de poder causar dor, prurido e restrição de movimento, o principal motivo da procura de assistência médica para queloide é devido ao aspecto cosmético/estético, e as taxas de reincidência e falha terapêutica ainda são altas, sendo necessário conscientizar o paciente sobre o procedimento e seus efeitos. Conclusão: São muitos os tratamentos disponíveis para o queloide, sejam cirúrgicos ou não, todavia não há consenso sobre uma abordagem universalmente aceita. São necessários mais estudos, com a finalidade de definir a melhor conduta e atingir melhores resultados, visto a qualidade mediana das evidências apresentadas nos estudos.
Introduction: Keloids are characterized by an abnormal response to dermal trauma, resulting in fibroblast proliferation, excessive collagen production, and impairment of adjacent healthy tissue. The diagnosis is clinical, and many conservative and surgical methods can be used as treatments. However, data on the efficacy of these treatments are limited, and there is no consensus regarding the best treatment option. This gap needs to be filled by developing comprehensive evidence-based therapies. Methods: A non-systematic literature review of keloid scars was carried out using PubMed, Scielo, MEDLINE, UptoDate, and dermatology and dermatological surgery textbooks. Literature review: The search retrieved relevant information on surgical and adjuvant therapies used for keloids, including excision, intralesional injections, cryotherapy, laser therapy, silicone gel sheeting, radiation therapy, and pressure therapy. These data are crucial because, in addition to complaints of pain, itching, and restriction of movement, the main reason for seeking treatment for keloids is for cosmetic and aesthetic improvement, and the rates of recurrence and treatment failure are high, emphasizing the importance of creating awareness regarding the available procedures and their effectiveness. Conclusion: Many surgical and adjuvant therapies for keloids are available. Nonetheless, there is no consensus on a universally accepted treatment. Therefore, additional high-quality studies are needed to identify the most effective therapeutic approaches to achieve better results.
Assuntos
Humanos , História do Século XXI , Recidiva , Cirurgia Plástica , Terapêutica , Fator 1 de Crescimento de Fibroblastos , Fibroblastos , Procedimentos Cirúrgicos Dermatológicos , Queloide , Cirurgia Plástica/efeitos adversos , Cirurgia Plástica/métodos , Terapêutica/métodos , Ferimentos e Lesões , Ferimentos e Lesões/cirurgia , Ferimentos e Lesões/terapia , Fator 1 de Crescimento de Fibroblastos/análise , Fator 1 de Crescimento de Fibroblastos/efeitos adversos , Cicatriz , Cicatriz/complicações , Procedimentos Cirúrgicos Dermatológicos/métodos , Queloide/cirurgiaRESUMO
OBJECTIVE@#To determine the time course and potential mechanism of fibroblast growth factor-1 (FGF-1) in the regulation of adipogenesis. @*METHODS@#We cultured human Simpson-Golabi-Behmel syndrome (SGBS) pre-adipocytes with recombinant FGF-1 and harvested cells at various stages prior to and during differentiation; at cell proliferation (D-3), confluence (D0), early (D3), middle (D7) and mature (D14) stages of differentiation. We determined lipid accumulation in mature adipocytes by morphological observation and quantitative measurement of oil red O staining. We also examined the expression of adipogenic genes and related markers involved in the Wnt/β-catenin pathway using quantitative Real-time PCR and Western blot. @*RESULTS@#Compared to control SGBS cells, treatment with FGF-1 increased lipid accumulation; induced a sustained increase in the mRNA for peroxisome proliferater-activated receptor γ (PPARγ), glyceraldehyde-3-phosphate dehydrogenase (G3PDH), adiponectin and glucose transporter type 4 (GLUT4); and promoted a sustained decrease in expression of markers of the Wnt/β-catenin pathway, β-catenin and transcription factor 4 (TCF4). @*CONCLUSION@#The adipogenic effects of FGF-1 are apparent throughout the whole priming and differentiation period in human SGBS pre-adipocytes. Furthermore, our results suggest that FGF-1 promotes adipogenesis, at least in part, via a sustained decrease in activity of the Wnt/β-catenin pathway.
Assuntos
Humanos , Adipócitos , Metabolismo , Adipogenia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Metabolismo , Diferenciação Celular , Células Cultivadas , Fator 1 de Crescimento de Fibroblastos , Farmacologia , Proteínas Recombinantes , Farmacologia , Fator de Transcrição 4 , Fatores de Transcrição , Metabolismo , Via de Sinalização Wnt , beta Catenina , MetabolismoRESUMO
Biodentine™ é um biomaterial à base de silicato de cálcio produzido, segundo o fabricante, com o intuito de apresentar propriedades físico-químicas e biológicas superiores ao MTA. Nosso objetivo foi avaliar a resposta tecidual promovida por Biodentine™ (BDT) e MTA Angelus Branco em subcutâneo de ratos. Foram utilizados ratos adultos distribuídos em 3 grupos (n=20/grupo), segundo o material preenchendo os tubos de polietileno implantados no subcutâneo: BDT, MTA e GC (controle, tubos vazios). Após 7, 15, 30 e 60 dias, os implantes e os tecidos adjacentes foram processados para parafina. Cortes longitudinais das cápsulas adjacentes aos implantes foram corados com H&E, tricrômico de Masson, Picrosirius e submetidos ao Alcian Blue (AB) e reações imuno-histoquímicas para IL-6 e FGF-1. Obteve-se a densidade numérica de células inflamatórias (CI), de células imunomarcadas para IL-6 e FGF-1 e de mastócitos AB-positivos, além da porcentagem de colágeno birrefringente. Os dados foram submetidos à ANOVA e teste Tukey (p≤0,05). O número de CI e de células imunomarcadas para IL-6 e FGF-1 foi significantemente maior aos 7 dias em todos os grupos. Diferenças significantes no número de células IL-6-positivas não foram observadas entre BDT e MTA aos 30 e 60 dias; aos 60 dias, diferenças significantes no número de CI também não foram detectadas. O número de células FGF-1- positivas foi significantemente maior no grupo BDT em comparação ao grupo MTA, em todos os períodos. A densidade numérica de mastócitos e a porcentagem de colágeno aumentaram significantemente ao longo do tempo. Aos 60 dias, o número de mastócitos e o conteúdo de colágeno foram significantemente maiores nos grupos BDT e MTA, respectivamente. A redução significante do processo inflamatório, concomitante à redução na imunoexpressão para IL-6, indica que ambos os materiais são biocompatíveis. A acentuada imunoexpressão de FGF-1 aos 7 dias sugere que este fator deve ser responsável, pelo menos em parte, pela proliferação de fibroblastos e, consequentemente, estimula a formação de colágeno nas cápsulas. Os mastócitos devem ter um papel importante na remodelação das cápsulas, pois uma forte correlação foi detectada entre o aumento significante de mastócitos e de colágeno.
Biodentine™ is a new calcium silicate-based biomaterial which presents improved physicochemical and biological properties compared to MTA. The aim of this study was to evaluate the tissue reaction promoted by Biodentine™ (BDT) and MTA Angelus White in rat subcutaneous. Adult rats were distributed into 3 groups (n=20/group) according to the implanted materials: BDT, MTA or CG (Control group; empty tubes). After 7, 15, 30 and 60 days, the implants and adjacent tissues were fixed and embedded in paraffin. Longitudinal sections of the capsule adjacent to implants were stained with H&E, Masson's trichrome, Picrosirius and submitted to Alcian Blue (AB). Immunohistochemical reactions for IL-6 and FGF-1 were also performed. The number of inflammatory cells (IC), IL-6 and FGF-1 immunolabeled cells, AB-positive mast cells as well as birefringent collagen percentage were obtained. Data were statistically analyzed by ANOVA and Tukey test (p≤0.05). The number of IC and IL-6 and FGF-1 immunolabeled cells were significantly high at 7 days in all groups. At 30 and 60 days, significant differences in the number of IL-6-positive cells were not detected between BDT and MTA. At 60 days, statistical difference in the IC number was not observed between BDT and MTA groups. In all periods, the number of FGF-1-positive cells was significant higher in BDT group in comparison to MTA. The numerical density of mast cells and collagen percentage increased over time. At 60 days, mast cells number and collagen content were significantly high in BDT and MTA groups, respectively. A significant reduction of inflammatory process and IL-6 immunoexpression indicates that both materials are biocompatible. Intense FGF-1 immunoexpression at 7 days suggests that this factor may be responsible, at least in part, for fibroblast proliferation and subsequent collagen formation in the capsules. Since a strong correlation between mast cells number and collagen density was detected, it is conceivable to suggest that mast cells play a pivotal role in the capsules remodeling
Assuntos
Animais , Ratos , Teste de Materiais , Colágeno , Fator 1 de Crescimento de Fibroblastos , Mastócitos , Materiais Biocompatíveis , Calcarea Silicata , Análise de VariânciaRESUMO
<p><b>OBJECTIVE</b>To investigate the expression of acidic fibroblast growth factor (aFGF) in transgenic safflower and lay the foundation for the use of the plant bioreactor large-scale production aFGF.</p><p><b>METHOD</b>The haFGF gene was transformed into plant preference of the aFGF sequence as a basis for design of primers, plant preferences aFGF gene sequences was amplified by PCR. The vegetable body expression vector was constructed by using digested connection method and then transferred to Agrobacterium tumefaciens EHA105 by the freeze-thaw method. It transferred to safflowers by agrobacterium-mediated transformation method, and identified by PCR, southern blot and RT-PCR.</p><p><b>RESULT</b>The full-length aFGF gene sequences were amplified through PCR and constructed into plant expression vector with soybean oleosin and promoter, and transformed into safflower. Three independently transformed safflower plant units with point insertion were successfully obtained, which showed the same size of aFGF expression at the transcriptional level.</p><p><b>CONCLUSION</b>The plant oil body expression vectors were successfully constructed, and the optimal condition for genetic transformation was selected. The transgenic safflower plants were obtained.</p>
Assuntos
Humanos , Agrobacterium tumefaciens , Genética , Carthamus tinctorius , Genética , Fator 1 de Crescimento de Fibroblastos , Genética , Regulação da Expressão Gênica de Plantas , Vetores Genéticos , Proteínas de Membrana , Genética , Proteínas de Plantas , Genética , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , Transformação GenéticaRESUMO
Peptide RADA can undergo spontaneous assembly into well ordered nanofibers and hydrogels in its water solution. In this work, a variety of proteins, including IGF-1, aFGF and VEGF with different molecular weight and isoelectric points, were chosen and encapsulated within the RADA peptide hydrogel. UV-vis spectroscopy was used to determine the concentration of the released proteins in the solution. The release kinetics suggested that protein diffusion through nanofiber hydrogels depended primarily on the size of the protein and the density of the peptide nanofiber. Circular dichroism (CD) spectroscopy indicated that the encapsulation and release by RADA hydrogel did not affect the secondary structure of the proteins studied.
Assuntos
Dicroísmo Circular , Preparações de Ação Retardada , Fator 1 de Crescimento de Fibroblastos , Metabolismo , Hidrogéis , Química , Fator de Crescimento Insulin-Like I , Metabolismo , Nanofibras , Peptídeos , Química , Estrutura Secundária de Proteína , Fator A de Crescimento do Endotélio Vascular , MetabolismoRESUMO
This paper is to report the study of the pharmacokinetics of a fusion protein TAT-haFGF(14-154) for human acidic fibroblast growth factor and transcriptional activator protein in rat plasma, and the investigation of their penetration across blood-brain barrier in mice and rats, in order to provide a basis for clinical development and treatment of Alzheimer's disease. Enzyme-linked immunosorbent assay (ELISA) was used to determine concentration of TAT-haFGF(14-154) in rat plasma and in mouse brain homogenate; and immunohistochemistry was used to analyze the distribution in brain. The concentration-time curve fitted two-compartment open model which was linear kinetics elimination after a single intravenous injection of TAT-haFGF(14-154) in rat at the dose of 300 microg x kg(-1). The half life time was 0.049 +/- 0.03 h for distribution phase and 0.55 +/- 0.05 h for elimination phase, and the weight was 1/C2. The result showed that TAT-haFGF(14-154) could be detected in the brain by ELISA and immunohistochemistry, the elimination of TAT-haFGF(14-154) in rat was swift, and TAT-haFGF(14-154) could penetrate across the blood-brain barrier, distribute in pallium and hippocampus and locate in the nucleus.
Assuntos
Animais , Feminino , Masculino , Camundongos , Ratos , Barreira Hematoencefálica , Metabolismo , Encéfalo , Metabolismo , Núcleo Celular , Metabolismo , Córtex Cerebral , Metabolismo , Fator 1 de Crescimento de Fibroblastos , Farmacocinética , Produtos do Gene tat , Farmacocinética , Hipocampo , Metabolismo , Injeções Intravenosas , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão , FarmacocinéticaRESUMO
Acid fibroblast growth factor (aFGF) has great potential in clinical application, but it is very expensive. In order to reduce the cost of production and to make full use of the merits integrated with plant bioreator, we have explored the aFGF in transgenic Tobacco expression. AFGF gene was inserted into plant expression vector pBI121; the acquired plants contained aFGF gene expression vector pBI121-TOAB-aF. Using Agrobacterium-mediated gene transformation of Tobacco and using transgenic Tobacco containing kanamycin and cephalosporin culture medium, we obtained kanamycin resistant transgenic Tobacco plants. PCR detection, RT-PCR detection and Western blot detection confirmed that foreign genes were successfully expressed in Tobacco. These data could serve as a theoretical foundation on which to use the plant bioreactor for production of aFGF.
Assuntos
Agrobacterium , Genética , Fator 1 de Crescimento de Fibroblastos , Genética , Vetores Genéticos , Genética , Plantas Geneticamente Modificadas , Genética , Metabolismo , Proteínas Recombinantes , Genética , Nicotiana , Genética , MetabolismoRESUMO
<p><b>OBJECTIVE</b>To establish a high-frequency regeneration system of Astragalus and an aFGF transformation system.</p><p><b>METHOD</b>Cotyledon node of the Astragalus explants was used for organogenesis to establish a high-frequency regeneration system. GV3101 was used to transform cotyledon node, and aFGF gene was introduced into Astragalus, renewable strain was detected by PCR.</p><p><b>RESULT</b>All cotyledon node was explants, adventitious buds were induced in the medium of MS +2.0 mg x L(-1) BA +0.5 mg x L(-1) IBA, the root was taken in the medium of 1/2MS +5.0 mg x L(-1) NAA to give a high frequency regeneration system. All cotyledon node was precultured in medium for 3 days and infected with Agrobacterium (A600 0.3) for 10 min and then cocultured for 2 days. The aFGF gene was confirmed to transform into genome of Astragalus.</p><p><b>CONCLUSION</b>A high-frequency regeneration system of Astragalus and an aFGF transformation system were established.</p>
Assuntos
Humanos , Astrágalo , Genética , Metabolismo , Fator 1 de Crescimento de Fibroblastos , Genética , Metabolismo , Regulação da Expressão Gênica , Engenharia Genética , Métodos , Plantas Geneticamente Modificadas , Genética , Metabolismo , Transformação GenéticaRESUMO
The BPI23-haFGF fusion gene was subcloned to the yeast expression vector pPICZaA and the recombinant plasmid pPICZaA-BPI23-haFGF was constructed. After linearization by sac I, the construct was introduced into X-33 yeast cells. The efficient engineering strain was obtained by the resistance and phenotype selection and identified by specific PCR. SDS-PAGE and Western blot analysis indicated that a 43 KD protein band coincident with the anticipated fusion protein size expressed in the culture supernatant of the transformed yeast cells, which accounted for above 50% of the total proteins of the culture supernatant. About 90% purity of recombinant BPI23-haFGF fusion protein was obtained by affinity chromatography. The in vitro bioactivity testing showed that the purified fusion protein killed E. coli and promoted proliferation of NIH3T3 cells, suggesting that the recombinant BPI23-haFGF fusion protein possessed both of BPI and FGF functions.
Assuntos
Animais , Humanos , Camundongos , Peptídeos Catiônicos Antimicrobianos , Genética , Metabolismo , Proteínas Sanguíneas , Genética , Metabolismo , Proliferação de Células , Clonagem Molecular , Escherichia coli , Fator 1 de Crescimento de Fibroblastos , Genética , Metabolismo , Fusão Gênica , Vetores Genéticos , Células NIH 3T3 , Pichia , Genética , Metabolismo , Proteínas Recombinantes de Fusão , Genética , Metabolismo , FarmacologiaRESUMO
<p><b>OBJECTIVE</b>To explore the effect of basic fibroblast growth factor 1 (bFGF1) during embryonic development on hematopoiesis, to study the expression of FGF1, vascular endothelial growth factor receptor (KDR), CD133, CD34 and transcription factors Ihh, SCL, GATA-1, GATA-2 and PU. 1 in the yolk sac, and to learn about the role and relationships of FGF1, hematopoietic cells and transcription factors during embryonic hematopoiesis.</p><p><b>METHODS</b>10 microm sections and total RNA were prepared from 107 human embryos aged 3-12 weeks. Immunohischemical SP staining and RT-PCR were performed.</p><p><b>RESULTS</b>The yolk sac blood islands of human 3 approximately 12 weeks embryos consisted of peripheral vascular endothelial cells and central hematopoietic cells. The expression of FGF1 was firstly found in visceral mesoderm around periphery of yolk sac blood island at day 16, while was little inside it. KDR was not or lowly expressed and CD34 and CD133 were not expressed then. The expression increased, gray value decreased and staining enhanced at day 21. Strong staining of CD34+, CD133+ and KDR+ cells were found in blood island and mesoderm at day 30, their gray values changed from 156 +/- 16, 173 +/- 18 and 160 +/- 14 to 53 +/- 7, 52 +/- 6 and 69 +/- 8 respectively. FGF1 expression was strong positive, the gray value declined dramatically from 161 +/- 13 to 40 +/- 5. Some positive cells formed vessel-like structure along the periphery of blood island. Moderate expression of CD34+, CD133+, KDR+ cells increased at day 45, the cells aggregated into mass in blood island and FGF1+ cells did the same in blood island, while little in mesoderm. Its gray valve was increased. After 7 weeks, CD133+, KDR+, CD34+ cells significantly decreased their gray values increased, the staining became week. FGF1 was weakly expressed in yolk sac and its gray value increased to 179 +/- 22. RT-PCR showed Ihh, SCL, GATA-1 and GATA-2 were expressed at different time in yolk sac. PU. 1 were not expressed at day 16, and then expressed.</p><p><b>CONCLUSIONS</b>The hematopoietic properties of yolk sac may be dependent on signaling through FGF receptors and FGF1 plays an important role in hematopoietic stem cell homeostasis. The FGF pathway regulates primitive hematopoiesis by modulating transcription factors such as Gata1 expression level and activity.</p>
Assuntos
Humanos , Embrião de Mamíferos , Metabolismo , Fisiologia , Fator 1 de Crescimento de Fibroblastos , Metabolismo , Hematopoese , Técnicas In Vitro , Saco Vitelino , Metabolismo , FisiologiaRESUMO
A new type wound dressing-aFGF/collagen composite sponge was prepared by bovine tendon and aFGF. Its physical function, biocompatibility, and hemocompatibility in particular, were studied for full assurance of its biosafety. The acute toxicity and skin irritation tests of composite sponge of high and low doses were negative. Recalcification test demonstrated that the recalcification time of composite sponge was much longer than that of the control group. Hemolysis test revealed that the composite sponge did not lead to hemolysis. Platelet adhesion test showed that the surface of composite sponge had less platelet adhesion than did the surface of glass, and the composite sponge did not destroy platelets. The results indicate that aFGF/collagen composite sponge has good biocompatibility and possibility for clinical use.
Assuntos
Animais , Feminino , Masculino , Camundongos , Coelhos , Materiais Biocompatíveis , Química , Curativos Biológicos , Colágeno Tipo I , Química , Fator 1 de Crescimento de Fibroblastos , Química , Membranas Artificiais , Testes de ToxicidadeRESUMO
Angiogenesis gene therapy has long been sought as a novel alternative treatment for restoring the blood flow and improving the contractile function of the ischemic heart in selected clinical settings. Angiogenic fibroblast growth factor-1 [FGF-1] is a promising candidate for developing a promising gene therapy protocol due to its multipotent ability to stimulate endothelial cell [EC] growth, migration, and tube formation. Despite these advantages, however, FGF gene therapy has suffered setbacks mainly due to the inefficient delivery rate of the growth factor in vivo. Given the potent angiogenic effect of FGF-1, we reasoned that constitutively synthesized minute quantities of this polypeptide hormone, when empowered with the ability to escape the cellular constraint, could freely act in a paracrine/autocrine fashion on nearby existing capillary plexuses and lead to neovascularization and restoration of the blood flow to ischemic tissues for reparative purpose. We report the direct gene transfer of a retroviral-based mammalian expression vector encoding a secreted form of FGF-1 [sp-FGF-1] for the purpose of therapeutic angiogenesis into the porcine myocardium subjected to the surgical placement of an ameroid occluder to induce the chronic coronary occlusion of the left circumflex coronary artery [LCx] and regional myocardial ischemia. Coronary angiography, performed 3 weeks after surgery, confirmed the interruption of the blood flow in the LCx distal to the site of ameroid placement.: Immunohistochemical analysis using antibody specific to von Willebrand factor [vWF], an endothelial marker, showed a significant increase [p < 0.05] in myocardial vascularity in the sp-FGF-1 hearts compared to the control [vector alone]. Importantly, an assessment of the cardiac function by echocardiography, performed 3 weeks after surgery, demonstrated improved cardiac contractility due to increased left ventricular free wall contraction in the sp-FGF-1-treated animals only. These results suggest that the intramyocardial delivery of our chimeric secretory FGF-1 gene can enhance vascularity and improve cardiac contractility in a chronic ischemic heart. This protocol may serve useful for developing reparative angiogenesis strategies aimed at improving the pumping function of the ischemic hearts in human patients
Assuntos
Animais de Laboratório , Indutores da Angiogênese , Fator 1 de Crescimento de Fibroblastos/genética , Fator 1 de Crescimento de Fibroblastos , Neovascularização Fisiológica , Isquemia Miocárdica , Trombose Coronária , Doença das Coronárias , Contração Miocárdica , Terapia Genética , Ecocardiografia , Técnicas de Transferência de Genes , Resultado do TratamentoRESUMO
Introdução: Existe pouco entendimento a respeito dos mecanismos moleculares que levam à formação dos quelóides e cicatrizes hipertróficas, assim como não existe um modelo animal adequado para seu estudo, visto que só ocorrem em humanos. A teoria mais aceita propõe que um aumento na regulação do TGF-b1 leva à formação dessas cicatrizes. Objetivo: Criar um modelo animal de quelóide utilizando células geneticamente modificadas codificando o TGF-b1. Método: Fibroblastos dérmicos humanos foram geneticamente modificados para aumentarem a expressão de TGF-b1 na forma selvagem ou latente ou na forma mutante ou ativa. Os fibroblastos transfectados foram transplantados intradermicamente em camundongos atímicos, e a formação de tecido fibroso analisada em diferentes intervalos de tempo por meio de histologia e imunohistoquímica. Resultados: Após a injeção intradérmica nos camundongos atímicos, apenas os fibroblastos expressando a forma ativa do TGF-b1 formaram nódulos quelóide-like, contendo colágeno e fibronectina. Estas estruturas persistiram microscopicamente por mais tempo que os implantes dos fibroblastos controle e os expressando a forma latente do TGF-b1. Conclusões: A injeção de fibroblastos transfectados codificando o TGF-b1 levou à formação de nódulos semelhantes ao quelóide na pele de camundongos atímicos. O TGF-b1 precisa estar em sua forma ativa para produzir os nódulos fibróticos.
Assuntos
Ratos , Cicatriz Hipertrófica , Fator 1 de Crescimento de Fibroblastos , Fibroblastos , Técnicas In Vitro , Queloide , Modelos Animais , Fator de Crescimento Transformador beta , Técnicas Genéticas , Imuno-Histoquímica , MétodosRESUMO
The expression levels and changes of endogenous acid fibroblast growth factor (aFGF) in microwave burn wound tissues were detected in order to investigate how to get better therapeutic effects by using the exogenous aFGF for repairing trauma. A burnt-wound animal model was established by NS-F II multifunction spectrum therapeutics equipment, and reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry assay were applied to detect the expression levels of endogenous aFGF mRNA in microwave burn wound tissues. The expression level of endogenous aFGF mRNA was significantly increased in the burn wound tissues 12 h after burn, reached the peak at 48 h, and gradually deceased 96 h after burn. The expression of endogenous aFGF mRNA after tissue damage was reversible, and its intensity was in accordance with the repair process of tissue damage, suggesting endogenous aFGF may take part in the cell metabolism and proliferation, and then promote the repair of the burn wound.
Assuntos
Queimaduras/metabolismo , Fator 1 de Crescimento de Fibroblastos/genética , Fator 1 de Crescimento de Fibroblastos/metabolismo , Micro-Ondas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Cicatrização/fisiologiaRESUMO
<p><b>OBJECTIVE</b>To explore measures to prevent motor endplate degeneration and muscular atrophy after motor nerve injury.</p><p><b>METHODS</b>Thirty Sprague-Dawley rats were randomized into 3 equal groups. In two of the groups, the right common peroneal nerves of the rats were transected and immediately sutured with implantation of collagen gel carrier of acidic fibroblast growth factor (aFGF) or the empty carrier into the denervated tibialis anterior muscles. In the control group, the transected nerves were sutured without implantation. Six weeks after the operation, morphological and electrophysiological examinations were performed.</p><p><b>RESULTS</b>In the control rats and those with empty collagen gel carrier implantation, obvious motor endplate degeneration and muscular atrophy occurred, which were not obvious in rats receiving aFGF carrier implantation. The decrement of repetitive nerve stimulation was significantly greater in the former two groups than in the latter.</p><p><b>CONCLUSION</b>Implantation of collagen gel carrier of aFGF may prevent motor endplate degeneration and facilitate functional recovery of the neuromuscular junction after motor nerve injury.</p>
Assuntos
Animais , Feminino , Masculino , Ratos , Eletrofisiologia , Fator 1 de Crescimento de Fibroblastos , Farmacologia , Usos Terapêuticos , Placa Motora , Ferimentos e Lesões , Denervação Muscular , Métodos , Atrofia Muscular , Patologia , Degeneração Neural , Regeneração Nervosa , Nervo Fibular , Ferimentos e Lesões , Ratos Sprague-DawleyRESUMO
<p><b>AIM</b>To compare the effects of the non-mitogenetic human acidic fibroblast growth factor (nmhaFGF) and the human acidic fibroblast growth factor (haFGF) on the proliferation and MAPK signal transduction pathway of the malignant tumor cell and to study the clinical safety of nmhaFGF.</p><p><b>METHODS</b>The mammary tumor cells (MCF-7) were treated with haFGF and nmhaFGF separately. The mitogenic activities of both haFGF and nmhaFGF were detected by MTT method and the cell cycle was analyzed by flow cytometer (FCM). The expression levels of the signal proteins, Grb2 (growth factor receptor bound 2) and ERK1/2 (extracellular signal-regulated kinase 1/2), were detected by semi-quantitative Western blotting method.</p><p><b>RESULTS</b>The mitogenic activity of nmhaFGF was obviously lower than that of haFGF. The activity of nmhaFGF was weaker than that of the haFGF. The ratio of G1/G0, G2/M of haFGF was markedly lower than that of nmhaFGF and control group, and was reverse in S phase. The expression levels of both Grb2 and ERK1/2 of the nmhaFGF treated group were lower than that of the haFGF treated group and approaching the control group.</p><p><b>CONCLUSION</b>The mitogenic activity of the nmhaFGF decreased remarkably. Its mechanism probably via down-regulation of the expression of the signal moleculars, MAPK-ERK1/2 and Grb2.</p>
Assuntos
Feminino , Humanos , Neoplasias da Mama , Metabolismo , Patologia , Ciclo Celular , Linhagem Celular Tumoral , Regulação para Baixo , Fator 1 de Crescimento de Fibroblastos , Genética , Farmacologia , Proteína Adaptadora GRB2 , Metabolismo , Proteína Quinase 1 Ativada por Mitógeno , Metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Metabolismo , Mitose , MutaçãoRESUMO
Human acidic fibreblast growth factor (haFGF) was a kind of cell growth factor with wide bio-activity on cell from mesectoderm and neuro-ectoderm.In this paper, the effect of acetate concentration on the growth and expression of recombinant human acidic fibroblast growth factor mutant system E.coli BL21(DE3)/pET3C-haFGF was investigated. Four fed-batch modes: batch-fed, batch-DO static balance, DO static balance-glucose starvation, and pH-static state were investigated. The accumulation of acetate during the fermentation course was effectively inhibited. The OD600nm value was about 22, after purification, the soluble rhaFGF yielded 450mg/L. During the fermentation, no special ways such as pure oxygen, pressure were adopted, thus the established process would be easily scaled up for industry purpose.
Assuntos
Humanos , Ácido Acético , Metabolismo , Reatores Biológicos , Microbiologia , Técnicas de Cultura de Células , Métodos , Meios de Cultura , Escherichia coli , Genética , Metabolismo , Fermentação , Fator 1 de Crescimento de Fibroblastos , Genética , Proteínas Mutantes , Genética , Proteínas Recombinantes , GenéticaRESUMO
<p><b>AIM</b>To study the effect of non-mitogenic human acidic fibroblast growth factor (nm-haFGF) on retinal injury induced by N-methyl-N-nitrosourea (MNU) in Sprague-Dawley rats and its mechanism.</p><p><b>METHODS</b>Female rats of 50-days-old were injected with MNU (60 mg x kg(-1)) intraperitoneally, and three doses of nm-haFGF (1.25 microg, 2.5 microg and 5 microg in one eye of each rat) were injected, separately, into vitreous body of one eye of each rat twice a day at 0 and 12 h after MNU treatment. 24 h later, apoptotic index of photoreceptor cells was detected by TUNEL labeling and the expressions of Bcl-2 and Bax were analyzed by Western blotting. At the 7th day, retinal injury was evaluated based on retinal thickness.</p><p><b>RESULTS</b>Compared with model group, apoptotic index of photoreceptor cells was significantly reduced in nm-haFGF groups at the dose of 1.25 microg and 2.5 microg in one eye of each rat at 24 h, and the total retinal thickness as well as the outer retinal thickness markedly increased 7 days after MNU, respectively. The expressions of Bcl-2 increased and that of Bax decreased adversely after being injected with different doses of nm-haFGF.</p><p><b>CONCLUSION</b>nm-haFGF partially suppressed retinal injury induced by MNU in Sprague-Dawley rats. The mechanism could be related to up-regulation of Bcl-2 and down-regulation of Bax.</p>
Assuntos
Animais , Feminino , Ratos , Apoptose , Fator 1 de Crescimento de Fibroblastos , Genética , Farmacologia , Metilnitrosoureia , Células Fotorreceptoras de Vertebrados , Patologia , Substâncias Protetoras , Farmacologia , Proteínas Proto-Oncogênicas c-bcl-2 , Metabolismo , Distribuição Aleatória , Ratos Sprague-Dawley , Retina , Patologia , Retinose Pigmentar , Metabolismo , Patologia , Proteína X Associada a bcl-2 , MetabolismoRESUMO
PURPOSE: Several naturally-derived biopolymers have been introduced as implantable biomaterial for tissue reconstruction, and some of them are already being applied in various clinical fields. They serve as backbones for the regeneration of damaged tissue. It is well known that growth factors play an important role in this process and some of the naturally- derived biopolymers were known to contain several kinds of growth factors, but there are few reports about any growth factors in the various biopolymers. We tried to confirm the existence of growth factor receptors and neurogenic factors through the expression of the mRNA from the biopolymers. MATERIALS AND METHODS: Bladders were harvested from adult pigs. Vesical submucosa was obtained with using mechanical decellulization technique. Acellular matrices were made from pig bladder with using a cell lysis technique, and they were examined by performing scanning electron microscopy. Expression of the mRNA of vascular endothelial growth factor (VEGF) receptor, fibroblast growth factor (FGF)-1 receptor, neuregulin and brain-derived neurotrophic factor (BDNF) in the acellular matrix and the submucosa of porcine bladder were examined for via reverse transcription- polymerase chain reaction (RT-PCR). RESULTS: The ultrastructure of the acelluar matrix shows collagen fibers with many pores. The mRNA's of VEGF receptor, FGF-1 receptor and neuregulin were expressed in the porcine vesical acellular matrix, whereas BDNF was not expressed. On the other hand, none of the mRNA's examined was expressed in the porcine vesical submucosa. CONCLUSIONS: This study's results shows that several growth factors are detected in the acellular matrix of the porcine bladder. These growth factors may play an important role for the growth of tissue when it is implanted in vivo. It is also assumed that there are more growth factors in naturally-derived biopolymers, and it would be useful to investigate the undefined components of naturally-derived biopolymers for developing new and advanced polymers.