RESUMEN

BACKGROUND/AIMS: Renal vascular injury accounts for the poor outcomes of patients with IgA nephropathy (IgAN). In this study, we investigated whether endostatin, a potent inhibitor of angiogenesis, is associated with IgAN. METHODS: Serum endostatin levels were detected in patients with IgAN, disease controls, and healthy controls, and the correlation among endostatin and clinicopathologic manifestations, as well as prognosis in patients with IgAN, was analyzed. In addition, serum endostatin levels were compared in patients "before" and "after" treatment. Data on endostatin expression in the renal interstitium of patients with IgAN were downloaded and analyzed from the GSE35489 array in the GEO database. The poly-IgA1 (pIgA) immune complex is widely recognized as the "trigger" of IgAN initiation. pIgA in the plasma of patients was extracted and used to stimulate human glomerular endothelial cells (GECs). Endostatin, IL-6, and CXCL1 in the cell supernatant were detected by ELISA kits. RESULTS: We found that serum endostatin levels were significantly increased in patients with IgAN, as was endostatin expression in the renal interstitium. Patients with IgAN were divided into 2 groups according to the median value. The high endostatin expression group had significantly higher levels of serum creatinine and BUN and more severe tubular/interstitial damage. Moreover, patients with arteriolar injury and endothelial cell proliferation had higher serum endostatin levels. Patients with high serum endostatin levels had poor prognosis. According to the in vitro experiment, the GEC apoptosis rate and the supernatant levels of endostatin, IL-6, and CXCL1 were significantly increased following pIgA stimulation. CONCLUSION: Our study found that elevated endostatin expression was associated with disease severity and poor prognosis in patients with IgAN and can be upregulated by pIgA, but how it participates in the pathogenesis of IgAN deserves further exploration.

Asunto(s)

Endostatinas/sangre , Glomerulonefritis por IGA/sangre , Inmunoglobulina A/sangre , Adulto , Células Cultivadas , Endostatinas/inmunología , Femenino , Glomerulonefritis por IGA/diagnóstico , Glomerulonefritis por IGA/inmunología , Glomerulonefritis por IGA/patología , Humanos , Inmunoglobulina A/inmunología , Riñón/inmunología , Riñón/patología , Masculino , Persona de Mediana Edad , Pronóstico , Índice de Severidad de la Enfermedad , Adulto Joven

RESUMEN

PEGylated recombinant human endostatin (M2ES) exhibited prolonged serum half-life and enhanced antitumor activity when compared with endostatin. A pre-clinical study was performed to evaluate the safety of M2ES in rats. After intravenous (IV) infusions of M2ES at a dose level of 3, 15 and 75 mg/kg in Sprague Dawley (SD) rats, M2ES was well tolerated in animals, with no observable changes in clinical observation, body weight, food consumption, urine analysis, hematology and serum biochemical analysis. The increase of kidney weights, and slight to severe vacuolation and necrosis of proximal tubule epithelial cells in kidney were observed in 15 and 75 mg/kg M2ES groups, but this adverse-effect was reversible. In summary, the major toxicity target organ of M2ES might be kidney, and the no observed adverse effect level (NOAEL) of M2ES in rats was 3 mg/kg in this study. These pre-clinical safety data contribute to the initiation of the ongoing clinical study.

Asunto(s)

Endostatinas/toxicidad , Polietilenglicoles/toxicidad , Animales , Anticuerpos/sangre , Evaluación Preclínica de Medicamentos , Endostatinas/química , Endostatinas/inmunología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Femenino , Humanos , Túbulos Renales Proximales/citología , Masculino , Necrosis/inducido químicamente , Nivel sin Efectos Adversos Observados , Polietilenglicoles/química , Ratas Sprague-Dawley , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/toxicidad , Pruebas de Toxicidad Subcrónica

RESUMEN

Corneal transplantation is the most common solid organ transplantation. The immunologically privileged nature of the cornea results in high success rates. However, T cell-mediated rejection is the most common cause of corneal graft failure. Using antiangiogenesis treatment to prevent corneal neovascularization, which revokes immune privilege, prevents corneal allograft rejection. Endostatin is an antiangiogenic factor that maintains corneal avascularity. In this study, we directly test the role of antiangiogenic and immunological signals in corneal allograft survival, specifically the potential correlation of endostatin production and T cell recruitment. We report that 75% of the corneal allografts of BALB/c mice rejected after postoperative day (POD) 20, whereas all syngeneic grafts survived through POD60. This correlates with endogenous endostatin, which increased and remained high in syngeneic grafts but decreased after POD10 in allografts. Immunostaining demonstrated that early recruitment of allospecific T cells into allografts around POD10 correlated with decreased endostatin production. In Rag(-/-) mice, both allogeneic and syngeneic corneal grafts survived; endostatin remained high throughout. However, after T cell transfer, the allografts eventually rejected, and endostatin decreased. Furthermore, exogenous endostatin treatment delayed allograft rejection and promoted survival secondary to angiogenesis inhibition. Our results suggest that endostatin plays an important role in corneal allograft survival by inhibiting neovascularization and that early recruitment of allospecific T cells into the grafts promotes destruction of endostatin-producing cells, resulting in corneal neovascularization, massive infiltration of effector T cells, and ultimately graft rejection. Therefore, combined antiangiogenesis and immune suppression will be more effective in maintaining corneal allograft survival.

Asunto(s)

Neovascularización de la Córnea/inmunología , Trasplante de Córnea , Endostatinas/metabolismo , Rechazo de Injerto/inmunología , Linfocitos T/inmunología , Traslado Adoptivo , Inhibidores de la Angiogénesis/farmacología , Animales , Córnea/inmunología , Córnea/metabolismo , Neovascularización de la Córnea/metabolismo , Endostatinas/inmunología , Endostatinas/farmacología , Femenino , Rechazo de Injerto/metabolismo , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la Polimerasa , Trasplante Homólogo

RESUMEN

We have recently demonstrated that a 4-in-1 gene therapy strategy that contains two anti-angiogenic genes [endostatin and pigment epithelium-derived factor] and two cytokine genes [granulocyte macrophage colony-stimulating factor and interleukin 12] has a considerable antitumor effect on large tumors in a woodchuck hepatoma model. The current study further investigates the underlying mechanisms for the antitumor effect observed by using small rodent models. We found that immunotherapy alone increased immunosuppressive cells in large tumors over time, whereas the anti-angiogenic therapy contained in the 4-in-1 strategy alleviated immunosuppression and made tumors vulnerable to immunotherapy, thus resulting in a synergistic antitumor effect.

Asunto(s)

Endostatinas/genética , Endostatinas/inmunología , Proteínas del Ojo/genética , Proteínas del Ojo/inmunología , Terapia Genética/métodos , Inmunoterapia/métodos , Neoplasias Hepáticas Experimentales/terapia , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/inmunología , Serpinas/genética , Serpinas/inmunología , Adenoviridae/genética , Animales , Apoptosis/genética , Apoptosis/inmunología , Línea Celular Tumoral , Terapia Combinada , Endostatinas/biosíntesis , Proteínas del Ojo/biosíntesis , Humanos , Neoplasias Hepáticas Experimentales/irrigación sanguínea , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Ratones Endogámicos BALB C , Neovascularización Patológica/genética , Neovascularización Patológica/inmunología , Neovascularización Patológica/terapia , Factores de Crecimiento Nervioso/biosíntesis , Serpinas/biosíntesis , Linfocitos T Reguladores/inmunología , Microambiente Tumoral/inmunología

RESUMEN

We report the preparation and characterization of a matrix-free carboxylated surface plasmon resonance (SPR) sensor chip with high sensing efficiency by functionalizing a bare gold thin film with a self-assembled monolayer of 16-mercaptohexadecanoic acid (SAM-MHDA chip). The self assembled monolayer surface coverage of the gold layer was carefully evaluated and the SAM was characterized by infrared reflection absorption spectroscopy, X-ray photoemission spectroscopy, atomic force microscopy, X-ray reflectivity-diffraction, and SPR experiments with bovine serum albumin. We compared the SPR signal obtained on this chip made of a dense monolayer of carboxylic acid groups with commercially available carboxylated sensor chips built on the same gold substrate, a matrix-free C1 chip, and a CM5 chip with a ~100 nm dextran hydrogel matrix (GE Healthcare). Two well-studied interaction types were tested, the binding of a biotinylated antibody (immunoglobulin G) to streptavidin and an antigen-antibody interaction. For both interactions, the well characterized densely functionalized SAM-MHDA chip gave a high signal-to-noise ratio and showed a gain in the availability of immobilized ligands for their partners injected in buffer flow. It thus compared favourably with commercially available sensor chips.

Asunto(s)

Ácidos Carboxílicos/química , Oro/química , Ácidos Palmíticos/química , Resonancia por Plasmón de Superficie/instrumentación , Animales , Antígenos/inmunología , Biotinilación , Bovinos , Endostatinas/análisis , Endostatinas/inmunología , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Microscopía de Fuerza Atómica , Unión Proteica , Albúmina Sérica Bovina/análisis , Albúmina Sérica Bovina/metabolismo , Estreptavidina/metabolismo , Propiedades de Superficie , Espectroscopía de Absorción de Rayos X

RESUMEN

BACKGROUND: Although therapeutic angiogenesis is a most promising strategy for the treatment of myocardial infarction (MI), it remains unknown if and how endogenous angiogenesis inhibitors, such as endostatin, regulate angiogenesis in MI. In the present study the role of endostatin in left ventricular (LV) remodeling and heart failure was tested in a rat MI model. METHODS AND RESULTS: When exposed to hypoxia, rat cardiomyocytes showed increased expression of endostatin. After MI induction in the rat MI model, endostatin expression was upregulated in cardiomyocytes, and serum endostatin levels were significantly elevated. Anti-endostatin antibody treatment resulted in significantly higher mortality of MI rats than controls. The MI rats with endostatin neutralization displayed adverse LV remodeling and severe heart failure compared with control MI rats. Although angiogenesis was increased, tissue remodeling and interstitial fibrosis were further exaggerated in post-MI hearts by endostatin neutralization. Furthermore, the expression and protease activity of matrix metalloproteinases -2 and -9, and of angiotensin-converting enzyme were markedly elevated by endostatin neutralization. CONCLUSIONS: Neutralization of endostatin worsens the symptoms and outcomes of MI in a rat model. The results imply that endogenous endostatin/collagen XVIII may suppress aberrant LV remodeling and heart failure after MI. (Circ J 2010; 74: 109 - 119).

Asunto(s)

Colágeno Tipo XVIII/antagonistas & inhibidores , Endostatinas/antagonistas & inhibidores , Insuficiencia Cardíaca/fisiopatología , Infarto del Miocardio/fisiopatología , Remodelación Ventricular/fisiología , Animales , Células Cultivadas , Colágeno Tipo XVIII/inmunología , Colágeno Tipo XVIII/fisiología , Modelos Animales de Enfermedad , Endostatinas/inmunología , Endostatinas/fisiología , Insuficiencia Cardíaca/metabolismo , Inmunoglobulina G/farmacología , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Infarto del Miocardio/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/fisiología , Peptidil-Dipeptidasa A/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Wistar

RESUMEN

AIM: To determine the in vitro and in vivo bioactivity of recombinant human endostatin (rhEndostatin) and to analyze its pharmacokinetics and immunogenicity in rhesus monkeys and patients. METHODS: The physical chemical characteristics of rhEndostatin were detected according to Pharmacopoeia of the People's Republic of China (2005 edition, part III). Its in vitro and in vivo bioactivities were assayed via proliferation-inhibition on human umbilical vein endothelial cells and their inhibitory effect on tumor-bearing mice models. Serum concentrations of rhEndostatin in monkeys and patients were determined by an enzyme immunoassay method. RESULTS: The corresponding specific in vitro activities of rhEndostatin obtained from the cell counting method, 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and lactate dehydrogenase assay, respectively, were 6.4 x 10(7), 6.7 x 10(7), and 3.8 x 10(8) U/mg, and the in vivo antitumoral potency was 4.04 x 10(7) U/mg. In rhesus monkeys, there were no gender differences in all pharmacokinetic parameters. Serum anti-rhEndostatin immunoglobulin (Ig)G antibodies were generated quickly after intravenous (iv) administration and decreased rapidly when therapy was stopped. In phase I clinical trials, linearity in the pharmacokinetics of rhEndostatin was indicated by dose-proportionate increases in the area under the curve and the maximum serum concentration. Serum rhEndostatin reached a steady-state level after 7 d of successive administration with the average concentration at a steady state of 272.44+/-91.98 ng/mL. Neither IgG nor IgM antibodies against rhEndostatin were observed in patients. CONCLUSION: RhEndostatin exhibited a definite proliferation- inhibition effect on HUVEC, and significant antitumoral activity in mice. The immunoreactivity of rhesus monkeys to rhEndostatin is common, and rhEndostatin showed no immunogenicity in patients in this trial. The results provide a basis for further clinical trials.

Asunto(s)

Inhibidores de la Angiogénesis/uso terapéutico , Antineoplásicos/uso terapéutico , Endostatinas/uso terapéutico , Inhibidores de la Angiogénesis/inmunología , Inhibidores de la Angiogénesis/farmacocinética , Animales , Antineoplásicos/inmunología , Antineoplásicos/farmacocinética , Endostatinas/inmunología , Endostatinas/farmacocinética , Células Endoteliales/efectos de los fármacos , Femenino , Humanos , Macaca mulatta , Masculino , Ratones , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapéutico , Caracteres Sexuales , Sales de Tetrazolio , Tiazoles

RESUMEN

PURPOSE: The half-life of the antiangiogenic molecule endostatin that has been used in clinical trial is short ( approximately 2 h). In addition, approximately 50% of the clinical grade endostatin molecules lack four amino acids at their NH(2) termini. Lack of these amino acids gives rise to a molecule that is devoid of zinc, resulting in no antitumor activity. Our goal was to develop a new version of endostatin that does not show such deficiency. EXPERIMENTAL DESIGN: A recombinant human endostatin conjugated to the Fc domain of IgG was constructed and expressed in mammalian cell culture. The presence of Fc has been shown by previous investigators to play a major role in increasing the half-life of the molecule. Fc-endostatin was tested in tumor-bearing mice, and its half-life was compared with the clinical grade endostatin. RESULTS: The antitumor dose of Fc-endostatin was found to be approximately 100 times less than the clinical grade endostatin. The half-life of Fc-endostatin in the circulation was found to be weeks rather than hours, as observed for endostatin alone. In addition, a U-shaped curve was observed for antitumor activity of endostatin as a function of endostatin concentration delivered to the animals. CONCLUSION: Fc-endostatin is a superior molecule to the original clinical endostatin. Due to its long half-life, the amount of protein required is substantially reduced compared with the clinically tested endostatin. Furthermore, in view of the U-shaped curve of efficacy observed for endostatin, we estimate that the requirement for Fc-endostatin is approximately 700-fold less than endostatin alone. The half-life of endostatin is similar to that of vascular endothelial growth factor-Trap and Avastin, two other antiangiogenic reagents. We conclude that a new clinical trial of endostatin, incorporating Fc, may benefit cancer patients.

Asunto(s)

Endostatinas/inmunología , Endostatinas/farmacocinética , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Melanoma Experimental/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Recombinantes de Fusión/farmacocinética , Animales , Apoptosis , Ensayo de Inmunoadsorción Enzimática , Semivida , Humanos , Técnicas para Inmunoenzimas , Etiquetado Corte-Fin in Situ , Masculino , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Mutación/genética , Neoplasias Pancreáticas/patología , Proteínas Recombinantes/uso terapéutico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

PURPOSE: We evaluated the effects of celecoxib treatment on tumor-infiltrating lymphocyte (TIL) subsets [CD3(+), CD4(+),CD8(+), CD25(+), and T cell receptor (TCR)-zeta-expressing cells] and tryptase-positive mast cells in cervical tumors. Circulating levels of cytokines [interleukin (IL)-1beta, IL-10, tumor necrosis factor-alpha, IL-6, and IL-12] and angiogenesis-modulating factors (vascular endothelial growth factor and endostatin) have also been analyzed. EXPERIMENTAL DESIGN: Cervical tumor biopsies and blood samples were obtained at the time of diagnosis and after 10 days of celecoxib treatment (400 mg b.i.d., at 8:00 a.m. and 8:00 p.m.) in 27 cases. Immunohistochemistry and ELISA assays were used to assess the expression of biological factors in tumor tissue and circulating levels of cytokines and angiogenic molecules. RESULTS: We showed a statistically significant increase in the percentage of TIL expressing the TCR-zeta chain after celecoxib treatment: indeed, in cases exposed to celecoxib, the percentage of TCR-zeta(+) cells ranged from 5.0 to 50.0 (median, 22.5) with respect to baseline expression (range, 3.0-50.0; median, 10.0; P = 0.0016). There was no significant treatment-related difference in the percentage of CD3(+), CD4(+), CD8(+), and CD25(+) TIL as well as in tryptase-positive cells. IL-12 levels were significantly reduced in posttreatment samples with respect to baseline levels (P = 0.002). We also found a reduction in the circulating levels of vascular endothelial growth factor, and a statistically significant increase of serum endostatin levels (P = 0.035). CONCLUSIONS: We reported the first evidence in humans that celecoxib restores zeta expression by TIL in primary cervical tumors, suggesting that a positive modulation of immune function may serve as an additional mechanism supporting the antitumor effect of this class of drugs.

Asunto(s)

Regulación de la Expresión Génica/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Pirazoles/farmacología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Sulfonamidas/farmacología , Neoplasias del Cuello Uterino/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Celecoxib , Citocinas/efectos de los fármacos , Citocinas/metabolismo , Endostatinas/efectos de los fármacos , Endostatinas/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Regulación de la Expresión Génica/genética , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Cooperación del Paciente , Pirazoles/efectos adversos , Sulfonamidas/efectos adversos , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/inmunología , Neoplasias del Cuello Uterino/diagnóstico , Neoplasias del Cuello Uterino/tratamiento farmacológico , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/inmunología

RESUMEN

Circulating autoantibodies to self-antigens overexpressed by cancer cells are common in cancer patients. As specific proteins are expressed during neoangiogenesis, a similar phenomenon might occur with particular antigens of tumour vessels. Collagen XVIII, from which endostatin is cleaved, is highly expressed in the perivascular basement membrane of tumour-associated blood vessels and autoantibodies to endostatin have been reported in cancer patients. The present study analyses the incidence of naturally occurring autoantibodies to endostatin in the sera of breast cancer patients and their relation to endostatin serum levels and patient clinical outcome. Serum samples from 36 patients with localised breast cancer and 59 patients with a fully documented history of metastatic breast cancer were used. The immunoreactivity of serum samples was tested against purified recombinant human endostatin and endostatin levels were determined by immunoassay. We could detect anti-endostatin antibodies in the sera of 66% of the patients with localised disease and 42% of the patients with metastatic disease (P=0.03). There was no correlation between the presence of antibodies to endostatin and circulating levels of endostatin. The detection of autoantibodies to endostatin was associated with better prognosis in metastatic breast cancer patients (median survival time: 20 vs 8 months, P = 0.03), as was the presence of low levels of serum endostatin (median survival time: 20 vs 9 months, P = 0.007). These results show that a natural immune reaction against endostatin can occur in breast cancer patients. This could have important therapeutic implications with regard to endostatin therapy and raises the question of a possible role of this humoral reaction against endostatin in the neoplastic process.

Asunto(s)

Autoanticuerpos/análisis , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Endostatinas/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Formación de Anticuerpos , Neoplasias de la Mama/sangre , Neoplasias de la Mama/genética , Femenino , Predisposición Genética a la Enfermedad , Humanos , Persona de Mediana Edad , Metástasis de la Neoplasia , Pronóstico , Análisis de Supervivencia

RESUMEN

Endostatin, a 20-kDa proteolytic fragment of collagen XVIII, is a potent inhibitor of angiogenesis and tumor growth. The anti-angiogenic effects of endostatin include inhibition of endothelial cell migration and proliferation, and inhibition of the activity of MMP2. Structure-function analysis of endostatin that implies this contravention function buried in separate fragments of endostatin introduces new issues into the understanding of the structure-function relationship of endostatin. We developed and characterized a novel murine MAb, 4E7, to human endostatin, which antagonizes the function of endostatin. As we show here, MAb 4E7 blocks the anti-migration/adhesion effects of endostatin in vitro and the anti-angiogenesis effect of endostatin in vivo, but the inhibition effect of endostatin on endothelial cell proliferation is not affected by MAb4E7. These results suggest that the anti-migration and anti-proliferation functions of endostatin may have distinct structural foundations.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Endostatinas/antagonistas & inhibidores , Endostatinas/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Neovascularización de la Córnea/inducido químicamente , Endostatinas/genética , Endostatinas/metabolismo , Células Endoteliales , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoprecipitación , Ratones , Ratones Endogámicos BALB C , Hidróxido de Sodio/toxicidad , Relación Estructura-Actividad

RESUMEN

Intravenous endostatin gene transfection results in tumor suppression in a murine pulmonary metastasis model. We transfected the endostatin gene at different times, in order to achieve an optimal protective effect. pST2-Endo encoding murine endostatin was injected in a complex with cationic lipid. Pulmonary metastases were caused by intravenous injection of murine fibrosarcoma cells. Mice were observed for 14 days following fibrosarcoma cell inoculation (FSI). In the study groups, the animals were transfected with pST2-Endo at three different times: 2 days before and 3 and 7 days after FSI. In the group transfected with pST2-Endo 2 days before FSI, the weights of the lungs and tumor-occupied area ratio were significantly less than in the other groups. Significant inhibition of tumor neovascularization was documented by means of CD31 immunohistochemistry. The effect of repeated endostatin transfection on survival after FSI was determined. Animals repeatedly transfected with the endostatin gene survived significantly longer than the groups treated with a single endostatin gene transfection. A stable endostatin-expressing fibrosarcoma transfectant was created and tested for migration and invasion. Compared with controls, endostatin expression reduced migration and invasion by 15%. It is concluded that endostation gene transfection before FSI and repeated transfection thereafter results in significant tumor suppression.

Asunto(s)

Endostatinas/genética , Fibrosarcoma/genética , Terapia Genética , Lípidos , Neoplasias Pulmonares/genética , Transfección , Animales , Supervivencia Celular/genética , Endostatinas/biosíntesis , Endostatinas/inmunología , Fibrosarcoma/patología , Fibrosarcoma/terapia , Vectores Genéticos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Masculino , Ratones , Ratones Endogámicos C3H , Neovascularización Patológica/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/biosíntesis , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/inmunología , Transfección/métodos

RESUMEN

AIM: To prepare monoclonal antibody against human endostatin for the in depth study on the mechanism of anti-tumor effect of endostatin. METHODS: Monoclonal antibody specific for endostatin was prepared using hybridoma technique and screened with ELISA. Ascites fluids were produced in BALB/c mice following in sequentical intraperitoneal injection of pristine and hybridoma cells. The mAb was purified by affinify chromatography with protein A sepharose CL-4B. RESULTS: One hybridoma cell line 4E7 was established, which could produce mAb against human endostatin. The titers of mAb 4E7 in culture supernatant and ascites fluid were 1:128-1:516 and 1:10(-4)-1:10(-6), respectively. The mAb 4E7 belonged to IgG1 (lambda type). Western blot analysis showed that the mAb 4E7 could react to the human endostatin expressed by yeast and E.coli, but it had no cross reaction to other cytokines such as bFGF. CONCLUSION: The mAb 4E7 is able to react specifically with human endostatin and can be used for further investigation.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Endostatinas/inmunología , Animales , Especificidad de Anticuerpos , Femenino , Humanos , Hibridomas/inmunología , Ratones , Ratones Endogámicos BALB C