RESUMEN
BACKGROUND: Factor VII (FVII) is a plasma glycoprotein that participates in the coagulation process leading to generation of fibrin. It is converted to factor VIIa that plays an important role in the coagulation cascade. The aim of this study was isolating and cloning the genes of human factor VII and hepsin and subsequent co-transfection of the constructs to Chinese hamster ovary (CHO) cell line to obtain rFVIIa. METHODS: Factor VII and hepsin cDNAs were isolated from HepG2 cell line and cloned into pcDNA3.1 (+) vector. The constructs were co-transfected to CHO cell line. A cell line that permanently expressed recombinant factor VII (rFVII) and hepsin was established. The expression of rFVII was confirmed by reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay and Western blot analysis. Biological activity of rFVII was evaluated by prothrombin time assay. RESULTS: The results showed that the genes of FVII and hepsin were successfully cloned and expressed. Stable CHO cells co-transfected with pcNDA3.1-FVII and pcNDA3.1-hepsin expressed FVII and hepsin mRNA, but there was no expression in the CHO cells transfected with insert free pcDNA3.1. FVIIa protein was secreted to medium of CHO cells co-transfected with pcNDA3.1-FVII and pcNDA3.1-hepsin. The expected band of rFVII was detected in Western blot analysis. A three- to fourfold decrease in clotting time was observed when human FVII-depleted plasma was used in combination with human thromboplastin in the presence of rFVII, confirming the biological activity of rFVII. CONCLUSION: As we are aware, this is the first report of establishing a cell line expressing FVIIa using genetic engineering methods.
Asunto(s)
Factor VII/genética , Factor VIIa/metabolismo , Ingeniería Genética/métodos , Animales , Western Blotting , Células CHO , Línea Celular Tumoral , Clonación Molecular , Cricetinae , Cricetulus , ADN Complementario/genética , Ensayo de Inmunoadsorción Enzimática , Factor VII/biosíntesis , Factor VII/metabolismo , Humanos , Tiempo de Protrombina , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidasas/biosíntesis , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Transfección/métodos , Células Tumorales CultivadasRESUMEN
Electromagnetic fields with high energy same as ionizing radiation inserts their destructive effects via free radical production. Using antioxidants or herbal plants with antioxidants components could diminish hazardous effects of EMF. Polygonum aviculare has a high amount of phenolic and flavonoid and proved that has antioxidants effects. The aim of this study was to evaluate the effects of Polygonum aviculare herbal extract on sperm parameters after EMF exposure in mouse. Twenty four male mice, 8 weeks divided to 4 groups (one control and three experimental groups). Control group didn't receive EMF exposure. EMF group mice received 3 mT EMF during 2 months, 4 h daily and 5 days weekly. Polygonum aviculare group received 50 mg kg(-1) herbal extract during 2 months and poly -EMF group received 3 mT EMF during 2 months, 4 h daily and 5 days weekly and 50 mg kg(-1) herbal extract during 2 months. After 2 months the mice sacrificed with cervical dislocation and sperm obtained from tail of epididymis and motility and morphology of them were analyzed. Sperm analysis results showed that in group with Polygonum aviculare, morphology and motility of sperm developed (p < 0.05). Present results showed that EMF can reduce motility of sperm and treatment of Polygonum aviculare after EMF exposure developed sperm quality after EMF exposure.