RESUMEN
The highly virulent PRRSV isolate strain HN-1/06 was cultivated on Marc-145. To study the viral entry mechanisms, the GP5 gene of PRRSV isolate was amplified by RT-PCR and cloned into pcDNA3.0 to generate the expressing plasmid pcDNA-GP5. pcDNA-GP5 was transfected into 293T by the calcium phosphate precipitation method. Analysis of flow cytometry confirmed that the GP5 proteins were expressed in surface of the 293T cells. Then 293T cells were transfected with pcDNA-GP5, pHIT60 and pHIT111 plasmids to generate pseudotyping virus. The pseudotyping virus supernatant was harvested 48 hours post-transfection and was detected by Western blotting and infection assay. Western blotting indicated that the GP5 glycoproteins were incorporated into the retroviral pseudotyped virus. Infection assay showed that the pseudotyped virus infected 293T and Mark-145 cell. The pseudotyped virus could be used to further study infectious mechanism of PRRSV.
Asunto(s)
Animales , Ratones , Línea Celular , Clonación Molecular , Células Endoteliales , Biología Celular , Metabolismo , Virología , Virus de la Leucemia Murina , Genética , Metabolismo , Virus del Síndrome Respiratorio y Reproductivo Porcino , Química , Genética , Proteínas Recombinantes , Genética , Porcinos , Transfección , Proteínas del Envoltorio Viral , Genética , Virión , Genética , MetabolismoRESUMEN
Objective:To investigate the feasibility of using marrow stromal cells (MSC) as seed cells and three-dimensional porous nano-hydroxylapatite as scaffolds for constructing tissue-engineered bone.Methods:MSC from rat were cultured and induced to differentiate into osteoblasts in vitro. And then these induced cells were identified and seeded onto three-dimensional porous nano-hydroxylapatite scaffolds, cultured for 15 days in vitro. Scanning electron microscopy was used to evaluate the growth of these induced cells on three-dimensional porous nano-hydroxylapatite scaffolds. Results: The activity of alkaline phosphatase (ALP) and the secretion of osteocalcin of MSC from rat appeared and were increased gradually along the culture. The cells seeded on three-dimensional porous nano-hydroxylapatite scaffolds could adhere and proliferate well, and come into being many tiny calcium nodules and collagenous fibers. Conclusion:The data demonstrated that the new developed culture method is conducive to MSC’s differentiating and proliferating into osteoblasts that have a fine activity and the three-dimensional porous nano-hydroxylapatite may be considered as a suitable scaffold for the seeded cells. Using marrow stromal cells as seed cells and three-dimensional porous nano-hydroxylapatite as scaffolds is advantageous for constructing tissue-engineered bone.
RESUMEN
Objective The purpose of this study was to develop a new cultural method, so that the rat bone marrow stromal cells (MSCs) can well differentiate into osteoblasts in vitro and the cultured osteoblasts can be identified. Methods MSCs of rats were isolated and cultured, then the cultured cells were identified with inverted microscopy and undergone an HE stain for observing the morphology of living cells, and the cells were histochemically stained for examining the activity of cellular alkaline phosphatase (ALP), as well as were radio-immunologically analyzed for detecting the secretion of osteocalcin. Results The MSCs were typical fibroblast-like and possess a strang proliferative capability after cultured in vitro.The cellular ALP activity and osteocalcin secretion achieved the peak value from the fortieth day and maintained the status until the sixtieth day after cultured in vitro.Conclusion The data demonstrated that the new developed culture method has the advantages of short time, less risk of contamination and higher efficiency, and conducive to MSC's differentiating and proliferating into osteoblasts that do function well.
RESUMEN
In order to screen genes transregulated by core protein of hepatitis C virus, cDNA microarray technology was employed to detect the gene expression change between HepG2 cells transfected with pcDNA3 1(-) core and the empty vector, respectively. Among 1152 genes, there were 95 genes with different expressions, of which 45 genes were upregulated and 50 genes were downregulated in HepG2 cells transfected with core protein expression plasmid. These genes transregulated by HCV core protein included human genes encoding proteins involved in cell proliferation, differentiation, apoptosis, signal transduction and immune regulation. Therefore, the results provided some new clues for further clarifying the molecular biology mechanism of pathogenesis and tumorigenesis of HCV core protein