RESUMEN
miR-122 is the most abundant miRNA in adult human liver. To study the functions of miR-122 in liver disease, we amplified the precursor of human miR-122 gene by polymerase chain reaction (PCR) from the HepG2 genomic DNA, and then constructed miR-122 expression vector pLMP-miR-122. pLMP-miR-122 could overexpress mature miR-122 when human normal liver cells L-O2 and the hepatoma cells HepG2 were transfected with it. When HepG2 cells were co-transfected with HBV1.3 and pLMP-miR-122, we found that miR-122 could down-regulate the expression of HBs and HBe antigen. These results showed that the human liver specific miR-122 expression vector was constructed successfully, and it could regulate the replication and the expression of HBV genes. The plasmid pLMP-miR-122 will facilitate further studies of the functions of miR-122 in the development of liver virus infection diseases and HCC.
Asunto(s)
Humanos , Secuencia de Bases , Carcinoma Hepatocelular , Genética , Patología , Línea Celular Tumoral , Vectores Genéticos , Genética , Hígado , Metabolismo , Neoplasias Hepáticas , Genética , Patología , MicroARNs , Genética , Metabolismo , Datos de Secuencia Molecular , Plásmidos , Genética , Reacción en Cadena de la Polimerasa , MétodosRESUMEN
We cloned human U6 promoter from pAVU6 + 27 vector into pXSN to transcripe small RNA. Meanwhile, a shRNA targeting GDF-8 was cloned down-stream of the hU6 promoter to construct recombinant vector. Then the packing cell GP-293 was co-transfected the recombinant with pVSV-G to gernarate virus particle. Resistant C2C12 cell pools were screened using G418. Levels of mRNA and protein of GDF-8 were tested by Real-Time PCR and western blotting. Cell proliferation and cell cycle were analyzed using MTT and FACS. The expression of GDF-8 was dramatically decreased by the retrovirus-based system in C2C12 cells. Cells proliferated effectively after integrating the recombinant. The cells in G0/G1 phase decreased by 13.7%, while cells in S phase increased by 14.9%. In conclusion, the retrovirus-based RNAi could be used to stably silence GDF-8. It can be a powerful tool in curing muscle atrophy.
Asunto(s)
Humanos , Proliferación Celular , Células Cultivadas , Regulación hacia Abajo , Regulación de la Expresión Génica , Genética , Mioblastos , Biología Celular , Metabolismo , Miostatina , Genética , Interferencia de ARN , ARN Mensajero , Metabolismo , ARN Interferente Pequeño , Genética , Retroviridae , Genética , MetabolismoRESUMEN
This paper reflected Physician-Patient Relationship from the "Jing Liang-affair" and concluded four views:Part of medical professionals also belong to vulnerable groups,medical organizations belong to strong groups;compared to those reasonless patients who have over expectations on medicine,medical professionals belong to vulnerable groups too;The medical system should be the root of the deterioration of Physician-Patient Relationship;The burnout of medical professionals is very serious.