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The human resistin expression vector was constructed and transfected into cells to observe its effects on glucose uptake in adipocytes,cell proliferation and differentiation.The results suggested that human resistin impaired glucose uptake in adipoeytes via stimulating proliferation of preadipocytes and suppressing adipocyte differentiation.Metformin reversed the inhibition imposed by resistin on adipogenesis.
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Objective To construct the vector for ?-galactosidase (?-gal) gene expression regulation with tetracycline-regulated gene expression system,and to control ?-galactosidase gene expression in hepG2 cells with the vector.So that offer a experimental base for regulating gene expression for liver cancer gene therpy with this system.Methods The primers for explanding two tetracycline operator (TetO2) gene according to the gene nucleotide sequences of TetO2 gene and pcDNA3.1 vector were designed.TetO2 gene was amplified by PCR with pcDNA3.1 plasmid as template.The TetO2 PCR products were cloned into pcDNA3.1 and the vector was named as pcDNA3.1-TetO2.The pcDNA3.1-Teto2 with TetO2 PCR products sequence was analyzed by ABI3130 sequencing analysis.Then ?-gal gene was cloned into pcDNA3.1-TetO2,the vector was named as pcDNA3.1-TetO2-?-gal.The pcDNA6/TR plasmid vector with tetracycline repressor(TR) was transfected into HepG2 cells by Lipotap,and the stable transfection cells were screened by blasticidin.TR gene expression was detected by RT-PCR in HepG-2 cells with and without pcDNA6/TR plasmid transfection.The pcDNA3.1-TetO2-?-gal plasmid vector was transfected into HepG2 cells with and without pcDNA6/TR plasmid transfection,and 3 to 4 d later, these transfected gene cells were treated with doxycline(4 mg?L-1).After 48 h,?-gal gene expression was detected with ?-gal cell staining.Results The pcDNA3.1-TetO2 sequencing analysis results showed that a cassette was made for a cytomegalovirus-type 2 tetracycline operator (TetO2)-TetO2 promoter in pcDNA3.1-Teto2.The double digestion reslut of pcDNA3.1-TetO2-?-gal plasmid vector demonstrated that ?-gal gene was successfully cloned into pcDNA3.1-TetO2 vector .The RT-PCR result of TR gene showed TR gene could be expressed in HepG2 cells with pcDNA6/TR plasmid transfection and TR gene didn’t express in HepG2 cells without pcDNA6/TR plasmid transfection.?-gal gene expressed in HepG2 cells without pcDNA6/TR plasmid transfection,but didn’t express in HepG2 cells with pcDNA6/TR plasmid transfection.However,?-gal gene expression could be induced with doxycline in HepG2 cells with pcDNA6/TR plasmid transfection.Conclusion The tetracycline-regulated gene expression system vector for regulating ?-gal gene expression is successfully constructed and the vector system can control ?-gal gene expression in HepG2 cells.
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AIM: To study apoptotic injury induced by aeactive oxygen species - hydrogen peroxide (H2O2) on cardiac myocytes. METHODS: Cultured rat neonatal cardiac myocytes were treated with H2O2 of various concentra- tion to observe apoptoitic injury of cardiomyocytes by agarose gel electrophoresis, Giemsa - stained smears of cell, and flow cytometry, meanwhile lactate dehydrogenas (LDH) and malondialdehyde (MDA) were determined to assess the effect of H2O2 on lipid peroxidation and permeability of the plasma membrane. RESULTS: 5 mmol/L H2O2 caused culted cardiomyocytes apoptotic morophological characteristics, including nucleosomal DNA fragmentation in my- ocytes by agarose gell electrophoresis (DNA ladder), cell shrinkage, nuclear condensation, and chromatin margin by Giemsa-stained cell smears, and aneuploid peak (AP) - apoptotic bodies occurence by flow cytometry. CONCLU- SONS: H2O2 - induced apotosis in myocytes was a the - and concentration - dependent process, Treatment with low concentration of H2O2(10 mmol/L) rapidly induced a necrotic form of death characterized by smeared patterns of DNA digestion on agarose gel electrophoresis and lethal membrane disuption (as measured by LDH release). Exposure of 5 - 10 mmol/ L H2O2 induced cardiomyocytes apoptosis concurrently with biochemical changes of LDH and MDA increase in the medium.