Background The fat derived protein adiponectin plays an important role in the regulation of glucose metabolism. The aim of this study was to provide the experimental basis for further investigating on adiponectin （A...Background The fat derived protein adiponectin plays an important role in the regulation of glucose metabolism. The aim of this study was to provide the experimental basis for further investigating on adiponectin （ADPN） function. Its eukaryotic recombinant was constructed and expressed in precursor cells of 3T3-L1 adipocytes. The effects of dexamethasone on peroxisome proliferator activated receptor-gamma （PPAR-y） mRNA expression in 3T3-L1 cells with human recombinant adiponectin were assessed. Methods The recombinant plasmid pMD18-T-hADPN and eukaryotic expression vector pcDNA3.1^＋ were digested by two restrictive endonucleases and adiponectin and linear pcDNA3.1^＋ were obtained. Then, they were ligated and translated into JM109. The recombinant pcDNA3.1^＋-hADPN so obtained was identified by digestion by restrictive endonuclease and nucleotide sequencing. The 3T3-L1 precursor cells were transfected using SuperFect Transfection Reagent （Qiagen）. Furthermore, 3T3-L1 cells with human recombinant adiponectin incubated with dexamethasone （0.5 mmol/L） for 24 hours, cells were collected and total RNA was extracted. The PPAR-γ mRNA expression was quantified by semiquantitative reverse transcription-polymerase chain reaction （RT-PCR）. Results After eukaryotic recombinant was digested by Hind Ⅲ and EcoR Ⅰ, fragments of 800 bp and 5.4 kb were identified by nucleotide sequence scanning and consistent with theoretical values. Electrophoretogram of RT-PCR in 3T3-L1 precursors showed only one band in front of 250 bp, which was consistent with theoretical value 234 bp. In the 3T3-L1 cells, 3T3-L1 cells with plasmid and 3T3-L1 cells human recombinant adiponectin, treatment with dexamethasone （0.5 mmol/L） decreased PPAR-y mRNA expression compared to untreated controls （P〈0.01）. Effect of dexamethasone on PPAR-γ mRNA expression in 3T3-L1 cells was reversed by stably transfected human recombinant adiponectin. Conclusion The 3T3-L1 cells stably transfected human recombinant adiponectin had increased PPAR-γ mRNA expression. Dexamethasone suppressed PPAR-γ mRNA expression in the 3T3-L1 cells. Effect of dexamethasone on PPAR-γ mRNA expression in 3T3-L1 cells was reversed by stably transfected human recombinant adiponectin.展开更多
文摘Background The fat derived protein adiponectin plays an important role in the regulation of glucose metabolism. The aim of this study was to provide the experimental basis for further investigating on adiponectin （ADPN） function. Its eukaryotic recombinant was constructed and expressed in precursor cells of 3T3-L1 adipocytes. The effects of dexamethasone on peroxisome proliferator activated receptor-gamma （PPAR-y） mRNA expression in 3T3-L1 cells with human recombinant adiponectin were assessed. Methods The recombinant plasmid pMD18-T-hADPN and eukaryotic expression vector pcDNA3.1^＋ were digested by two restrictive endonucleases and adiponectin and linear pcDNA3.1^＋ were obtained. Then, they were ligated and translated into JM109. The recombinant pcDNA3.1^＋-hADPN so obtained was identified by digestion by restrictive endonuclease and nucleotide sequencing. The 3T3-L1 precursor cells were transfected using SuperFect Transfection Reagent （Qiagen）. Furthermore, 3T3-L1 cells with human recombinant adiponectin incubated with dexamethasone （0.5 mmol/L） for 24 hours, cells were collected and total RNA was extracted. The PPAR-γ mRNA expression was quantified by semiquantitative reverse transcription-polymerase chain reaction （RT-PCR）. Results After eukaryotic recombinant was digested by Hind Ⅲ and EcoR Ⅰ, fragments of 800 bp and 5.4 kb were identified by nucleotide sequence scanning and consistent with theoretical values. Electrophoretogram of RT-PCR in 3T3-L1 precursors showed only one band in front of 250 bp, which was consistent with theoretical value 234 bp. In the 3T3-L1 cells, 3T3-L1 cells with plasmid and 3T3-L1 cells human recombinant adiponectin, treatment with dexamethasone （0.5 mmol/L） decreased PPAR-y mRNA expression compared to untreated controls （P〈0.01）. Effect of dexamethasone on PPAR-γ mRNA expression in 3T3-L1 cells was reversed by stably transfected human recombinant adiponectin. Conclusion The 3T3-L1 cells stably transfected human recombinant adiponectin had increased PPAR-γ mRNA expression. Dexamethasone suppressed PPAR-γ mRNA expression in the 3T3-L1 cells. Effect of dexamethasone on PPAR-γ mRNA expression in 3T3-L1 cells was reversed by stably transfected human recombinant adiponectin.
