The present study established a porcine intramuscular preadipocyte in vitro model to provide a new experimental method for the study of intramuscular fat metabolism. The 5-day old crossbred pig was purchased and intramuscular tissue was digested by collagenase type Ⅱ, digesta were passed through 400 screen mesh filter to isolate digested cells. Cells were subjected to centrifugation at 1 500 r/min, then resuspend the cells in DMEM/F12 supplemented with 10％ fetal bovine serum (complete medium), and the intramuscular preadipocytes were separated by differential velocity adherent technique: A media change after 2 h of seeding to remove the non-adherent cells. Subcultured cells after 48 h of constituted a confluent monolayer, complete medium supplemented with 0.5 mmol/L 3-isobutyl-1-methyl xanthine (IBMX), 1 μmol/L dexamethasone (DEX), 10 mg/L insulin (INS) induced cultured for 48 h, then changed the medium to complete medium supplemented with 10 mg/L insulin induced cultured for 48 h, at last, changed the complete medium cultured for up to 90％ of the cells secreted lipid droplets. Results: The cells adhered to the wall showed a short spindle or a prism appearance, and had a high rate of lipid accumulation. Their dynamic morphological changes, growth curve and stained with oil red O, all verified their intramuscular preadipocyte identity. And the expression of adiponectin was detected by both general PCR and Real-time flurescent quantitive PCR. This study successfully cultured porcine intramuscular preadipocyte by differential velocity adherent technique, and under controlled conditions, the preadipocytes replayed their hyperplasia process in vitro.
国家科技期刊平台
NSTL
万方数据
维普
